UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

Comparative studies on several catalytic properties of biosynthetic L-threonine dehydratase (Deaminating).. Kripps, Robert Stephen 1972

You don't seem to have a PDF reader installed, try download the pdf

Item Metadata

Download

Media
[if-you-see-this-DO-NOT-CLICK]
UBC_1972_A6_7 K75.pdf [ 9.4MB ]
Metadata
JSON: 1.0101499.json
JSON-LD: 1.0101499+ld.json
RDF/XML (Pretty): 1.0101499.xml
RDF/JSON: 1.0101499+rdf.json
Turtle: 1.0101499+rdf-turtle.txt
N-Triples: 1.0101499+rdf-ntriples.txt
Original Record: 1.0101499 +original-record.json
Full Text
1.0101499.txt
Citation
1.0101499.ris

Full Text

COMPARATIVE STUDIES ON SEVERAL CATALYTIC PROPERTIES OF BIOSYNTHETIC L-THREONINE DEHYDRATASE (Deaminating) IN SEVEN SPECIES OF UNICELLULAR MARINE PLANKTONIC ALGAE  by  ROBERT STEPHEN KRIPPS B.H.E., University of British Columbia, 1971  A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in the division of " HUMAN NUTRITION SCHOOL OF HOME ECONOMICS We accept this thesis as conforming to the required standard.  THE UNIVERSITY OF.BRITISH COLUMBIA August, 1972  In p r e s e n t i n g an  this  thesis  advanced degree at  the  Library  I further for  shall  by  his  of  this  written  fulfilment of  University  of  make i t f r e e l y  agree that  scholarly  the  in p a r t i a l  permission  p u r p o s e s may  representatives. thesis  for  be  available  granted  gain  permission.  Department  Date  ^,  1-,  \\W c  r  for  for extensive by  the  It i s understood  financial  The U n i v e r s i t y o f B r i t i s h V a n c o u v e r 8, Canada  British  Columbia  shall  requirements  Columbia,  Head o f my  be  I agree  r e f e r e n c e and copying of  that  not  the  that  study.  this  thesis  Department  copying or  for  or  publication  allowed without  my  i  ABSTRACT  Several aspects of L-threonine dehydratase from seven species of unicellular marine planktonic algae were investigated; (1) the disulfide group requirement for a c t i v i t y of the enzymes from two cryptomonads, (2) monovalent inorganic cation requirement for enzyme a c t i v i t y , (3) substrate s p e c i f i c i t y and substrate analog inhibitions, (4) a l l o s t e r i c activation and i n h i b i t i o n and diverse effects from other amino acids, (5) pH optima of the algal enzymes with particular emphasis on the elucidation of the unique pH-activity response of the enzyme from Hemiselmis virescens. The threonine dehydratases from Chroomonas salina and Hemiselmis virescens require disulfide groups for enzyme a c t i v i t y as exemplified by the specific i n h i b i t i o n exerted by a l l t h i o l reagents tested, which inhibition could be p a r t i a l l y reversed or prevented by the appropriate treatments. Sulfhydryl group requirement: for enzyme a c t i v i t y was confirmed and i t was demonstrated that these^ groups are essential for feedback i n h i b i t i o n from L-isoleucine. A l l algal enzymes appear to require monovalent alkali-metal cations for f u l l expression of a c t i v i t y , more s p e c i f i c a l l y K and NH*. +  was exceptional i n showing maximal stimulation from L i . +  Anacystis marina  Organic cations were 2+  2+  without effect whereas some inhibition from certain divalent cations (Zn , Cu ) 2 - 2 and anions (N0 , I , C10 ) were observed, whilst HPC^ and SO^. were stimulatory. 3  3  Aside from L-threonine, the algal enzymes extended substrate a c t i v i t y to L-serine and L-aliothreonine.  In addition to i t s known threonine dehydratase,  Chroomonas salina" appeared to produce a serine.dehydratase which accounted f o r the r e l a t i v e l y high substrate - a c t i v i t y observed toward L -serine with this species. Inhibition from substrate analogs was limited to L-homoserine and L-serine despite the substrate a c t i v i t y of the l a t t e r .  ,  The mechanism for the peculiar  ii  mode'Of  inhibition evinced by L-homoserine remains unknown whereas that of  L-serine appears to result from inactivation of the enzyme. With the exception of Cyclotella nana and to a lesser extent Hemiselmis virescens, a l l the algal enzymes were subject to feedback inhibition from Lisoleucine, which inhibition was pH dependent, subject to reversal by L-valine, and could be duplicated by the analog L-O-methyl threonine.  Several other  amino acids (L-leucine, L-norvaline, L-valine) were able to inhibit most enzymes when present at high concentration.  It was proposed that the mode of inhibition  by these latter amino acids may occur via interaction at the site specific for allosteric inhibition.  L-Valine at low concentration effected pronounced  activation of the enzymes and was thusly assigned the role of allosteric activator, acting at a site distinct from that of L-isoleucine or L-threonine. Hemiselmis virescens was distinctly unique in that, unlike the other algal enzymes, i t displayed two pH-activity optima. The investigation of this phenomenon was pursued in two ways (i) examination of enzyme response to various potential effectors (nucleotides, L-methionine, L-aspartate,^ L'-cystathionine) at a pH intermediate between the two optima, (ii) examination of enzyme response to known effectors (L-valine, L-isoleucine) at the two pH optima. It was concluded from these studies that Hemiselmis virescens may produce a culture-dependent mixture of two threonine dehydratases, one of which is generally similar to the other algal enzymes, the other of which is insensitive to the usual allosteric regulation yet is not a standard biodegradative isozyme.  iii  ACKNOWLEDGEMENTS  I wish to express my sincere appreciation to: Dr. Naval J. Antia, Fisheries Research Board of Canada, Vancouver Laboratory, for his priceless advice, criticism and above a l l , moral support during the research and preparation of this dissertation. Dr. Indrajit D. Desai, Division of Human Nutrition, School of Home Economics, University of British Columbia, for accepting me as a graduate student and arranging the financial support which made this investigation possible. Rinske, for the understanding and devoted love she unfailingly gave me during my academic pursuits.  iv  TABLE OF CONTENTS Page ABSTRACT  i  ACKNOWLEDGBENr  iii  LIST OF TABLES  vii  LIST OF FIGURES  viii  INTRODUCTION TO THE PRESENT INVESTIGATION  1  REVIEW OF LITERATURE Differentiation of biosynthetic and biodegradative threonine dehydratases  4  Bacterial biosynthetic threonine dehydratases  5  Yeast biosynthetic threonine dehydratases  6  Plant and fungal biosynthetic threonine dehydratases...  7  MATERIALS AND METHODS Algal cultures  8  Algal extracts  8  Reaction mixtures  9  Assay procedure  9  Chemicals PART I.  10  Substrate Specificity Studies and Inhibition from Substrate Analogs.  Evidence for Additional L-Serine Dehydratase Activity  in the Cryptomonad C. salina  12  RESULTS Substrate specificity  12  Optimum pH of the deaminase reaction in relation to substrate  13  V  Page Sensitivity to L-isoleucine in relation to substrate  13  Substrate-related response of C. salina enzyme activity to various reagents and effectors  14  Inhibition of algal TDH activity by structural analogs of L-threonine  DISCUSSION PART II.  15  17  Feedback inhibition, Allosteric Activation and Diverse Effects from Other Amino Acids  27  RESULTS Response to L-isoleucine at saturating substrate concentration  27  pH dependency of L-isoleucine inhibition  28  Response to L-valine at saturating substrate concentration  29  Reversal of L-isoleucine inhibition by L-valine  29  Alteration of substrate saturation kinetics by L-valine and L-isoleucine  30  Effects of other amino acids on the algal enzymes  31  DISCUSSION  32  PART III. Re-investigation of pH Optima with Strict Buffer-Ion Control. Evidence for 2 pH Optima with differential Sensitivity to Allosteric Effectors i n the Cryptomonad H. virescens  44  RESULTS pH optima of the deaminase reaction  44  vi  Page Response of H. virescens enzyme to potential effectors at pH intermediate between the 2 optima  45  Differential effects from allosteric compounds at the 2 pH optima DISCUSSION LITERATURE CITED  46 47 55  APPENDIX A.  The Common Locus of Threonine Dehydratase i n Branched-chain Amino Acid Biosynthesis  B.  63  Schematic Representation of Threonine Biosynthesis and the  C.  Major Peripheral Regulatory Circuits  64 *  Standard Calibration Curve for a-Ketobutyrate and Pyruvate  65  SUPPLEMENTS I.  L-Threonine Deaminase in Marine Planktonic Algae. Disulfide and sulfhydryl group requirements of enzyme activity" i n two cryptophytes  II.  66-94  L-Threonine Deaminase i n Marine Planktonic Algae. Stimulation of activity by monovalent inorganic cations and diverse effects from other ions  95-129  vii  LIST OF TABLES  TABLE  Page  . 1  Culture and standard assay conditions of the algal species used  2  Algal deaminase activity observed from structural analogs of Lthreonine tested as substrate  3  23  Effects of various amino acids on algal threonine dehydratase activity  7  22  Effects of substrate analogs on algal TDH activity towards Lthreonine  6  21  Substrate-related response of C. salina deaminase activity to various reagents and effectors  5  20  Sensitivity of algal deaminases to L-isoleucine inhibition in relation to substrate  4  11  37  Effects of certain nucleotides and amino acids on enzyme activity of H. virescens at pH 8.5  51  viii  LIST OF FIGURES FIGURE 1  Page pH-activity profile of the deaminase reaction in relation to substrate  2  A.  Effects of L-serine and L-homoserine concentration on  threonine dehydratase activity of A. marina and A. quadruplicatum.. B.  40  Reversal of L-isoleucine inhibition of the algal enzymes by graded concentrations of L-valine  8  =. 39  Effect of L-valine concentration on algal threonine dehydratase activity  7  38  Influence of pH on the effects from L-isoleucine and L-valine on threonine dehydratase activity  6  26  Effect of L-isoleucine concentration on algal threonine dehydratase activity  5  25  Effect of L-homoserine and L-serine on the rate of L-threonine deamination by the enzymes from T. maculata and C_. salina  4  25  Effects of L-homoserine and L-serine on the substrate  saturation kinetics of the A. marina enzyme 3  24  41  Effects of L-valine and L-isoleucine on the substrate saturation kinetics of the enzymes from T. maculata, C. nana and A. marina  42  ix  FIGURE 9  Page Effect of L-O-methyl threonine concentration on algal threonine dehydratase activity  43  10  pH-activity profiles for six algal threonine dehydratases  52  11  pH-activity profiles for the enzymes from three nutritionally different cultures of H. virescens  12  53  pH-activity profiles i n the presence of L-isoleucine for the enzymes from three nutritionally different cultures of H. virescens  13  53  Effects of L-homoserine, L-serine and L-valine on the H. virescens enzyme at pH 8 and pH 9  54  1  INTRODUCTION TO THE PRESENT INVESTIGATION  It was the purpose of this investigation to examine several interesting features of algal biosynthetic threonine dehydratase, some of which were indicated, but not adequately investigated i n the earlier study from our laboratories (23). This earlier work involved the preliminary characterization of the enzyme from seven species of unicellular marine planktonic algae (belonging to five taxonomic divisions).  These species were reported to exhibit considerable differences i n  specific activity as well as significant effects from the nutritional conditions of algal culture.  The enzymatic production of a-ketobutyrate from L-threonine was  confirmed by the isolation and chromatographic identification of i t s 2,4,-dinitrophenylhydrazbne. -The algal enzymes showed pH optima i n the range of 8.5-9.5 and sigmoid or paraboloid kinetic response to L-threonine concentration.  With the  exception of Cyclotella nana, a l l the algal enzymes were strongly inhibited by L-isoleucine; L-valine, EDTA, AMP, ADP, and cyclic 3',5'-AMP were reported to have no significant effect. Several carbonyl reagents strongly inhibited enzyme activity and this inhibition was reversed to varying degrees by pyridoxal 5'phosphate.  Excepting iodoacetamide, a l l the reagents known to modify protein  sulfhydryl groups inhibited the activity and these inhibitions were partially reversed by dithiothreitol.  The enzymes of Chroomonas salina and Hemiselmis  virescens were also markedly inhibited by dithiothreitol.  These results character-  ized the algal threonine dehydratases generally as isoleucine-regulated, pyridoxal phosphate requiring, allosteric enzymes similar to the corresponding TDH (BS) previously reported for bacteria, fungi and higher plants.  In addition, the  algal enzymes appeared to require sulfhydryl groups for the expression of activity. The C. nana enzyme appeared to be insensitive to isoleucine regulation, and the  2  cryptophyte enzymes appeared to require disulfide groups for activity. When this present investigation was initiated, i t s scope was intended to include the study of the following features of the algal enzymes 1) the particular disulfide and sulfhydryl group requirements of the enzymes from two cryptophytes,  2) the effects of cations and anions on the activity of a l l the  algal enzymes previously examined, 3) substrate specificity and substrateanalog inhibitions, 4) specificity of feedback regulation by L-isoleucine and structurally related amino acids, 5) the causes of the previously reported peculiar isoleucine-induced feedback inhibition of the enzyme from H. virescens. From this l i s t , the study of the f i r s t two features yielded such f r u i t f u l results that they called for priority of publication. These have now been prepared as papers and submitted for publication, and are appended to this dissertation as Supplements I and II.  Their results w i l l be summarized i n the  next two paragraphs. Disulfide and sulfhydryl group requirements of enzyme activity i n two cryptophytes (see Supplement I).  A systematic investigation of the cryptophyte s "disulfide 1  requirement" revealed that both C. salina and H. virescens were sensitive to inhibition from a l l thiols tested (dithiothreitol, cysteine, etc.) but showed no effect from ascorbic acid or reduced NAD.  By contrast, the enzyme activities  from the five non-cryptophycean algae were generally not affected by any of these reagents.  The thiol-reagent inhibition of the cryptomonad enzymes (i) achieved  saturation with 60-70 % reduction i n activity, (ii) was considerably reduced by pretreatment of the enzymes with L-threonine and L-isoleucine, and ( i i i ) was partially reversed by subsequent treatment with arsenite and exposure to a i r . It was deduced that such inhibitions were caused by thiol-specific reduction of enzyme-protein disulfide groups essential for the f u l l expression of activity  3  and that these groups were susceptible to ready reductive cleavage and oxidative restoration.  The additional activity-requirement of the cryptomonad enzymes  for sulfhydryl groups was confirmed a) by the study of their sensitivity to inhibition from mercurials and disulfide-sulfhydryl exchanging reagents, and b) by the partial reversal of these inhibitions from subsequent treatment with dithiothreitol.  Both cryptophyte enzymes were desensitized to feedback inhibition  from L-isoleucine by prior exposure to subinhibitory concentrations of HgCl or 2  dithiodipyridine. Stimulation of TDH activity by monovalent inorganic cations and diverse effects from other ions (see Supplement II).  Threonine dehydratases from the five  classes of algae (seven species) were a l l activated to varying degrees by monovalent inorganic cations.  The activation was generally the strongest (3-5 fold)  with K and NH*, whilst L i , Rb , and C s showed intermediate orders varying +  with algal species.  +  +  +  Anacystis marina was exceptional i n showing strongest  stimulation (5-fold) from L i and more pronounced activation from Na than Cs , +  +  +  whilst Tetraselmis maculata showed another type of response with the least effect from L i * and markedly greater activation from Rb than NH*. Activation +  was hyperbolic i n response to ion concentration and specific for monovalent inorganic cations with indications of a coenzyme type of role. 2*f*  were inert and the divalent cations Mg or without effect.  24-  , Ca  2-t*  , Zn  Organic cations  2-f-  , Cu  were either inhibitory  Among the anions tested, chloride, bromide, fluoride,  bicarbonate showed no effect; iodide, nitrate, chlorate were inhibitory, whilst phosphate and sulfate were slightly stimulatory.  It was concluded that the  algal threonine dehydratases may have an absolute K or NH* requirement for +  in vivo expression of activity.  4  REVIEW OF LITERATURE Differentiation of biosynthetic and biodegradative threonine dehydratases L-Threonine dehydratase (L-threonine hydro-lyase, deaminating; EC 4.2.1.16) catalyses the conversion of L-threonine to a-ketobutyric acid and ammonia.  The  product, a-ketobutyrate may be further metabolized in two ways 1) sequential enzymic alteration ultimately leading to the biosynthesis of L-isoleucine (see Appendix A for details), 2) oxidative catabolism to yield energy.  Threonine  dehydratases (TDH) involved in the former process are appropriately termed "biosynthetic" (BS) and for the latter process, "biodegradative" (BD).  Identi-  fication of the isozymes i s based primarily on certain regulatory characteristics; TDH (BS) being subject to feedback inhibition by L-isoleucine and TDH (BD) being isoleucine insensitive but activated by AMP  (72) or ADP  (78). With two exceptions  (46), the pH optima for the TDH (BS) have been reported within the range 8.09.5 whereas the TDH (BD) range widely in their optima from pH 6.2-10.5. Molecular weights for these enzymes have been reported as low as 147,000 (72) although they have generally been established in the v i c i n i t y of 200,000 (7,21, 28,35,38,56,73) with a dimeric or tetrameric subunit composition (35,38,56). The requirement of pyridoxal phosphate as a bound cofactor and a general sulfhydryl group requirement has been conclusively established.  Although the  simultaneous occurrence of both isozymes in one organism has been reported (46, 48,74,77), the usual circumstance i s the presence of only one type. The ensuing descriptions therefore, pertain only to biosynthetic threonine dehydratases from various sources which have hitherto been studied and represents a condensation of the major physico-chemical and regulatory characteristics of this enzyme.  5  Bacterial TDH  (BS)  Since the i n i t i a l work on threonine dehydratase of Escherichia c o l i (76,77), numerous reports have appeared concerning this enzyme from a variety of bacterial sources (3,7,10,12-16,18,19,24,25,33-37,39,45,54-59,66,67,73). Sensitive feedback inhibition from isoleucine was observed in a l l but a few instances (28,45) wherein high concentration of isoleucine was required to effect inhibition and then only with low levels of substrate concentration.  On  the other hand, mutant as opposed to the wild type TDH have frequently shown resistance to isoleucine (4,21,52).  Feedback inhibition has shown pH dependency  (3,12,19,22,26,54-56,73) with optimum effects in the v i c i n i t y of pH 8 and virtual abolition of response within pH 9.5-11. Valine has been implicated as an allosteric effector since low concentration has been observed to activate the deaminating reaction (3,7,12,19,29,34) in the presence of sub-saturation levels of substrate.  At high concentration of valine,  however, inhibition of the enzyme reaction has been observed (3,18,19,39,54-56, 73), which inhibition has shown an analogous pH dependency to that of isoleucine (3,54).  Valine has also displayed the a b i l i t y to partially reverse the isoleucine-  induced inhibition of TDH  (7,12,19,26,39,67,73).  Other amino acids reported to  inhibit the enzyme at high concentration are leucine (3,15,56,73), norvaline (3, 15,73), norleucine (3,73) and a-aminobutyrate (3).  The a b i l i t y of these latter  amino acids to either overcome isoleucine inhibition or activate at low substrate concentration has been occasionally described (15,29,56). Substrate saturation kinetics have usually exhibited sigmoidal rather than hyperbolic profiles although the latter situation has also been reported (3,24, 28,38,58,59,67,72).  In a l l instances, the presence of isoleucine either increased  or conferred sinuosity upon the substrate saturation curves whereas valine  6  either increased or conferred hyperbolicity upon these curves (12,19,34,69). The opposing nature of the alterations to substrate saturation kinetics by isoleucine and valine has prompted the suggestion that the i n i t i a l profile with threonine alone may reflect the relative amounts of contaminating isoleucine:valine in the enzyme preparation (37) or may be the net result of an inadequately stabilized enzyme (31,32). Aside from L-threonine, L-serine i s the only other amino acid able to serve as substrate, albeit with substantially lower reactivity relative to threonine (10,15,28,33,55,56,73).  Conversely, the substrate analogs allo-  threonine (15,29,59) and homoserine (15,29) appear to function as inhibitors of the enzyme activity toward threonine; such inhibition has also been reported from serine despite i t s capacity to function as a substrate (28,56,66). Systematic investigations of any ionic requirements of the bacterial TDH have been infrequently performed, although high ionic strength i s commonly employed to maintain enzyme stability.  From reports which have studied this  aspect, i t appears that monovalent cations may assist in the f u l l expression of enzyme activity (7,73). Yeast TDH  (BS)  Threonine dehydratase has been studied i n yeast (5,6,9,22,41,42,49) and has exhibited gross similarity to the bacterial counterparts.  Feedback inhibition  from isoleucine has been reported in a l l cases and on one occassion, low concentration of isoleucine stimulated enzyme activity (22).  Feedback inhibition was  demonstrated to be pH dependent (9,22,41) and subject to reversal by valine (9,22), norleucine or a-aminobutyrate (9). At low concentration, valine serves to activate the enzyme (6,9,49) although inhibition from high concentration has not been reported.  Other amino acids reported as inhibitory are allothreonine, homoserine,  7  and leucine (9,41).  Substrate saturation kinetics were sigmoidal. for a l l cases  studied and attained a hyperbolic profile in the presence of valine (6,9,22,49) or the amino acids which reversed isoleucine inhibition (9). Monovalent cations appear to be required for the optimal expression of yeast TDH activity, particul a r l y ammonium, which has been implicated as an "end-product activator" (9,42). Plant and.Fungal TDH (BS) Threonine dehydratases of plants (26,46:48,53,60,69,70,74) and fungi (47) undergo feedback inhibition from isoleucine, which inhibition i s pH dependent (26) and subject to reversal from valine (26,60,69,70). An interesting feature of these enzymes is that the role of allosteric activator has been assigned not only to valine (69,70), but also to norvaline, norleucine, aspartate (46,48,74) and in the case of the fungal TDH, only phenylalanine (47).  Amino acids reported to  inhibit the enzyme at high concentration are leucine (60), valine (26,69,70) and homoserine (47,74).  Substrate saturation curves have been reported as  hyperbolic (60,69,70) or sigmoidal (46-48,74); in the latter case, the presence of allosteric effector evinced a hyperbolic response to substrate concentration. Compounds other than L-threonine capable of functioning as substrates with lowered reactivity are L-serine (26,60,69) and for a rose tissue culture enzyme, Lallocystathionine (26).  Monovalent cations (more specifically potassium and  ammonium) appear to be required for the f u l l expression of TDH activity (26,29).  8  MATERIALS AND METHODS  Algal cultures Table 1 l i s t s the algal strains employed in the investigations together with other relevant data.  Details of the algae, their source and maintenance  have been communicated (1) as have the techniques of mass culture, harvesting and freeze drying (23).  Protein content of each algal culture was  determined  by micro-Kjeldahl N-determinations and were previously reported (23).  Excepting  the 2 cryptophytes, a l l the algae used i n this study were grown photoautotrophically with added vitamins.  C_. salina was cultured under 3 sets of conditions,  (i) photoautotrophic (vitamins, light), (ii) photoheterotrophic (glycerol, vitamins, light), ( i i i ) chemoheterotrophic  (glycerol, vitamins, darkness) (17).  Being  unable to use nitrate as N-source or to grow i n darkness on organic substrates hitherto tested, H. virescens was cultured phototrophically with the same vitamins under 3 other conditions (i) with urea (2 mM) as N-source, (ii) with glycine as N-source, ( i i i ) with glycine (4mM) glycerol concentration was 0.25 M.  and glycerol (2,17).  (4mM)  Where present, the  Unless otherwise stated, the enzyme tests  concerning C. salina and H. virescens were normally made with the glycerollight grown culture of the. former and the glycine-light grown culture of the latter. Algal extracts Suspensions of algal powder in appropriate buffer were subjected to ultrasonic oscillation (5 min, 0-4 C) in a Raython 10 Kcycle magnetostrictive oscillator at a maximum output of 1.1 A.  9  Reaction mixtures Unless otherwise indicated, a l l enzyme incubation mixtures contained unmodified whole algal sonicate (0.5 ml i n 0.2 M potassium N-tris(hydroxymethyl) methylglycine (K-Tricine), pH 8.5), pyridoxal phosphate (0.1 jimole), and Lthreonine (80 umoles) i n a final volume of 1 ml.  For studies which involved  the effects of pH, the following buffers were substituted for K-Tricine: tris(hydroxymethyl)methylamine (Tris-HCl), pH 7.0-8.7; 2-methyl-2-aminopropanol (Map-HCl), pH 9.0-10.0. When these latter buffers were employed, KC1 (final concn 0.1 M) was included to satisfy the enzyme requirement for monovalent cation. The mixtures were normally preincubated f i r s t with the test reagent o o (15 min, 22 C), then with pyridoxal phosphate (5 min, 37 C), and f i n a l l y incubated with threonine at the latter temperature for periods varying with the algal species (usually as indicated in Table 1).  Identical preincubations were  effected on controls taken without the test reagent. unincubated and f u l l y incubated controls.  A l l the tests included.both  The enzyme.reaction was terminated by  the addition of aqueous trichloroacetic acid (0.5 ml, 50 % w/v) followed by centrifugation (3,000 g, 20 min). Assay procedure Keto acid produced was assayed according to the method of Friedemann and Haugen (30) with minor modifications.  A 1 ml aliquot of the reaction mixture  supernate was treated with 2,4,-dinitrophenylhydrazine (1 ml, 1 % i n 2 N HCl) and allowed to react (5 min, 22°C) after which absolute ethanol (1 ml) and benzene (3 ml) were added and the mixture vortex-mixed at high speed (1 min) and centrifuged (2,000 g, 5 min).  A 2 ml aliquot of the organic layer was then  vortex-mixed for 1 min with sodium carbonate (3 ml, 10 % w/v) followed by  10  centrifugation (2,000 g, 5 min). A 2 ml aliquot of the aqueous layer was then combined with sodium hydroxide (2 ml, 1.5 N) and absorbance determined at 435 nm on a Beckman DU-2 spectrophotometer.  From the corrected optical density, mymoles  keto acid produced was calculated by means of a conversion factor obtained from standard calibration curves of a-ketobutyrate and pyruvate (see Appendix C). Chemicals Chromatographically homogeneous L-threonine, other L-amino acids, and dithithreitol were obtained from Calbiochem (Los Angeles, Calif.); D-amino acids and L-O-methyl threonine from Sigma (St. Louis, Mo.); AMP, ADP, ATP and cyclic 3',5'-AMP from P-L Biochemicals (Milwaukee, Wis.). A l l other reagents used were of the highest purity grade commercially available.  11  Table 1.  Culture and standard assay conditions of the algal species used.  Alga  Culture conditions  Assay conditions  Light  Dry alga  Added organic nutrients  (mg)  Incubation (min  Q  37° C)  CHLOROPHYTA Tetraselmis maculata (Te.ma.)  nil  40  nil  30  BACILLARIOPHYTA +  Cyclotella nana (Cy.na.) CRYPTOPHYTA  nil  4  20  glycerol  2  IS  glycerol  2  10  +  glycine  2  15  +  Jglycine jglycerol  4  15  4  20  Chroomonas salina(Ch.sa.(NA)) (Ch.sa.) (Ch.sa.CGly.  D))  Hemiselmis virescens fJHe.vi.) (He.vi. (Gly.L)) +  (He.vi.(Urea) RHODOPHYTA Porphyridium cruentum (Po.cr.) CYANOPHYTA Agmenellum quadruplicatum (Ag.qu.) Anacystis marina (An.ma.)  urea  nil  30  nil  2  10  nil  2  15  ^ .Abbreviations in parentheses are used to denote these species in figures and tables. ' Recently redesignated Thalassiosira pseudonana (see refs. 18 § 20 of Supplement I). * Presence (+) or absence (-) of continuous illumination (ca. 16,500 lux). ** In addition to the vitamins normally added to culture medium (see methods for concentrations used).  12  PART I.  Substrate Specificity Studies and Inhibition from Substrate Analogs.  Evidence for Additional L-Serine Dehydratase Activity  in the Cryptomonad C. salina  RESULTS Substrate specificity Several compounds of structural similarity to L-threonine were tested as to their substrate reactivity with the algal threonine dehydratases, the results of which are aummarized in Table 2.  Of the amino acids tested, L-serine and L-  allothreonine were deaminated by a l l species, the response of the former to graded concentration being sigmoidal with similar Km but lowered Vmax relative to L-threonine.  D-Threonine, D-serine, L-homoserine and L-O-methyl threonine  were not acted upon to any significant extent.  The slight activity obtained  with L-allothreonine (4-12 3 ) did not appear to be due to L-threonine contamination because the keto acid produced did not show the expected increase (as a constant % of the possible L-threonine impurity) on examining the saturation kinetics with L-allothreonine as substrate. Furthermore, the complete inhibition of this activity by L-isoleucine confirmed that the reaction was due to algal TDH acting inefficiently on L-allothreonine as substrate analog. With the exception of C. salina, a l l species showed substantially less reactivity with L-serine as compared to L-threonine (17-26 % at equimolar concentration).  C_. salina showed double the activity from L-serine relative  to L-threonine and appeared, therefore, to contain a serine dehydratase in addition to i t s known TDH.  (SDH)  Such simultaneous occurrence of both dehydratases  has been previously reported for a pseudomonad (18).  It was  subsequently  13  observed that another nutritionaly different culture of this algal species (C. salina (NA)) behaved analogously to the standard culture in that a l l amino acids tested were ineffective as substrates excepting L-allothreonine and Lserine which showed activities of 7 % and 236 % respectively when compared to equimolar concentrations of L-threonine.  Evidently, the a b i l i t y of C. salina  to deaminate L-serine is a species peculiarity and not a nutritionally-induced characteristic.  To this end, a series of comparative studies involving several  known properties of the cryptomonad and other algal TDH was initiated to compare the responses obtained in relation to the substrate used (ie. L-threonine or L-serine). Optimum pH of the deaminase reaction in relation to substrate The pH optima for the deamination of L-threonine by the algal TDH had been previously obtained (23) but were re-investigated under rigorously controlled conditions (see Part III). Examination of the pH optima when L-serine was used as substrate (Fig. IB) yielded identical activity profiles when compared to those obtained using L-threonine as substrate (Fig. 1 A ) , excepting the case of C. salina.  With this species, the optimum with L-serine (pH 8) did not coincide  with that using L-threonine  (pH 8 . 5 ) , which was indeed suggestive of an enzyme  other than TDH assuming responsibility for L-serine deamination. Sensitivity to L-isoleucine in relation to substrate Feedback inhibition from L-isoleucine is a characteristic feature of a l l except one algal TDH  (see Part II), a property alien to. serine dehydratase ( 1 8 ) .  In the event that'the C. salina enzyme preparation contained an active SDH in addition to the TDH, the overall dehydratase activity of this preparation on L-serine as substrate would be expected to be relatively insensitive to feedback  14  inhibition from L-isoleucine.  The data presented in Table 3 clearly demonstrates  that only C. salina (from both nutritionally different cultures) displayed any difference i n response to L-isoleucine from L-serine versus L-threonine as substrate.  It may be noted that the C. salina enzyme preparation did show some  inhibition (12 %) when L-serine was used as substrate, which inhibition appears to be due to the L-isoleucine-sensitive activity of the TDH component known to be present.  Based on this premise, the keto acid difference between the values  obtained without and with L-isoleucine may be reasonably assumed as due to the action of the TDH component only.  It was thus inferred that 12 % of the total  keto acid produced from 50 mM L-serine as substrate was the result of TDH action, the remainder  (88 %) being contributed from the activity of the SDH component.  Stoichiometric computations, equating the keto acid contribution (12 I ) from TDH action on L-serine to the known amount of keto acid produced from equivalent L-threonine by the same enzyme, give an estimate of 24 % conversion of L-serine relative to L-threonine, due to the TDH component only.  Referring to Table 2,  i t i s apparent that this estimated a b i l i t y of the C. salina TDH to deaminate Lserine i s i n agreement with the values obtained for the other algal enzymes (1726 % ) . Substrate-related response of C. salina enzyme activity to various reagents and effectors The C. salina TDH has shown certain characteristic properties, such as monovalent cation (see Supplement II) and disulfide group (see Supplement I) requirements and feedback regulatory effects from L-isoleucine and L-valine (see Part II). It was of interest to examine these and other effects on the the deamination obtained from L-serine by the C. salina enzyme preparation in order to further establish the presence of SDH activity in this system.  15  Table 4 shows the different responses obtained from such an examination comparing L-serine versus L-threonine as substrate.  In the f i r s t place, K was the more +  effective monovalent cation for L-serine deamination whilst NHI" was most effective towards L-threonine as previously observed, secondly, the known inhibitory effects of L-isoleucine, L-valine and dithiothreitol with respect to L-threonine deamination were markedly decreased with L-serine as substrate.  Moreover, the maximal  inhibitions induced by these reagents with L-serine as substrate followed the same relative pattern (although substantially reduced) as was obtained when Lthreonine functioned as the substrate.  These observations confirm the occurrence  of SDH i n C. salina and also lend support to the previous.inference that part of the total keto acid produced through the deamination of L-serine was the result of TDH action. EDTA had been previously shown to be without effect on the algal TDHs (23) but was tested with L-serine as substrate in view of a previous report (8) of a ferrous ion requirement for a bacterial SDH, which requirement appeared to be exceptional i n that no definite divalent cation involvement has been recorded for other serine dehydratases from bacterial, plant or animal sources (27,40, 65).  The ineffectiveness of EDTA on the deamination of L-serine by the C. salina  enzyme preparation suggests that the algal enzyme is no exception to the other SDHs and has no recognizable divalent cation requirement. Inhibition of algal TDH activity by structural analogs of L-threonine In view of previously reported inhibition (presumably via substrate antagonism) of other threonine dehydratases by L-serine, L-aliothreonine or L-homoserine, these substrate analogs were tested for effects on the activity of the algal TDHs towards L-threonine.  The results summarized i n Table 5 show that L-serine and  L-homoserine inhibit the enzyme reaction markedly whilst L-allothreonine has no  16  effect.  It is interesting to note that whereas both L-allothreonine and L-serine  could function as substrates to varying degrees, only the latter was inhibitory. To elucidate the nature of these inhibitions, the following enzymatic kinetics were examined (i) the effects of gradient concentrations of L-serine and L-homoserine at saturating concentrations of substrate, (ii) the response of graded substrate concentration to the presence of the analogs at concentration levels which were 50 I inhibitory at substrate saturation. The typical kinetic profiles obtained from this 2-fold examination are depicted i n Figs. 2A and 2B, which profiles were closely comparable for a l l the algal species examined. These results showed a distinct difference i n the nature of the inhibitory effect obtained from the two substrate analogs.  In response to increasing concentration  of the inhibitors, the enzyme activity drops rapidly with L-serine to attain a "saturating" degree of inhibition whereas with L-homoserine, there appears to be a threshold concentration prior to which no inhibition was evident- and beyond which an apparently linear decrease in activity was observed (see Fig. 2A). Although the net inhibition evinced by 50 mM L-serine and L-homoserine approached comparable magnitude for each algal enzyme, the latter amino acid required approximately  3-fold greater concentration to effect 50 % inhibition of the  enzyme. Substrate saturation kinetics for A. marina  have been shown to be sigmoidal  in the absence of allosteric effector (see Part II). This pattern was found to be altered differently by the inhibitions obtained from L-serine and L-homoserine. As expected from their concentrations used, both compounds produced 50 % reduction of Vmax at substrate saturation levels ( see Fig. 2B).  At low substrate levels  however, L-serine produced an even more pronounced inhibition whilst L-homoserine tended to weaken i t s inhibitory grasp with declining substrate concentration.  17  These effects on the substrate saturation kinetic profiles may be interpreted as caused by an overall decrease in Vmax with both substrate analogs but different effects on Km, which appears to be increased by L-serine but hardly altered by L-homoserine. When the deamination of L-threonine was measured as a function of the incubation period and compared with those obtained from simultaneous addition of either L-serine or L-homoserine, a difference i n the mode of inhibition from the substrate analogs (as exemplified by Fig. 3A) was again observed.  Apart  from the overall 50 I inhibition obtained, the rectilinear profile of the Lthreonine deamination reaction was not altered by the presence of L-homoserine, whereas i t was radically changed to a hyperbolic profile from the presence of L-serine.  This latter profile reflected a similar course of the deamination  of L-serine taken as a control without the L-threonine.  In the case of C_. salina  (Fig. 3B), the deamination course was linear for L-threonine, L-homoserine + Lthreonine and L-serine but the slope was considerably greater for L-serine relative to L-threonine, providing additional evidence for the simultaneous of an active serine dehydratase.  DISCUSSION Biosynthetic threonine dehydratases from a variety of surces have been genera l l y reported to extend substrate specificity to L-serine (10,15,26,28,33,55,56, 60,69,73) with a reactivity considerably less than that for L-threonine. A few reports have appeared describing TDHs which may deaminate other amino acids, notably allocystathionine for an enzyme from rose tissue culture (26) and allothreonine in the case of a biodegradative TDH from sheep liver (65). The algal enzymes, being able to act upon both L-serine and L-allothreonine (although the  18  latter was affected to a very small extent), may be likened to the animal, rather than bacterial or plant enzymes in this respect.  Further substantiation of this  resemblance arises from the observation that the algal enzymes, like that from sheep l i v e r , were not subject to the competitive inhibition from allothreonine previously reported for yeast (9) and bacterial (15,29,59) biosynthetic TDHs. The plant enzymes do not appear to have been sufficiently investigated in this respect. Among the algae studied in the present investigation, the cryptomonad C. salina was unique in showing evidence of an enzyme (other than TDH) capable of readily deaminating L-serine.  That the enzyme in question was an SDH was substantiated  by studies wherein certain known properties previously characterized for the C. salina TDH were compared to those manifested when L-serine replaced L-threonine as substrate; (i) shift in pH optimum from 8.5  (for L-threonine) to 8.0  (for L-  serine), (ii) abolition of the characteristic effects from dithiothreitol, Lisoleucine and L-valine (known for TDH activity) on substitution of L-serine for L-threonine,  ( i i i ) reversal of the order of activation by two monovalent cations  on exchanging the two substrates.  In addition, apparent linearity of L-serine  deamination and the failure of L-homoserine to function as substrate negate the possibility of an active homoserine dehydratase which could conceivably deaminate L-serine (43).  These findings are in agreement with previously reported proper-  ties of certain SDHs occurring either singly or in conjunction with a TDH (18,43,65). Previous investigators have reported inhibitory effects from L-serine (28, 56,65,66) and L-homoserine (29,47,65,74) in spite of the former amino acid's a b i l i t y to undergo deamination by TDH.  Irreversible inactivation of the enzyme  has been proposed to account for this paradoxical L-serine inhibition and this explanation may apply to the algal enzymes as well in view of the conversion of  19  the L-threonine deamination velocity from a rectilinear to a hyperbolic profile on simultaneous incubation with L-serine, which response is strikingly similar to that reported for the sheep liver TDH (65). This phenomenon of inactivation has been described for several bacterial biosynthetic TDHs (28,56,66) and animal biodegradative TDHs (44,65). Little is known regarding the mechanism of inactivation involved although i t has been hypothesized that L-serine, by binding to the enzyme in a "tighter" manner than L-threonine, affects the binding constant of the cofactor (pyridoxal phosphate), ultimately leading to alteration in the balance of active holoenzyme and inactive apoenzyme (44). The question of L-homoserine inhibition however, poses a completely different situation from that of L-serine since the inhibition became evident only at high substrate levels.  This indicates that the L-homoserine inhibition may not be  competitive with L-threonine for direct interaction at the substrate binding sites as has been suggested (29) since, i f this were the case, inhibition would undoubtedly be greater, not nonexistent at low levels of substrate. Because of the immediate involvement of homoserine in the biosynthesis of threonine (see Appendix B), i t is tempting to speculate that homoserine may be interrelated with threonine dehydratase in some presently unknown precurssor-product mechanism.  20  Table 2.  Algal deaminase activity observed from structural analogs of L-threonine tested as substrate?  Enzyme activity (I of control) Substrate (50 mM)  Ch.sa.  He.vi.  Ag.qu.  An.ma.  Po.cr.  Te.ma.  Cy.na.  L-threonine  100  100  100  100  100  100  100  6  8  9  12  9  4  6  L-O-methyl threonine  <2  <2  <2  <2  <2  <2  <2  D-threonine  <2  <2  <2  <2  <2  <2  <2  L-serine  197  24  19  17  18  20  26  D-serine  <2  <2  <2  <2  <2  <2  <2  L-homoserine  <2  <2  <2  <2  <2  <2  <2  L-allothreonine  *When tested, the structural analogs were added to the reaction mixture in place of the L-threonine normally used in the standard enzyme incubation procedure.  21  Table 3.  Sensitivity of algal deaminases to L-isoleucine inhibition in relation to substrate.*  Enzyme activity (% of control) Alga  L-Ileu. (5 mM)  Ch.sa.  Ch.sa.(NA)  He.vi.  Ag.qu.  An.ma.  Po.cr.  Te.ma.  L-Serine  Substrate (50 mM)  L-Threonine  -  100  100  +  88  4  -  100  100  +  84  6  -  100  100  +  48  46  -  100  100  +  8  10  -  100  100  +  6  8  -  100  100  +  5  4  -  100  100  +  11  — -  • -10  * Method involved pretreatment with L-isoleucine (15 min, 22 C) where indicated, prior to the regular assay procedure commencing with pyridoxal phosphate addition.  50 mM L-serine or L-threonine  replaced the substrate normally used.  22  Table 4.  Substrate-related response of C. salina deaminase activity to various reagents and effectors.*  Enzyme activity % [ of control) Compound  Concn Substrate (50 mM)  tested  (mM)  L-Serine  L-Threonine  100  100  0.01  99  88  0.10  92  36  1.0  88  5  10  86  4  0.10  101  112  1.0  100  100  10  96  48  100  89  11  1.0  93  26  10  92  25  EDTA  100  96  102  KCl  100  428  310  100  345  400  -  nil L-isoleucine  L-valine  dithiothreitol  f  NH.C1  +  * Method involved pretreatment with the test compound (15 min, 22 C) prior to the regular assay procedure commencing with pyridoxal phosphate addition. 50 mM L-serine or L-threonine replaced the substrate normally used. * Relative to controls of Tris-HCl buffer without added cation.  23  Table 5.  Effects of substrate analogs on algal TDH activity towards L-threonine.*  Substrate  Enzyme activity % [ of control)  analog  — — —  (50 nW)  Ch.sa.  He.vi.  Ag.qu.  An.ma.  Po.cr.  Te.ma.  Cy.na.  L-serine  -  40  .13  16  22  18  .27  L-allothreonine  99  101  103  104  103  98  99  L-homoserine  12  45  19  30  32  25  39  * Method involved pretreatment with the analog (15 min, 22 C) prior to the regular assay procedure commencing with pyridoxal phosphate addition.  24  Fig. 1.  pH-activity profile of the deaminase reaction in relation to substrate; (A) L-threonine and (B) L-serine.  Algal extracts were prepared  at the appropriate pH (see methods) prior to the regular assay procedure commencing with the addition of pyridoxal phosphate.  50 mM  L-serine or L-threonine was substituted for the substrate normally used.  25  Fig. 2. A. Effects of L-serine and L-homoserine concentration on threonine dehydratase activity of A. marina and A. quadruplicatum.  Method involved pretreatment  with the analog (15 min, 22°C) prior to the regular assay procedure commencing with pyridoxal phosphate addition.  Fig. 2. B. Effects of L-homoserine and L-serine on the substrate saturation kinetics of the A. marina enzyme. Method involved pretreatment with the required analog (15 min, 22°C) prior to the regular assay procedure commencing with pyridoxal phosphate addition. Appropriate concentration of L-threonine was substituted for that normally used.  '.CC  An.ma. Ag.qu.  c  -a-  o U  o NO  >-  > < ^ >-  20  N  z  LU  5  •.  25  50  SUBSTRATE ANALOG (mM) i  B  !  !  i "  5  An.ma.  c r_ vg.  ~§ 200  —  D  T O a  o •4—  + /-homoserine ( 3 7 m M ) '.CC -  +/-serine (73mM)  ^^^"^  i  ^C  6  1  25  CC  L-THREONINE (mM)  26  Fig. 3. Effect of L-homoserine and L-serine on the rate of L-threonine deamination by the enzymes from (A) T. maculata and (B) C_. salina.  Substrates were added  alone or simultaneously with the analog as indicated.  s  Te. m a .  i  i  \  TIME (min)  nr  27  PART II.  Feedback Inhibition, Allosteric Activation and Diverse Effects from Other Amino Acids  L-isoleucine is well known as the natural feedback inhibitor of the deaminating reaction catalysed by L-threonine dehydratase (16).  Likewise, L-valine has  been implicated as an allosteric activator of the same reaction (61).  In view  of the conflicting information reported in the literature concerning the effects of amino acids either analogous to, or structurally unrelated to L-isoleucine and/or L-valine, a systematic investigation was devised to elucidate the behaviour of the algal TDH in response to some of these amino acids. RESULTS Response to L-isoleucine at saturating substrate concentration With the exception of C. nana and H. virescens, a l l species showed sensitive inhibition from L-isoleucine (Fig. 4).  A. .marina and;A..-.quadruplicatum showed rela-  tively higher threshold concentrations but the inhibitions were extremely rapid once the threshold was surpassed so that by 5 mM L-isoleucine, the degree of inhibition was comparable to that shown by the remaining species. was the most sensitive to L-isoleucine.  T. maculata  The response of the algal enzymes was  found to be extremely rapid in that maximal inhibition could be effected in less than 60 sec indicating that the enzyme is non-hysteretic, similar to the TDH of Escherichia c o l i (34), but in contrast to that of Bacillus subtilis (36).  The  C. nana enzyme was insensitive to L-isoleucine and the suggested probability of i t being a biodegradative type of TDH seemed unlikely in view of i t s lack of response to the nucleotides previously tested (23).  The H. virescens enzyme  was only 50 % inhibited by L-isoleucine and was unaffected by nucleotides  like  28  the C_. nana enzyme, but was unique of a l l the algal enzymes in that i t displayed 2 pH optima (see Part III for details). pH dependency of L-isoleucine inhibition Since the feedback inhibition from L-isoleucine has been shown to be pH dependent in the case of several previously studied threonine dehydratases  (3,  9,26), i t was considered important to examine this property for the algal enzymes, particularly with a view to understand the exceptional cases of C. nana and H. virescens.  The algal enzymes were tested at 2 L-isoleucine concentrations  (giving maximal and 50 % inhibitions previously observed at pH 8.5) by introducing the inhibitor into the reaction mixture at the pH i n question (Fig. 5).  The  results showed that optimal L-isoleucine effect occurs within the range of pH 7.0-8.5, beyond which a gradual decline occurs, eventually culminating in the complete lack of inhibition toward pH 10.0.  Furthermore, within this effective  pH range, a "finer" profile of pH-dependent inhibition maximum was discerned in the case of H. virescens known to give only 50 % inhibition with saturating L-isoleucine concentration.  This "fineness" of inhibition pH optimum may  apply to a l l the algal enzymes at submaximal inhibitor concentration since the C_. salina enzyme showed a similar profile when tested at L-isoleucine concentration giving 50 % inhibition.  However, the C. nana enzyme gave no evidence  of any physiologically significant increase i n sensitivity to L-isoleucine over the entire pH range tested, confirming previous suggestions (23) of a "desensitized" type of TDH in this alga.  It is interesting to note that a l l  species s t i l l manifest considerable activity toward L-threonine at pH 9.510.0 (see Part III) although sensitivity toward the feedback inhibitor is considerably diminished.  This type of behavior has led to previous inferences  on the existence of separate binding sites for the substrate and feedback  29  inhibitor in the case of other threonine dehydratases ( 2 0 ) . Response to L-valine at saturating substrate concentration When tested in the presence of saturating L-threonine, low concentration of L-valine was slightly stimulatory (5-30 I ) with four algal enzymes arid at high concentration, inhibitory with a l l algae excepting C. nana(Fig. 6 ) . The inhibition profiles obtained from L-valine concentration were remarkably similar to those evoked by L-isoleucine (cf. Fig. 4) after allowing for the difference  (100-fold) observed between the two amino acids.  sensitivity  C. nana and H.  virescens behaved similarly toward inhibition from L-valine as they did toward L-isoleucine in that the former enzyme remained uninhibited and the latter was suppressed by only about 40 % at the highest concentration tested. As was the case with L-isoleucine, the inhibition exerted by L-valine was pH dependent and for concentrations selected to invoke 50 % inhibition, the pH-response profiles were identical.  Conversely, the stimulation obtained from  low concentration of L-valine appeared to be unaffected by pH (cf. Fig. 5 ) . Reversal of L-isoleucine inhibition by L-valine The tests (made in this study) of the effects of L-valine on the algal enzymes, already inhibited by L-isoleucine, indicated that the former amino acid is able to partially reverse the inhibitory effect of the latter, and that these reversals were prescribed by certain conditions: a) the L-valine concentration must be sufficiently low to be non-inhibitory, b) the L-isoleucine concentration must be below that causing maximal inhibition.  Figure 7 portrays the degree  of reversal effected by a concentration gradient of L-valine from the algal enzymes half-maximally inhibited by L-isoleucine.  No reversals were observed  from similar tests of the effects of L-valine concentration on the maximally-  30  inhibited algal enzymes (L-isoleucine "saturated"), nor was the residual activity of the latter suppressed by the highest levels (50 mM) of L-valine tested, suggesting that after L-isoleucine "saturation" the algal enzymes were indifferent to L-valine. Alteration of substrate saturation kinetics by L-isoleucine and L-valine In order to acquire a fuller understanding of the principle by which Lvaline and L-isoleucine exerted their effects, the substrate saturation kinetics of the algal enzyme was studied in the presence of these amino acids.  This  aspect was investigated for three algal species chosen as representing three "shades" of isoleucine-valine interrelated effects hitherto observed: (i) A. marina (maximal L-isoleucine inhibition, no L-valine stimulation),  (ii) T. macu-  lata (maximal L-isoleucine inhibition, significant L-valine stimulation), C. nana ( l i t t l e L-isoleucine inhibition, greatest L-valine stimulation).  (iii) The  results depicted in Fig. 8 appear to elucidate the mode of stimulation from Lvaline.  The saturation kinetics were sigmoidal with L-threonine alone and  hyperbolic in the presence of L-valine.  C. nana was found to be extremely sensi-  tive to L—valine, since its saturation profile, which was highly "abnormal" with L-threonine alone, acquired a "normal" hyperbolic shape in i t s presence. The kinetic response of A. marina to low (0.1 mM) concentration of L-valine provides an explanation for the lack of stimulation observed earlier when tested in the presence of high L-threonine concentration; the activating effect of this level of L-valine, being manifest primarily at subsaturating levels of substrate, serves to decrease the Km while leaving the Vmax essentially unchanged.  That  certain species did exhibit stimulation from L-valine at the higher substrate concentration also becomes understandable from a similar explanation indicating that the f u l l Vmax potential had not been attained by this substrate concentration  31  without L-valine, as exemplified by the response of T. maculata depicted in F i g . 8. Further insights into the inhibitions caused by L-isoleucine and L-valine (high concentration) were gained from these kinetic studies.  The data (Fig. 8)  show that L-isoleucine retains the sinuosity of the original substrate saturation curve, decreasing Vmax and elevating the Km, whereas inhibitory levels of Lvaline eliminate the sinuosity and decrease both Vmax and Km.  In fact, such  L-valine levels s t i l l exert a considerable degree of stimulation at low substrate concentrat ion. Effects of other amino acids on the algal enzymes Several amino acids, some of which are closely related structurally to either L-isoleucine or L-valine, were tested for any capacity to activate or inhibit the enzyme reaction.  From the data in Table 6 i t is evident that aside from L-  valine and L-isoleucine, L-leucine, L-norvaline and L-a-amino-n-butyrate showed inhibition at high concentration (with decreasing effectiveness as listed) while L-O-methyl threonine was equal in its inhibition to L-isoleucine at a comparable concentration. ever.  D-Isoleucine, L-alanine and L-glycine exerted no effects whatso-  L-Aspartate, which could only be tested at low concentration because of  solubility problems, showed slight stimulation (ca. 10 %) towards some species, similar to that reported for the TDH of Bacillus licheniformis  (55).  The pronounced inhibition produced by 5 mM L-O-methyl threonine prompted a closer inspection of the concentration-sensitive response provoked by this structural analog of L-isoleucine.  The profiles of this response, depicted in  in Fig. 9, showed the effects of L-O-methyl threonine to be v i r t u a l l y indistinguishable from those of L-isoleucine. Since the algal TDH were inhibited to varying degrees by L-leucine, L-norvaline and L-a-aminobutyrate, i t was decided to verify whether any of these amino acids  32  were capable of mimicking the previously observed stimulation effects from Lvaline.  Substrate saturation curves i n the presence of these compounds at low  concentration (0.1 mM) were determined with T. maculata, which had shown a sensitive response to L-valine.  These tests showed no recognizable activation  or kinetic alteration of the saturation profile which was observed to maintain a virtually identical sigmoidality i n the absence or presence of either Lnorvaline, L-leucine or L-a-aminobutyrate.  It was thus inferred that the pre-  viously noted activation was highly specific for L-valine.  DISCUSSION One of the prime c r i t e r i a for the classification of a threonine dehydratase as biosynthetic i s i t s sensitivity to feedback inhibition from isoleucine according to the model originally developed by Changeux (16). A l l of the algal enzymes conform to this criterion, with the exception of C. nana, and to a lesser extent, H. virescens.  However, evidence for the absence of effect from nucleotides (23)  and presence of allosteric response to L-valine negates the possibility of a biodegradative TDH i n the former species and likens i t to the desensitized biosynthetic TDH of Rhodospirillum rub rum (28). In the case of the latter algal species, the situation i s unique in that L-isoleucine inhibits enzyme activity by approximately 50 %, and the algal preparation displays a double activity-pH optima (suggestive of 2 enzymes) yet i s nevertheless insensitive to nucleotides (see Part III).  Apart from these exceptions, the L-isoleucine inhibition showed  (i) considerable inter-species variation with respect to threshold concentration, ( i i ) non-hysteretic response.and, ( i i i ) effects on substrate saturation kinetics tending to increase homotropic enzyme-substrate interactions (indicated by increased Km).  33  As is the case with the algal enzymes, previous investigations had revealed that inhibition by L-isoleucine is pH dependent (3,9,26).  That the inhibition  exerted by higher concentrations of L-valine follows an identical pH dependency as that of L-isoleucine suggests that its inhibitory effect may be due to an a b i l i t y to mimic isoleucine and occurs at the site specific for allosteric inhibition (3).  It appears from structural similarity considerations that the  trends of inhibition obtained from L-leucine and L-norvaline may be due to similar mechanistic reasons and that this inhibition tends to decrease in proportion to structural similarity.  The effect of L-a-aminobutyrate may not be the result of  binding at the isoleucine site, but through a degree of competition with Lthreonine for the substrate s i t e , which mode of inhibition has been ascribed to this amino acid i n certain biodegradative threonine dehydratases (63,64). Few investigation have been performed with respect to the absolute stereospecific nature of isoleucine inhibition.  Certain compounds such as thiaisoleucine  (4), L-O-methyl threonine (75), L-0-ethyl.threonine (71) and isoleucine hydroxamate (51) have been examined for their effect upon the growth of isoleucinerequiring autotrophs and concomitant biosynthesis of TDH following the incorporation of these antimetabolites into the growth media. work of Changeux designed to  reveal  However, excepting the early  the specificity of substrate and inhibitor  sites (15), the a b i l i t y of isoleucine analogs to function as feedback inhibitors of the enzyme in vitro has been virtually unexplored.  A recent publication has  reported that both L-O-methyl threonine and thiaisoleucine do in fact markedly inhibit TDH activity but at 10-fold greater concentrations than the isoleucine achieving the same degree of inhibition (75).  The algal enzymes appear to be  incapable of distinguishing between the natural feedback inhibitor (L-isoleucine) and L-O-methyl threonine as the development of inhibition from graded concen-  34  trations of both compounds were identical.  It may be inferred, therefore, that  the prime c r i t e r i a determining the a b i l i t y of a compound to mimic an allosteric role is the overall similarity in molecular configuration and volume to the natural agent. From the evidence obtained with algal threonine dehydratases, i t appears that the activating effect of L-valine is exerted via a site distinct from either the substrate or inhibitor sites resulting in the abolition of cooperative interaction between threonine molecules (19), rather than functioning as a substrate analog as has been suggested (29,34).  An alternate explanation of  valine activation equates the observance of sigmoidal substrate saturation curves without effector to the presence of contaminating isoleucine, the inhibition from which is reversed by the addition of valine; thus, the apparent activation (37).  The validity of this explanation is questionable in view of sigmoidal  substrate saturation kinetics subject to normalization by valine reported for highly purified enzymes, such as the TDH of yeast (49). In contrast to the majority of threonine dehydratases wherein valine was found to activate, certain plant enzymes have had L-norvaline (46,48,74) and to a lesser extent L-norleucine, Lraspartate (46) assume the role of allosteric ~ activator.  A fungal enzyme was specifically activated by phenylalanine  (47). *  For the algal enzymes, only L-valine is implicated as allosteric activator, which role was not imitated by any of the amino acids tested including Lnorvaline.  Even when a 50 % inhibitory (at saturating substrate concentration)  concentration of L-valine was incorporated, with.the enzyme, considerable activation occurred at non-saturating substrate concentrations, an effect previously observed with the TDH of E. c o l i K-12 (29). The fact that C. nana, the isoleucine-insensitive  algal TDH, was greatly  35  activated by L-valine imparts clear evidence of a distinct site for valine. The observed lack of inhibition of this isoleucine-insensitive  enzyme from  high concentration of L-valine may be extrapolated to the other algal enzymes in support of the premise that the inhibitory effect observed in these isoleucinesensitive enzymes is likely the result of interaction from excess L-valine at the isoleucine site (39).  Furthermore, the partial a b i l i t y of L-valine to  reverse L-isoleucine inhibition lends weight to the mechanistic view of an additional distinct site for valine (11).  An earlier suggested reversal mecha-  nism that valine may simply act by displacement of isoleucine at the latter site (4) appears unlikely from the overall evidence obtained with the algal enzymes. An alternate explanation of the unusual responses of the C. nana TDH cannot be ignored.  The substrate saturation curve with L-threonine alone  appears to be essentially "inhibited" in that appreciable activity was. not evident :prior to approximately 35 mM L-threonine, after which the increase i n activity appeared to be linear.  Since L-valine dramatically altered the  substrate saturation profile (14-fold stimulation at 25 mM L-threonine) and showed no sign of inhibition at high concentration, the impression is obtained that the enzyme lacks an active isoleucine site.  However, i t is conceivable  that this site may be present already "filled" by endogenous isoleucine since the substrate saturation profiled is not too unlike that of the TDH from Bacillus stearothermophilus in the presence of near-maximal inhibitory concentration of isoleucine (73); this possibility implies that the crude enzyme preparation of C. nana may already have bound endogenous isoleucine unlike the other algal enzymes.  Thus, the extremely sensitive stimulation of the C. nana TDH from L-  valine may be due to a net reversal of this "native" isoleucine inhibition.  36  On an overall basis, therefore, the algal threonine dehydratases conform to the K-system of enzymes as developed by Monod (61), characterized by; a) velocity plots exhibiting homotropic interactions for substrate  (sigmoidal  response to concentration), b) increased sinuosity of the i n i t i a l velocity plots i n the presence of allosteric inhibitor, and c) conversion of the i n i t i a l velocity plots to a hyperbola in the presence of allosteric effector.  37  Table 6.  Effects of various amino acids on algal threonine dehydratase activity.*  Amino acid tested  Concn  Enzyme activity % [ of control)  (mM)  Ch.sa.  He.vi.  Ag.qu.  An.ma.  Po.cr.  Te.ma.  Cy.na.  L-isoleucine  5  4  46  10  8  4  11  94  L-O-methyl threonine  5  6  47  7  7  5  10  98  L-valine  50  27  63  89  35  32  18  134  L-leucine  50  40  69  72  57  36  49  102  L-norvaline  50  60  81  83  70  59  61  97  L-a-amino-n-butyrate  50  77  89  92  80  73  70  96  L-aspartate  5  106  110  104  109  111  107  100  L-alanine  50  99  103  102  104  100  99  97  L-glycine  50  100  104  100  101  98  103  106  D-isoleucine  50  101  102  97  100  100  102  103  * Method involved pretreatment with the amino acid (15 min, 22 C) prior to the regular assay procedure commencing with pyridoxal phosphate addition.  38  Fig. 4. Effect of L-isoleucine concentration on algal threonine dehydratase activity. Method involved pretreatment with L-isoleucine (15 min, 22°C) prior to the regular assay procedure commencing with pyridoxal phosphate addition.  39  Fig. 5. Influence of pH on the effects from L-isoleucine and L-valine on threonine dehydratase activity. Algal extracts at the appropriate pH (see methods) were pretreated with the required concentration of effector (15 min, 22°C) prior to the regular assay procedure commencing with pyridoxal phosphate addition.  7  8  9  PH  10  40  Fig. 6.  Effect of L-valine concentration on algal threonine dehydratase activity. Method involved pretreatment with L-valine (15 min, 22°C) prior to the regular assay procedure commencing with pyridoxal phosphate addition.  41  Fig. 7.  Reversal of L-isoleucine inhibition of the algal enzymes by graded concentrations of L-valine. Method involved pretreatment with half-maximal inhibitory L-isoleucine concentration (1 min, 22°C) followed by preincubation with L-valine (15 min, 22°C) prior to the regular assay procedure commencing with pyridoxal phosphate addition.  42  Fig. 8.  Effects of L-valine and L-isoleucine on the substrate saturation kinetics of the enzymes from T. maculata, C. nana and A. marina. Where required, the algal extracts were pretreated with the indicated concentrations of effector (15 min, 22°C) prior to the regular assay procedure commencing with pyridoxal phosphate addition. Appropriate L-threonine was substituted for the concentration normally used.  .  5  •.:  ic  _-7-.<iC.\.x^  (mM)  43  Fig. 9. Effect of L-O-methyl threonine concentration on algal threonine dehydratase activity. Method involved pretreatment with L-O-methyl threonine (15 min, 22°C) prior to the regular assay procedure commencing with pyridoxal phosphate addition.  n  I  -5  i  1  1  I  —-.•  L  -3 1 0  ':0  L-O-METHYL THREONINE [AA  44  PART III. Re-investigation of pH Optima with Strict Buffer-Ion Control. Evidence for 2 pH Optima with Differential Sensitivity to Allosteric Effectors in the Cryptomonad Hemiselmis virescens.  RESULTS pH optima of the deaminase reaction With the establishment of the algal TDHs requirement for monovalent inorganic cations (see Supplement II), i t became necessary to re-evaluate the previously obtained pH profiles as they were performed without adequate control of cation concentration (23).  From this point of view, i t was considered desireable to  use buffers (such as Tris-HCl and Map-HCl) which contained no alkali-metal cations and to supplement these with a constant level of KC1 (100 mM),  thereby eliminating  the activity deviations previously obtained (23) from unpredictable cation variations such as present in K-phosphate, K-Tricine, K-bicarbonate etc. (in spite of their molarity equivalence).  Preliminary tests, in addition to those  reported in Supplement II, have shown that the activating effect from monovalent cations is unaffected by pH in the range 7-10.  Figure 10 shows the pH-activity  profiles for six algal enzymes, a l l of which exhibited a single optimum in the pH range 8.5-9.0. However, the examination of H. virescens showed a "shoulder" in the vicinity of pH 8 in addition to the expected optimum at pH 9 (Fig. 11). To verify this observation, two other nutritionally different cultures of this species were similarly examined. It was found that both the urea-grown and glycerol-grown cultures showed two distinct pH optima, one peak being in the vicinity of pH 8 (corresponding to the "shoulder" observed in the standard culture), the other in the vicinity of pH 9.  Identical pH-activity studies with  the enzymes from nutritionally different cultures of C. salina and P. cruentum  45  showed no difference as compared to the profiles observed for the standard cultures of these species. Response of H. virescens enzyme to potential effectors at pH intermediate between the two optima (8.5) In view of the ability of the H. virescens enzyme (from three nutritionally different cultures) to undergo only 50 % inhibition from L-isoleucine, the observation of two pH optima was highly suggestive of the simultaneous presence of a biodegradative TDH.  However, when several nucleotides were tested in the presence  or absence of dithithreitol* at pH 8.5, no activation was observed with either high or low concentration of substrate (Table 7).  The same lack of effect was  noted when the nucleotides were tested at pH 8 and 9, a l l of which cast doubt on the possibility of a biodegradative TDH in the enzyme preparation.  Because of the  relationships of aspartate, methionine and cystathionine to the metabolism of threonine (see Appendix B), these amino acids were tested with the H. virescens enzyme in order to assess the possibility of (i) unknown precurssor-product interactions with this TDH and, (ii) contaminating enzymes capable of e l i c i t i n g a substrate response with L-threonine (eg. cystathionine synthase (62), capable of deaminating threonine).  The results of these examinations (Table 7) showed  no untoward effects from L-aspartate, L-methionine or L-cystathionine when tested at low and high concentration at two substrate concentrations.  Finally, the  previously performed studies of substrate specificity (Part I), clearly indicated that, unlike the other cryptophyte C. salina, the simultaneous presence of an SDH or even homoserine dehydratase potentially capable of deaminating L-threonine  *Several investigators have reported the necessity of sulfhydryl-protective reagents for f u l l expression of nucleotide activation (46).  46  is improbable. Differential effects from allosteric compounds at the two pH optima The prior observations that the inhibition obtained from L-isoleucine, L-valine, L-leucine, and L-norvaline were invariably about half of those generally observed with the other algae (excepting C_. nana), prompted the of L-isoleucine inhibition at the two pH optima.  investigation  However, since i t was already  demonstrated that feedback inhibition is pH dependent and approaching elimination by pH 10 (Part II), l i t t l e could be gained from simply incorporating L-isoleucine at each pH and comparing the results.  For this reason, the algal extract was  f i r s t treated with maximally inhibiting levels of L-isoleucine in buffer of low strength (0.01 M) at pH 7.5 (the optimal inhibitory pH) and after allowing an interaction interval, the mixture was readjusted to the required pH with buffer of high strength (0.1 M).  From the results (Fig. 12), i t is apparent that  because the inhibition by L-isoleucine was effected at pH 7.5, the enzymes from C. salina and P. cruentum remained fully inhibited irregardless of the pH at which the subsequent incubation with L-threonine was performed.  On the other  hand, the enzymes from the three nutritionally different cultures of H. virescens showed progressively less inhibition from L-isoleucine as the more alkaline optimum was approached.  An interesting culture-dependent correlatiohship appears  between the activity profiles without L-isoleucine at the two pH optima and the inhibition profiles obtained with L-isoleucine in the v i c i n i t y of the pH 9 optimum. In the absence of L-isoleucine, each of the two cultures (Gly. L and Urea) showed comparable activity maxima at the two pH optima whereas the standard culture was considerably more active at the pH 9 optimum with a consequential "shoulder" (rather than a peak) at pH 8.  In the presence of L-isoleucine, the two former  47  cultures evinced more pronounced inhibition in the v i c i n i t y of the pH 9 optimum relative to the latter culture.  Such a correlationship appears to be more than a  coincidence'and suggests that (i) a l l three cultures may possess at least two enzymes, one sensitive to L-isoleucine with a pH optimum near 8 and another insensitive to L-isoleucine with a pH optimum near 9, ( i i ) these enzymes may occur in different proportions in the three cultures, ( i i i ) the isoleucineinsensitive enzyme (pH 9 optimum) may be present in the greatest proportion i n the standard culture. Since the activating effect of L-valine was shown to be independent of pH in the range 7-10 for the algal enzymes (Part II), examination of any differential effects to this amino acid at the two pH optima could be pursued without pH modification as was required for the above studies with L-isoleucine.  In  consequence, substrate saturation kinetics with L-threonine alone, and in combination with either L-valine, L-homoserine or L-serine were examined at pH 8 and 9, the results of which investigations are portrayed in Fig. 13 for the enzyme from the standard culture of H. virescens.  The data revealed the following  features (i) the response to inhibition from L-serine and L-homoserine were similar at each pH and analogous to that depicted for A. marina (cf. F i g . 2B, Part I) and, (ii) while substantial activation (through curvature alteration) by L-valine is evident at pH 8, no such effect was observed at pH 9.  DISCUSSION With few exceptions (18,46), the pH optima for biosynthetic threonine dehydratases have been reported within the range of 8-9.5.  In the case of the algal  enzymes, a single optimum pH for enzyme activity was within the narrow range of 8.5-9.0 excepting H. virescens.  The latter species showed an unusual response  48  to pH with two optima, one in the v i c i n i t y of pH 8, the other around pH 9.  The  sharpness of the pH optima appeared to depend on the conditions of algal culture. However, different culture conditions did not affect the pH optima of C. salina and P. cruentum enzymes, suggesting that the overall pH response of H. virescens may be independent of culture conditions.  Since the degree of this "two peak"  pH response does show some dependence on culture conditions, i t appears l i k e l y that this alga is producing a mixture of at least two TDHs.  This inference is  also supported by the culture-related degrees of inhibition obtained from Lisoleucine.  Assuming such a mixture, the two enzymes present may not differ  basically in their physiological function since no evidence was obtained of the "standard" type of biodegradative TDH. This two-enzyme hypothesis was extensively tested within the framework of the known characteristic behaviour of the other algal enzymes, as well as i n previously unexamined peripheral areas, with the expectation that possible unique differences in behaviour may be delineated at the two pH optima.  In designing  these tests, i t was rationalized that any real difference between the H. virescens and other algal TDHs or any real effects from potential substrates and unknown effectors would be manifested to a recognizable degree at a pH (8.5) between the two optima.  Once such differences or effects were recognized, i t was imperative  to submit them to a finer examination at the actual optima.  The results of such  tests and finer examination have already been stated earlier (Parts I § II, where such tests were concerned with routine examination of a l l algal species) and immediately prior to this section (Part III, Results, where the tests were more specifically concerned with H. virescens).  In reviewing these results with-  in the broad perspective of the above-proposed two-enzyme hypothesis for H. virescens, the overall picture obtained appears to further support this postulation  49  without any serious contraindication. The overall evidence so obtained from H. virescens i s summarized below: . 1) In contrast to the other algal enzymes, L-isoleucine, after ensuring i t s binding to the enzyme, gave 50 % inhibition at pH 8.5 and this inhibition was considerably enhanced at pH 7.5-8  but was markedly reduced at pH 9-9.5,  indicating clearly differentiable responses to this effector at the two optima. 2) Again in contrast to the other algal enzymes, high concentrations of L-valine, L-leucine and L-norvaline gave only half the degree of inhibition generally obtained from these enzymes at a pH intermediate between the two optima, leading to the inference that these amino acids were mimicking the pH-dependent L-isoleucine effects noted above. 3) Low concentration of L-valine converted the substrate saturation kinetic profile from sigmoidal to hyperbolic at pH 8, but had no effect on the saturation profile at pH 9, which again indicated a differential response from another effector at the two pH optima. 4) The substrate specificity study at pH 8.5 and the substrate analog inhibitions observed at the two pH optima showed no recognizable difference from the other algal species, confirming that the two enzymes of H. virescens were TDHs similar in this respect to one another as well as to the other algal enzymes. 5) The H. virescens enzyme showed no effects from certain nucleotides, nor from a number of amino acids (L-methionine, L-aspartate, L-cystathionine) that might be expected to bear an energy control or precurssor-product relationship to L-threonine in the presently known maps of metabolic pathways centering around this amino acid. The absence of effect from L-cystathionine also eliminated the possibility of nonspecific deaminations of L-threonine or specific formation of a-ketobutyric acid reported for certain enzymes  50  specifically acting on the former amino acid as substrate ( eg. cystathionine synthase (62), cystathionine-cysteine-lyase  (50)).  It is concluded from these considerations that H. virescens is exceptional among the algal TDHs in producing a K-type (according to Monod (61))  biosynthetic  TDH as well as an undefined type TDH insensitive to a l l know effectors tested (resembling certain bacterial mutant variants (4,21,52)) and whose physiological function remains unknown.  In fact, the case of H. virescens is strongly remini-  scent of a mutant of Rhodopseudomonas spheroides (19,20) reported to simultaneously contain two TDHs, both insensitive to nucleotides and showing similar substrate specificity, but differing in their sensitivity to effectors so that the one (PRTDI) enzyme is completely inhibited by L-isoleucine and activated by L-valine while the other (PRTDII) is 1000-fold less sensitive to L-isoleucine and not subject to activation by L-valine.  It is pointed out that this conclusion is largely  based on very indirect inferences drawn from the data obtained, and w i l l require large-scale attempts at purification and separation of the enzymes in question. Among enzymes other than threonine dehydratases that have shown two pH optima i n crude extracts, differential responses to inhibitors and activators have provided similar indirect evidence to indicate the occurrence of two enzymes in such extracts (eg. acid phosphatase in seminal fluid of rabbits (79)).  In the case  of aspartate transcarbamoylase (80) , the two pH optima also showed differential response to the principal allosteric effector involved, but these authors appear to have ignored the possibility of occurrence of more than one enzyme without adequate justification, presumably because the second and more pronounced pH optimum was observed at the high and relatively non-physiological pH of 10.5. Nevertheless,  in the absence of the final definitive proof, the question of two  threonine dehydratases, each with a single pH optimum, or one enzyme with two pH optima is s t i l l open to conjecture.  51  Table 7.  Effects of certain nucleotides and amino acids on enzyme activity of H. virescens at pH 8.5.*  Compound tested Concn (mM) nil  AMP ADP ATP cyclic-AMP L-methionine  DTT  L-Threonine  Enzyme activity  (0.01 mM)  (mM)  (% of control)  -  50  100  5  100  -  +  50  80  +  5  74  5  +  50  80  5  +  5  81  5  +  50  83  5  +  5  72  5  +  50  78  5  +  5  83  5  +  50  81  5  +  5  74  50  -  50  . 99  5  95  50  102  5  91  50  104  5  96  50  99  5  103  50  110  5  114  50 5 5 L-cystathionine  50 50 5 5  L-aspartate  5 5  Method involved pretreatment with DTT (15 min, 22°C) followed by preincubation with the test compound (15 min, 22°C) prior to the regular assay procedure commencing with pyridoxal phosphate addition. .substituted for that normally used. Dithiothreitol  Appropriate L-threonine concentration was  52  Fig. 10. pH-activity profiles for six algal threonine dehydratases.  Algal extracts were prepared  at the required pH (see methods) prior to the standard assay procedure commencing with the addition of pyridoxal phosphate.  53  Fig. 11. pH-activity profiles for the enzymes from three nutritionally different cultures of H. virescens. Algal extracts were prepared at the required pH (see methods) prior to the regular assay procedure commencing with pyridoxal phosphate addition.  Fig. 12. pH-activity profiles in the presence of L-isoleucine for the enzymes from three nutritionally different cultures of H. virescens.  C. salina,, P. _ cruentum, shown for  comparison. The algal extracts were prepared in 0.01 M Tris-HCl buffer (pH 7.5) and preincubated with 10 mM L-isoleucine (15 min, 22°C). The pH was then adjusted as required by the addition of appropriate buffer (see methods) prior to the regular assay procedure commencing with the addition of pyridoxal phosphate.  54  Fig. 13. Effects of L-homoserine, L-serine and L-valine on the substrate saturation kinetics of the H. virescens enzyme  at pH 8 and pH 9. Algal  extracts were prepared at the desired pH (see methods) and pretreated with the indicated concentration of effector (15 min, 22°C) prior to the regular assay procedure commencing with pyridoxal phosphate addition.  LITER\TURE CITED  1.  ANTIA, N . J . § CHENG, J . Y . 1970.  The survival of axenic cultures of  marine planktonic algae from prolonged exposure to darkness at 20 C. Phycologia, 9: 179-84. 2.  ANTIA, N . J . § CHORNEY, V. 1968.  Nature of the nitrogen compounds  supporting phototrophic growth of the marine cryptomonad Hemiselmis virescens. 3.  J . Protozool. 15: 198-201.  BALMGARTEN, J . § SCHLEGEL, H.G. 1971. Arthrobacter Stamm 23.  4.  Threonin-Desaminase aus  Arch. Mikrobiol. 75: 312-26.  BETZ, J . L . , HEREFORD, L.M. § MAGEE, P.T. 1971.  Threonine deaminases  from Saccharomyces cerevisiae mutationally altered in regulatory properties. 5.  Biochem. 10: 1818-24.  BRUNNER, A . , BEVTLLERS-MIRE, A. § DE ROBICHON-SZULMAJSTER,  H. 1969.  Regulation of isoleucine-valine biosynthesis in Saccharomyces cerevisiae.  Altered threonine deaminase in an is^ mutant  responding to threonine. 6.  European J . Biochem. 10: 172-83.  BRUNNER, A. § DE ROBICHON-SZULMAJSTER,  H. 1969.  Allosteric behavior  of yeast threonine deaminase under partially inactivating conditions. 7.  BURNS, R.O. § ZARLENGO, M.H. 1968. typhimuritgn.  8.  FEBS Letters, 2: 141-4. Threonine deaminase from Salmonella  J . Biol. Chem. 243: 178-85.  CARTER, J . E . § SAGERS, R.D. 1972.  Ferrous ion-dependent L-serine  dehydratase from Clostridium a c i d i u r i c i . 9.  CENNAMO, C , BOLL, M. § HOLZER, H. 1964. aus Saccharomyces cerevisiae.  10.  CENNAMO, C. § CARRETTI, D. 1966.  J . Bacteriol. 109: 757-63.  Uber Threonindehydratase  Biochem. Zeit. 340: 125-45. Kinetic studies with L-threonine  dehydratase from Salmonella typhimurium.  Biochim. Biophys.  Acta, 122: 371-3. 11.  CHANGEUX, J . - P . 1961.  The feedback control mechanism of biosynthetic  L-threonine deaminase by L-isoleucine. Quant. Biol. 26: 313-8.  Cold Spring Harbor Symp.  56  12.  CHANGEUX, J . - P . 1962.  Effet des analogues de l a L-threonine et  de l a L-isoleucine sur la L-threonine desaminase.  J . Mol.  B i o l . 4: 220-5. 13.  CHANGEUX, J . - P . 1964.  Sur les proprietes allosteriques de la L-  threonine desaminase.  I. Methcdes d'etude de l a L-threonine  desaminase de biosynthese. 14.  CHANGEUX, J . - P . 1964.  Bull. Soc. Chim. Biol. 46: 927-46.  Sur les proprietes allosteriques de l a L-  threonine desaminase de biosynthese.  II. Cinetiques d'action  de l a L-threonine desaminase de biosynthese vis-a-vis du substrat et de l'inhibiteur naturels.  Bull. Soc. Chim. Biol.  46: 947-61. 15.  CHANGEUX, J . - P . 1964.  Sur les proprietes allosteriques de l a L-  threonine desaminase de biosynthese.  III. Interpretation de  1'effet inhibiteur de l a L-isoleucine: empechement sterique ou effet allosterique. 16.  CHANGEUX, J . - P . 1965.  Bull. Soc. Chim. Biol. 46: 1151-73.  The control of biochemical reactions.  Sci.  Amer. 212: 36-45. 17.  CHENG, J . Y . £ ANTIA, N . J . 1970.  Enhancement by glycerol of phototrophic  growth of marine planktonic algae and i t s significance to the ecology of glycerol pollution. 18.  DART, R.K. 1968.  J . Fish. Res. Bd. Can. 27: 335-46.  The presence of threonine and serine dehydratase  activities in Pseudomonas. Biochem. J . 107: 29-30. 19.  DATTA, P. 1966.  Purification and feedback control of threonine  deaminase activity of Rhodopseudomonas spheroides.  J . Biol. Chem.  241: 5836-44. 20.  DATTA, P. 1969.  Effects of feedback modifiers on mutationally  altered threonine deaminases of Rhodopseudomonas spheroides.  J.  B i o l . Chem. 244: 858-64. 21.  DATTA, P. $ WAN LU, L. 1969.  Mutationally altered threonine deaminase  from a prototrophic revertant of Rhodopseudomonas spheroides. J . B i o l . Chem. 244: 850-7.  57  22.  DE ROBICHON-SZULMAJSTER, H. $ MAGEE, P.T. 1968.  The regulation of  isoleucine-valine biosynthesis in Saccharomyces cerevisiae. Threonine deaminase. 23.  I.  European J . Biochem. 3: 492-501.  DESAI, I . D . , LAUB, D. § ANTIA, N . J . 1972.  Comparative characterization  of L-threonine dehydratase in seven species of unicellular marine algae. 24.  Phytochem. 11: 277-87.  DESAI, I.D. § POLGLASE, W.J. 1967.  Kinetics of threonine deaminase  of Escherichia c o l i K-12 and a streptomycin-dependent mutant. Can. J . Biochem. 45: 1-9. 25.  DESAI, I.D. § POLGLASE, W.J. 1967.  End-product inhibition of threonine  deaminase of streptomycin mutants of Escherichia c o l i K-12. Can. J . Biochem. 45: 11-8. 26.  DOUGALL, D.K. 1970.  Threonine deaminase from Paul's Scarlet Rose  tissue cultures. 27.  Phytochem. 9: 959-64.  DUPOUROUE, D.W., NEWTON, A. § SNELL, E . E . 1966.  Purification and  properties of D-serine dehydrase from Escherichia c o l i .  J . Biol.  Chem. 241: 1233-8. 28.  FELDBERG, R.S. $ DATTA, P. 1971. rubrum.  L-Threonine deaminase of Rhodospirillum  Purification and characterization.  European J . Biochem.  21: 438-46. 29.  FREUNDLICH, M. $ UMBARGER, H.E. 1963.  The effects of analogues of  threonine and of isoleucine on the properties of threonine deaminase. Cold Spring Harbor Symp. Quant. Biol. 28: 505-11. 30.  FRIEDEMANN, T . E . : Determination of ct-keto acids, pp. 414-418.  In:  S.P. Colowick and N.O. Kaplan (ed.): Methods in enzymology, Vol. 3. 31.  New york:  HARDING, W.M. 1969.  Academic Press Inc. 1957.  Relationships between stability of threonine  deaminase and its apparent kinetics.  Arch. Biochem. Biophys. 129:  57-61. 32.  HARDING, W.M., TUBBS, J . A . $ MCDANIEL, D. 1970. valine and isoleucine on threonine deaminase.  Similar effects by Science, 167: 75-6.  58  33.  HATFIELD, G.W. 1970.  Ligand-induced maturation of threonine deaminase.  Science, 167: 75-6. 34.  HATFIELD, G.W. 1971.  Reaction velocity response of the Escherichia  c o l i biosynthetic L-threonine deaminase to repid changes i n substrate and modifier ligand concentrations.  Biochem. Biophys.  Res. Comm. 44:464-70. 35.  HATFIELD, G.W. $ BURNS, R.O. 1970. typhimurium.  Threonine deaminase from Salmonella  III. The intermediate substructure.  J . Biol. Chem.  245: 787-91. 36.  HATFIELD, G.W., RAY, W.J. § UMBARGER, H.E. 1970. from Bacillus s u b t i l i s .  Threonine deaminase  III. Pre-steady state kinetic properties.  J . Biol. Chem. 245: 1748-54. 37.  HATFIELD, G.W. $ UMBARGER, H.E. 1968. threonine deaminase.  38.  Biochem. Biophys. Res. Comm. 33:397-401.  HATFIELD, G.W. § UMBARGER, H.E. 1970. Bacillus subtilis.  A time-dependent activation of  Threonine deaminase from  I. Purification of the enzyme.  J . Biol. Chem.  245: 1736-41. 39.  HATFIELD, G.W. § UMBARGER, H.E. 1970. Bacillus subtilis.  Threonine deaminase from  II. The steady state kinetic properties.  J.  Biol. Chem. 245: 1742-7. 40.  HILL, H.M. § ROGERS, L . J . 1972. Bacterial origin of alkaline L-serine dehydratase in french beans.  41.  HOLZER, H . , BOLL, M. § CENNAMO, C. 1964. threonine deaminase.  42.  Phytochem. 11: 9-18. The biochemistry of yeast  Agnew. Chem. internat. Edit. 3: 101-7.  HOLZER, H . , CENNAMO, C. § BOLL, M. 1964.  Product activation of  yeast threonine dehydratase by ammonia.  Biochem. Biophys. Res.  Comm. 14: 487-92. 43.  HOSHINO, J . , SIMON, D. § KROGER, H. 1971.  Identification of one of  the L-serine dehydratase isoenzymes from rat liver as L-homoserine dehydratase.  Biochem. Biophys. Res. Comm. 44:872-8.  59  44.  HOSHINO, J . , SIMON, D. $ KROGER, H. 1972.  Influence of monovalent  cations on the activity of L-serine (L-threonine) dehydratase from rat liver.  The control of threonine-serine activity ratio.  European J . Biochem. 27: 388-94. 45.  HUGHES, M . , BRENNEMAN, C. £ GEST, H. 1964.  Feedback sensitivity of  threonine deaminases in two species of photosynthetic bacteria. J . Bacteriol. 88: 1201-2. 46.  KAGAN, Z . S . , SINELNIKOVA, E.M. § KRETOVICH, V . L . 1969.  L-threonine  dehydratases of flowering parasitic and saprophytic plants. Enzymologia 36: 335-52. 47.  KAGAN, Z . S . , SINELNIKOVA, E.M. $ KRETOVICH, V . L . Biosynthetic Lthreonine dehydratase of the meadow mushroom.  Biokhimiya, 34:  1279-87. 48.  KAGAN, Z . S . , SINELNIKOVA, E.M. § KRETOVICH, V . L . 1969.  Some kinetic  and allosteric properties of L-threonine dehydrase of cow-wheat, Melampyrum nemorosum L. 49.  Doklady Akademii Nauk SSSR, 185: 1372-5.  KATSUNUMA, T . , ELSASSER, S. § HOLZER, H. 1971.  Purification and  some properties of threonine dehydratase from yeast.  European  J . Biochem. 24: 83-7. 50.  KIDDER, G.W. § DEWEY, V . C . 1972.  Methionine or folate and phospho-  enolpyruvate in the biosynthesis of threonine in Crithidia fasciculata. 51.  J . Protozool. 19: 93-8.  KISUMI, M . , KOMATSUBARA, S., SUGIUR\, M. § CHIBATA, I. 1971. Isoleucine hydroxamate, an isoleucine antagonist.  J . Bacteriol.  107: 741-5. 52.  KISUMI, M . , KOMATSUBARA, S., SUGIURA, M. t\ CHIBATA, I. 1972. Isoleucine accumulation by regulatory mutants of Serratia marcescens: Lack of both feedback inhibition and repression. J . Bacteriol. 110: 761-3.  53.  KRETOVICH, V . L . , SINELNIKOVA, E . M . , KAGAN, Z.S. § BUTENKO, R.G. 1969. L-threonine dehydrase of a tobacco tissue culture. Akademii Nauk SSSR, 186: 1431-3.  Doklady  60  54.  LEIBOVICI, J . § ANAGNOSTOPOULOS, C. 1969.  Proprietes de la threonine  desaminase de l a souche sauvage et d'un mutant sensible a l a valine de Bacillus subtilis. 55.  Bull. Soc. Chim. Biol. 51: 691-707.  LEITZMANN, C. § BERNLOHR, R.W. 1968. Bacillus licheniformis.  Threonine dehydratase of  I. Purification and properties.  Biochim.  Biophys. Acta, 151: 449-60. 56.  LESSIE, T . G . $ WHITELEY, H.R. 1969.  Properties of threonine deaminase  from a bacterium able to use threonine as sole source of carbon. J . Bacteriol. 100: 878-89. 57.  LIEBERMAN, M.M. § LANYI, J . K . 1972. halophilic bacteria. dependence.  58.  Threonine deaminase from extremely  Cooperative substrate kinetics and salt  Biochem. 11: 211-6.  LOSEVA, L . P . , LYUBIMOV, V . I . , KAGAN, Z.S. § KRETOVICH, V . L . 1968. L-Threonine dehydratase of Azotobacter vinelandii.  Doklady  Akademii Nauk SSSR, 181: 997-1000. 59.  MAEBA, P. § SANWAL, B.D. 1966. Salmonella.  60.  Kinetic model for the native enzyme.  MODI, S.R. $ MAZUMDER, R. 1966. from spinach.  61.  The allosteric threonine deaminase of  A biosynthetic L-threonine dehydratase  Ind. J . Biochem. 3: 215-8.  MONOD, J . , CHANGEUX, J . - P . § JACOB, F. 1963. and cellular control systems.  62.  Biochem. 5: 525-36.  Allosteric proteins  J . Mol. B i o l . 6: 306-29.  MUDD, S . H . , FINKELSTEIN, J . D . , IRREVERRE, F. § LASTER, L. 1965. Threonine dehydratase activity in humans lacking cystathionine synthase.  63.  Biochem. Biophys. Res. Comm. 19: 665-70.  NAKAZAWA, A. $ HAYAISHI, 0. 1967.  On the mechanism of activation of  L-threonine deaminase from Clostridium tetanomorphum by adenosine diphosphate. 64.  J . Biol. Chem. 242: 1146-54.  NIEDERMAN, R.A., RABINOWITZ, K.W. $ WOOD, W.A. 1969.  Allosteric  control of biodegradative L-threonine dehydrase: Effect of AMP on an early step in the reaction mechanism. Res. Comm. 36: 951-6.  Biochem. Biophys.  61  65.  NISHMJRA, J . S . § GREENBERG, D.M. 1961.  Purification and properties  of L-threonine dehydrase of sheep l i v e r . 66.  RASKO, I. § ALFOLDI, L . 1971.  J . Biol. Chem. 236:2684-91.  Biosynthetic L-threonine deaminase as  the origin of L-serine sensitivity of Escherichia c o l i .  European  J . Biochem. 21: 424-7. 67.  REH, M. S, SCHLEGEL, H.G. 1969.  Die Biosynthese von Isoleucin und  Valin in Hydrogenomonas HI6. 68.  Arch. Mikrobiol. 67: 110-27.  SAYRE, F.W. § GREENBERG, D.M. 1956.  Purification and properties of  serine and threonine dehydrases. 69.  SHARMA, R.K. § MAZUMDER, R. 1970.  J . Biol. Chem. 220: 787-99.  Purification, properties, and feed-  back control of L-threonine dehydratase from spinach.  J . Biol.  Chem. 245: 3008-14. 70.  SHARMA, R . K . , MODI, S.R.  . MAZUMDER, R. 1967.  Studies on L-threonine  dehydratase from spinach (Spinacia oleracea). 4: 71.  Ind. J . Biochem.  61-4.  SHIGEURA, H.Y., HEN, A . C . , HIREMATH, C.B. § MAAG, T.A. 1969. Ethylthreonine: An antagonist of L-isoleucine. Biophys. 135:  72.  L-O-  Arch. Biochem.  90-6.  SHIZUTA, Y . , NAKAZAWA, A . , TOKUSHIGE, M. $ HAYAISHI, 0. 1969.  Studies  on the interaction between regulatory enzymes and effectors.  III.  Crystallization and characterization of adenosine 5'-monophosphatedependent threonine deaminase from Escherichia c o l i .  J . Biol.  Chem. 244: 1883-9. 73.  THOMAS, D.A. 5 KURAMITSU, H.K. 1971.  Biosynthetic  deaminase from Bacillus stearothermophilus. regulatory properties. 74.  TCMOVA, V . S . , KAGAN, Z.S. $ KRETOVICH, V . L . 1968.  TWAROG, R. 1972. Acinetobacter.  I. Catalytic and  Arch. Biochem. Biophys. 145: 96-104.  dehydratase from pea seedlings. 75.  L-threonine  L-Threonine  Biokhimiya, 33: 244-54.  Enzymes of the isoleucine-valine pathway in J . Bacteriol. I l l : 37-46.  62  76.  UMBARGER, H.E. § BROWN, B. 1956. coli.  I. D- and L-threonine deaminase activities of cell-free  extracts. 77.  J . Bacteriol. 71: 443-9.  UMBARGER, H.E. § BROWN, B. 1957. coli.  Threonine deamination in Escherichia  Threonine deamination in Escherichia  II. Evidence for two L-threonine deaminases.  J . Bacteriol.  73: 105-12. 78.  WHITELEY, H.R. § TAHARA, M. 1966. tetanomorphum.  Threonine deaminase of Clostridium  I. Purification and properties.  J . B i o l . Chem.  241: 4881-9. 79.  WIKSTROM, C. $ LINDAHL, P.E. 1971.  The effects of inhibitors and  activators of acid phosphatase in seminal fluid of rabbits in the pH range 5.1-6.5. 80.  YON, R . J . 1972.  Acta Chem. Scand. 25: 443-50.  Wheat-germ aspartate transcarbamoylase.  Kinetic  behaviour suggesting an allosteric mechanism of regulation. Biochem. J . 128: 311-20.  63  APPENDIX A. The Common Locus of Threonine Dehydratase in Branched-chain Amino Acid Biosynthesis  CH,  CH,  c=o  CH,  C=0  I  AHA  COOH PYRUVATE  3  CH.-C-OH 3  I  Rl  v C H -C-OH  v  ~?  I  v  DAD  I  3  C=0  *  COOH  JA_  I COOH  v  H-C-NH, COOH  a-KETOISOVALERATE  a,3-DIHYDROXYISOVALERATE  a-ACETOLACTATE  CH -C-H  3  H-C-OH  COOH  CH,  CH, I CH.-C-H I  VALINE  (ACTIVE ACETALDEHYDE)  3  C=0  AHA' I COOH a-KETOBUTYRATE  T  CH, I CH,-CH,-C-OH  CH, i ->  CH, I CH, I ^ -  3  c=o I  CH -CH -C-OH 3  Rl  H-C-OH I COOH  2  COOH a-ACETO a-HYDROXYBUTYRATE  CH, I CH.-CH.-C-H > I  CH, 3  I  3  3  DAD  a,3-DIHYDROXY3-METHYLVALERATE  CH,-CH,-C-H  1  C=0  I  TA  COOH a-KETO 3-METHYLVALERATE  > 7  2  I  H-C-NH, I COOH 1  ISOLEUCINE  TDH  Abbreviations for enzymes  CH3  1  HO-C-H TDH THREONINE DEHYDRATASE  C-NH, I COOH Z  AHA ACETOHYDROXY ACID SYNTHETASE Rl  REDUCTOISOMERASE  DAD DEHYDROXY ACID DEHYDRATASE  THREONINE  TA  TRANSAMINASE  Adapted from: ALLAUDEEN, H.S. $ RAMAKRISHNAN, T. 1968. 125: 199-209.  Arch. Biochem. Biophys.  APPENDIX B. Schematic Representation of Threonine Biosynthesis and the Major Peripheral Regulatory Circuits  ***********************************************  * * * * * * ******* LYSINE ********* * * * * * * * ** * *. ' * *  ASPARTATE  *  ' DIHYDRODIPICOLINATE  * *  *  t  *  X  * *. £ <****** ASPARTYL 8PHOSPHATE  i  * *  *  * * * * * * * * * * * * * * * * * * * *  * ***** ISOLEUCINE * * *  A.  * * *  * *  a-KETOBUTYRATE  *  * * *  * ********.[>: * * %************************** * * <*********** ASPARTATE 8V •> THREONINE -> HOMOSERINESEMIALDEHYDE  7!  ' *  * CYSTEINE-  <•«:***** ************ ** ** ** O-SUCCINYL *** ** HOMOSERINE ** ** * CYSTATHIONINE  (  ) single enzymatic step  (  ) more than one enzymatic step  (****) major regulatory sequences Adapted from: DATTA,P. 1969.  Science, 165: 556-62.  -> METHIONINE  65  APPENDIX C. Standard Calibration Curve for a-Ketobutyrate and Pyruvate.  SUPPLEMENT I  L-Threonine Deaminase in Marine Planktonic Algae.  Disulfide and  sulfhydryl group requirements of enzyme activity in,two cryptophytes  Submitted and accepted for publication in The Journal of Phycology  SUMMARY  The 'biosynthetic' L-threonine (deaminating) dehydratase of 2 cryptophytes (Chroomonas salina and Hemiselmis virescens) showed sensitive inhibition fran a l l thiols tested (dithiothreitol, cysteine, etc.) but no effect from ascorbic acid or reduced NAD.  By contrast, the  enzyme activities from 5 non-cryptophyceaen unicellular algae (2 cyanophytes, 1 rhodfophyte, 1 diatom, 1 chlorophyte) were generally not affected by any of these reagents. The thiol-reagent inhibition of the cryptophyte enzymes (i) achieved saturation with 60-70 % reduction i n activity, ( i i ) was considerably reduced by pretreatment of the enzymes with L-threonine and L-isoleucine, and ( i i i ) was p a r t i a l l y reversed by subsequent treatment with arsenite and exposure to air.  It was  deduced that such inhibitions were caused by thiol-specific reduction of enzyme-protein disulfide groups essential for the f u l l expression of activity and that these groups were susceptible to ready reductive cleavage and oxidative restoration.  This disulfide requirement, unique  to the cryptophytes, may be the f i r s t recorded case of such a property of threonine dehydratase from a l l forms of l i f e hitherto studied. The additional activity-requirement of the cryptophyte enzymes f o r sulfhydryl groups (which requirement was common to a l l the algal enzymes) was confirmed a) by the study of their sensitivity to inhibi t i o n from mercurials and disulfide-sulfhydryl exchanging reagents, and b) by the partial reversal of these inhibitions from subsequent treatment with dithiothreitol.  Both cryptophyte enzymes were desen-  sitized to feedback inhibition from L-isoleucine by prior exposure to subinhibitory concentrations of HgCl or dithiodipyridine. ?  6S  INTRODUCTION The occurrence and catalytic properties of the 'biosynthetic' type of L-threonine dehydratase (L-threonine hydro-lyase, deaminating; EC 4.2.1.16) in 7 marine unicellular algae from 5 taxonomic were reported in the previous communication  divisions  of this series (13).  In general, the algal enzymes showed broad resemblance in most properties (pH optima, substrate saturation kinetics, pyridoxal-phosphate and sulfhydryl-group requirements, isoleucine-sensitive allosterism) to the corresponding enzymes from bacteria, fungi, and higher plants. In particular, the alkali-metal cation (more specifically K or NH^ ) +  +  requirement of these algal enzymes indicated a closer property resemblance to the threonine dehydratases of yeast and higher plants (1). However, the algal enzymes themselves showed certain individual differences, one of which appeared to be taxonomically related.  Whereas the  enzyme activities from a chlorophyte, a diatom, a rhodophyte, and 2 cyanophytes were slightly stimulated or not affected by dithiothreitol [DTT], those from 2 cryptophytes were markedly inhibited by this thiol reagent (13). DTT, known to protect protein sulfhydryl groups (10), was expected to favour, or at least maintain, the activity of a l l the algal enzymes, since they had uniformly indicated sulfhydryl group requirement for the expression of activity (13). However, since DTT is also known to cause reductive cleavage of disulfides (31), this unexpected behaviour of the cryptophyte enzymes suggested that they may require both sulfhydryl  [-SH]  manifestation of activity.  and disulfide  [-S-S-]  groupings for the  This inference has now been confirmed by  further tests with a number of thiol and related reagents, the results  of which studies are reported below.  MATERIALS AND METHODS Algal species CHLOROPHYTA:  TetraseLmis maculata.  BACILLARIOPHYTA:  Cyclotella nana (clone 3H of Guillard and Ryther (18)  recently redesignated as Thalassiosira pseudonana (20)). v. l\TTOPHYTA:  Chroomonas salina; HemiseLmis virescens.  RHODOPHYTA:  Porphyridium cruentum.  CYANOPHYTA:  Agmenellum quadruplicatum; Anacystis marina.  Details of the algal strains used, their source and maintenance have been reported (2).  The algae were mass-cultured, harvested, and  freeze-dried as previously described (13). all  Excepting the 2 cryptophytes  the algae used i n this study were grown photoautotrophically with  added vitamins.  C. salina was cultured under 3 sets of conditions,  (i) photoautotrophic (vitamins, l i g h t ) ,  ( i i ) photoheterotrophic  (gly-  cerol, vitamins, l i g h t ) , ( i i i ) chemoheterotrophic (glycerol, vitamins, darkness) (9).  Being unable to use nitrate as N-source or to grow i n  darkness on organic substrates hitherto tested, H. virescens was cultured phototrophically with the same vitamins under 3 other conditions (all  presumably heterotrophic, but this has not yet been established),  (i) with urea (2 mM) as N-source, (ii) with glycine (4 mM) as X-source, ( i i i ) with glycine (4 mM) and glycerol (3,9).  Where present, the gly-  cerol concentration was 0.25 M. Unless otherwise stated, the enzyme tests reported below for C. salina and H. virescens were normally made with the glycerol-light grown culture of the former and the glycine-light grown culture of the l a t t e r .  70  Enzyme assay Suspensions of algal powder in appropriate buffer were submitted to ultrasonic oscillation and whole sonicates were assayed for enzyme activity as previously described (13), the assay method being based on that of Friedemann (15) for the colorimetric determination of keto acid produced. Unless otherwise stated, a l l enzyme incubation mixtures contained algal sonicate (0.5 ml i n 0.2 M potassium N-tris(hydroxymethy1)methyl glycine buffer, pH 8.5), pyridoxal phosphate (0.1 umole), and L-threonine (80 ymoles) in a final volume of 1 ml.  The mixtures were normally  preincubated f i r s t with the test reagent for 15 min at 22°C, then with pyridoxal phosphate for 5 min at 37°C, and f i n a l l y incubated with threonine at the latter temperature for periods varying with the algal species (usually as indicated by Desai e_t al_. (13)).  Identical pre-  incubations were effected on controls taken without the test reagent. A l l the tests included both unincubated and f u l l y incubated controls. A l l reagents used were of the highest purity grade commercially a v a i l -  able.  RESULTS Effects of t h i o l and reducing reagents The response of the 2 cryptophyte enzymes to an extensive range of concentrations (0.01-10.0 mM) of the following reagents was syste-  namide-adenine dinucleotide jNADHJ.  The results, depicted in Figs.  71  1 and 2, show that both cryptophyte enzymes are sensitively inhibited by a l l the thiol reagents but are not affected by ascorbic acid or NADrL,.  By contrast, the enzyme activities of the non-cryptophyceaen  algae were either slightly stimulated (10-20 % from DTT and DTE in the case of T. maculata) or not at a l l affected by the highest concentration (10 mM) of both the thiol and non-thiol reducing agents (Table 1).  The  concentrations of the thiol reagents required to produce 50 % inhibition (Figs. 1 and 2) revealed the following order of effectiveness of the reagents towards both cryptophyte enzymes: GSH > Cys.  DTT = DTE = BAL > L i p f S r f ^ >  This order of diminishing reactivity of the t h i o l reagents  recalled a similar order of diminishing electronegativity of their redox potentials  (10) and indicated that the thiol reagents were acting  on the cryptophyte enzymes by a process of reduction.  In view of the  total absence of effect from NADP^ known to possess comparable redox potential, i t appeared that the reductive action of the t h i o l reagents was more specifically linked to their -SH groups interchanging with sensitive disulfide groups of the cryptophyte enzymes. The inference that -SH sensitive disulfide groups were required for the activity of the cryptophyte enzymes was verified by tests designed to reverse the thiol reagent inhibition by a subsequent oxidative treatment effecting restoration of the original disulfide groups.  Minimal concentrations of DTT were used to produce 30-50 %  inhibitions, the excess thiol reagent was neutralized by specific complexing with sodium arsenite (31), and the p a r t i a l l y inactivated enzyme was reoxidized by prolonged incubation in presence of a i r .  This  mild reversal treatment was considered necessary for valid interpretation of the tests, since the algal enzymes were known to possess  -SH groups also required for activity (13), which should be l e f t undamaged under the conditions effecting the overall transformation ^-S-S-R^  R -SH + R -SH 1  2  R-^S-S-R^  .Air oxidation has been success-  fully used for reformation of the disulfide bonds of papaya lysoryme previously reduced by 2-mercaptoethanol (4).  The test results, summar-  ized i n Table 2, indicate that (i) 40-70 % reversal of inhibition was obtained with both cryptophyte enzymes under the different conditions examined, (ii) the reversal was favored by higher temperature and longer incubation in a i r .  It was established from appropriate controls  a) that sodium arsenite alone had no effect on the enzyme activity but was required in adequate concentration to ensure the reversal, and b) that active aeration (surface agitation of the enzyme mixture with an a i r stream) made no difference to the reversal.  Xo attempt was  made to obtain complete reversal of the t h i o l reagent inhibitions, since the more drastic reoxidation conditions indicated by these tests were themselves expected to have deleterious effects on the enzymes. Certain observations were made on the thiol-reagent induced i n h i bitions and their relationship to the cryptophyte enzyme substrate and allosteric effector, which might shed light on the nature of involvement of the essential disulfide groups in the mechanism of enzyme action.  In the f i r s t place, a l l the tested t h i o l reagents tended to  show saturation of inhibition with 60-70 % reduction in activity; the residual activity with 10 mM concentration of the various reagents ranged 25-29 % for C. salina and 29-37 % for H. virescens 2).  (Figs. 1 and  This residual activity from DTI inhibition was s t i l l sensitive to  feedback inhibition from isoleucine in the case of C. salina but was completely desensitized in the case of H. virescens  (Table 3).  It  75  appears l i k e l y that this difference between the 2 cryptophyte enzymes may be related to their original marked difference in sensitivity to the allosteric effector.  In the second place, i t was observed that the  inhibition from DTT was markedly prevented by prior treatment of both cryptophyte enzymes with threonine and isoleucine  (Table 4), implying  that protection of the sensitive disulfide groups from cleavage was obtained.  Such protection was afforded  best by the combination of both  substrate and allosteric effector and significantly less by either alone. Although known to be essential for activity of the enzymes, added pyridoxal phosphate showed no protective effect (Table 4), which result may be expected from our previous inference of bound pyridoxal phosphate in the algal enzymes (13).  An overall view of these observations  suggested that the sensitive disulfide groups are not directly involved i n the mechanism of enzyme action but appear to be in close proximity to the sites binding the substrate and allosteric effector, with the implication that such location enables them to favour enzyme action by f a c i l i t a t i n g enzyme-substrate binding. The possibility was considered that this unique disulfide-activity requirement observed from the cryptophyceaen algae may have been due to the special nutritional (photoheterotrophic) status of their cultures hitherto studied, in contrast to the photoautotrophic cultures of the other algae examined.  This implication was disproven by comparative  tests of the effects of 1-10 mM DTT on 3 nutritionally different cultures of each cryptophyte.  The results  (Table 5) showed that the  enzymes from the different cultures of the same species suffered the same degree of inhibition and retained the same order of residual activity on saturation of such inhibition.  74  Effects of reagents binding or modifying -SH groups The previous indications of -SH group requirement of the cryptophyte enzymes could not be satisfactorily verified at that time because of unforeseen d i f f i c u l t i e s created by the unexpected finding of thiol-reagent inhibition of these enzymes (13).  However, the i n -  teresting observation was then made that the organomercurial p_-chloromercuriphenyl sulfonate [FOIPS] showed a marked difference between the 2 cryptophyte enzymes (maximal inhibition:  49 I for C_. salina,  95 % for H. virescens), unlike the other -SH modifying agents.  This  difference was now confirmed by tests of the 2 enzymes with a range of concentrations of PCMPS, and comparable studies were made with another mercurial, HgC^, and the disulfide reagents, 2,2'-dithiodipyridine [DTDP] and 6,6'-dithiodinicotinic acid [DTDNA.] , both of which are known to modify protein -SH groups by exchange with their disulfide groups (16, 17).  The results  (Figs. 3 and 4) showed that, excepting the be-  haviour of PCMPS towards C. salina, a l l the reagents effected extremely sensitive inhibition of both cryptophyte enzymes, attaining 90-98 % inactivation at 0.1-0.5 mM reagent levels.  HgO^ was consistently the  most potent towards both enzymes, while the other reagents matched more evenly i n their degree of action.  Their overall 50 %-inhibition effec-  tiveness (inversely related to molar concentration) averaged 1 to 2 orders of magnitude greater than that of the t h i o l reagents (cf. Figs. 1, 2,3,4).  PCMPS produced marked difference in response of the 2 enzymes,  achieving saturation with 50 % inhibition in the case of C. salina but effecting close to 100 % inhibition of H. virescens. uniformly totally inhibitory towards both enzymes, this  Since HgC^ was difference  cannot be attributed to the mercurial nature of PCMPS but may indicate  the presence of 2 types of essential -SH groups in the enzyme protein of C_. salina, one type accessible to a l l the reagents (and presumably the only type in H. virescens) and another type inaccessible to PCMPS but approachable by the other reagents.  Such differences in reactivity of  an organomercurial towards different types of -SH groups on the same enzyme protein have been previously noted (19).  The specific involvement  of enzyme -SH groups i n these inhibitions was further verified by tests designed to reverse the observed inactivations by subsequent treatment with DTT.  The enzymes were f i r s t inactivated 40-60 % by PCMPS or DTDP  and then exposed to minimal excess of the reversal reagent, the concentration of which was circumscribed by its own inhibitory action on the activities.  The results (Table 6) showed that 50-60 % reversals were  obtained from the test conditions approaching optimum tolerable concentration of the reversal agent. The treatment with subinhibitory concentrations of HgC^ or p_chloromercuribenzoate has been previously used to obtain desensitization (to feedback inhibition from isoleucine) of several bacterial and plant threonine dehydratases (8,11,24,25,29,30).  Such desensitizations have  been generally attributed to the binding by the mercurial of -SH groups that are part of the isoleucine binding site on the enzyme. However, the yeast enzymes failed to show such desensitization despite their inactivation by mercurials (7,12).  It was therefore of interest  to conduct similar tests on the cryptophyte enzymes p a r t i a l l y inactivated from HgCl^, PCMPS, or DTDP by subsequent exposure to isoleucine. The results (Table 7) showed that the expected desensitization was obtained in a l l cases, excepting that of C. salina treated with PCMPS.  76 This exception may be consequential to the unique behaviour of PCMPS towards the C. salina enzyme noted above, suggesting that the very same -SH groups inaccessible to this reagent may govern the isoleucine binding s i t e . e r i a l and plant  Otherwise, the cryptophyte enzymes resemble their bactcounterparts, but differ from yeast, in their capacity  for desensitization towards isoleucine inhibition.  DISCUSSION These studies have established beyond doubt that the cryptophyte enzymes require both disulfide and sulfhydryl groups for the f u l l expression of catalytic activity.  The disulfide requirement is unique  to the cryptophytes i n that i t is not shown by the other algal enzymes nor has i t been previously recorded for any threonine dehydratase from a l l forms of l i f e hitherto studied. ment deserves a few comments.  The nature of this require-  Our observations indicated that the  thiol-inhibited enzymes retain 25-35 % of their maximal activity after complete cleavage of the sensitive disulfide groups, suggesting that the latter are not absolutely essential to the basic mechanism of enzyme action but that they promote the reaction rate presumably by f a c i l i t a t i n g the enzyme binding to the substrate.  Such an influence  may be exercised by assistance in maintaining the enzyme molecule i n its most favorable conformation state for activity, and, in this respect, the role of these disulfide groups may be similar to that of allosteric effectors activating enzymes by inducing conformational alterations  (26,27).  Viewed in this l i g h t , i t is tempting to specu-  late that these disulfide groups, with their high potential for reversible reduction, may serve to regulate in vivo activity of the  • ..  77  cryptophyte enzymes which regulation may be intertwined with the metabolic control of oxidation-reduction states of physiological thiols such as glutathione or even of pyridine nucleotides; in the latter case the regulatory interaction could be indirectly transmitted through the mediation of transhydrogenase-type enzymes such as the NADf^dependent disulfide reductase of Bacillus cereus (5).  In this connec-  tion i t must be pointed out that the extraordinary thiol-sensitivity of these disulfide groups of the cryptophyte enzymes distinguishes them from those normally involved i n bridging polypeptide chains of the enzyme molecule shown to be present in some purified bacterial threonine dehydratases (21,22) for which no disulfide group requirement was reported.  The redox sensitivity of the former disulfide  groups is reminiscent of similar sensitivity of the unusual type of disulfide bond "looping" 2 half-cysteine residues in the same polypeptide chain of insulin (6,14,23,28).  The close proximity of such  sensitive disulfide groups to the cryptophyte enzyme sites binding threonine and isoleucine i s suggested by our observations of the protection from thiol-induced inhibition afforded by both the substrate and allosteric  effector.  The sulfhydryl group requirement of the cryptophyte enzymes is common not only to a l l the algal species examined but appears to be a standard requirement of a l l threonine dehydratases hitherto tested (for citation of the literature, see ref. 13).  The generality of  this requirement and our observations, that reagents trapping or modifying such groups usually cause extremely sensitive and total i n a c t i vation, suggest that these particular -SH groups must be directly involved in the basic mechanism of enzyme action.  The "desensitiza-  tions" to feedback inhibition from isoleucine obtained in this studycertainly indicate their involvement at the enzyme site binding the allosteric  effector.  ACKNOU1EDGMENT One of us (I.D.D.) acknowledges financial support from the Fisheries Research Board of Canada and the Research Committee of the University of British Columbia.  79  TABLE 1.  Effects of thiol and reducing reagents on L-threonine dehydratase activity of 7 algal species.  Alga  Enzyme activity (% of control) Asc(OH)  NADrt,  DTE  GSH  Cys  salina  28  27  28  101  98  H. virescens  30  33  37  97  104  C. nana  105  104  105  99  101  T. maculata  112  107  108  99  96  P. cruentum  102  100  101  99  102  98  103  102  99  98  102  99  100  100  98  c.  A. quadruplicatum A. marina  2  Activity obtained from treatment with 10 mM concentration of the reagents shown, under standard assay conditions.  80  TABLE 2.  Reversal of DTT -induced cryptopnyte enzyme inhibition by treatment with sodium arsenite and prolonged air exposure *  DTT concn  (mM)  NaAs0  2  concn  (mM)  Enzyme activity  Preincubation with NaAsC^  H. virescens  C. salina  8•a  Si>  Temp. (°C)  Period (min)  inhib.  reversal  inhib.  reversal  %  %  0.44  -  37  30  50  -  49  -  0.44  1.0  37  30  23  54  14  72  0.44  2.5  37  30  23  54  19  60  0.44  5.0  37  30  21  58  18  60  0.44  -  22  30  49  -  48  -  0.44  5.0  22  30  28  44  26  46  1.0  -  22  30  73  -  67  -  1.0  5.0  22  30  36  51  39  42  0.44  -  22  60  49  -  50  -  0.44  5.0  22  60  15  70  12  71  1.0  -  22  60  74  -  67  -  1.0  5.0  22  60  27  64  25  63  * Method involved treatment with DTT (15 min, 22°C) followed by preincubation with NaAsC^ at the indicated temperature and period, prior to the regular assay procedure commencing with pyridoxal phosphate addition.  TABLE 3.  Effects of isoleucine on residual activity of the cryptophyte * enzymes obtained after maximal inhibition from DTT.  Reagent concn (mM) DTT  Isoleucine  1.0  -  1.0  Enzyme activity % { of control) C. salina  H. virescens  23  31  1.0  4  48  1.0  1  30  * Method involved treatment with DTT (15 min, 22°C) followed by preincubation with isoleucine  (1 min, 22°C), prior to the regular assay  procedure commencing with pyridoxal phosphate addition.  82  TABLE 4.  Protective effects of threonine and isoleucine on the cryptophyte enzymes from DTT-induced  * inhibition.  Concentration (mM) Pyridoxal phosphate  Threonine  Isoleucine  Enzyme activity % ( of control) DTT  C. salina  H. virescens  -  _  0.1  27  33  0.1  -  0.1  28  33  -  20  0.1  41  41  0.1  62  61  0.1  41  42  0.01  0.1  64  62  0.1  0.01 20  0.1  -  20  0.01  0.1  73  72  0.1  20  0.01  0.1  73  70  Method involved pretreatment with pyridoxal phosphate, threonine, isoleucine, or combinations thereof (1 min, 22°C) followed by preincubation with DTT (15 min, 22°C).  The regular assay procedure was modified to adjust for  the amounts of pyridoxal phosphate and threonine used in the pretreatment, such that the final concentrations i n the incubation mixture remained as usual.  TABLE 5. Effects of DTT on the activity of the cryptophyte enzymes from nutritionally different cultures. Alga  Culture conditions *  H. virescens  Light  Nutrients  +  glycine  +  T  DTT concn (mM)  Enzyme activity (% of control)  1  35  10  34  "glycine ")  1  36  glycerol \  10  35  1  38  10  35  1  28  10  26  1  26  10  25  1  24  10  25  <  +  C. salina  +  +  -  urea  nil  glycerol  glycerol  Presence (+) or absence (—) of continuous illumination (ca. 16,500 lux) from cool-white fluorescent lamps; when absent, complete darkness was used. These nutrients correspond to the added organic compounds (see text) i n addition to the vitamins normally included in a l l cultures.  84 TABLE 6.  Reversal by DTT of cryptophyte enzyme activity inhibition from  * -SH binding agents.  -SH binding agent  DTDP  PCMPS  DTT concn  Reversal of inhibition (%)  concn (mM)  (mM)  C. salina  H. virescens  0.02  0.02  13  0  0.02  0.05  67  33  0.02  0.10  58  31  0.02  0.02  20  15  0.02  0.05  62  53  0.02  0.10  52  39  0.05  0.05  12  0.05  0.10  29  Method involved pretreatment with -SH binding agents (15 min, 22°C) followed by preincubation with DTT (30 min, 22°C), prior to the regular assay procedure commencing with pyridoxal phosphate addition. ^Calculated by equating the inhibition from -SH binding agent alone to 0 % reversal, the inhibition from DTT alone to 100 % reversal, and scaling the net inhibition from the sequential action of both reagents between these extremes.  TABLE 7.  Desensitization of the cryptophyte enzymes to isoleucine * feedback inhibition by -SH binding agents.  -SH binding agents concn (mM)  HgCl  DTDP  2  (mM)  Enzyme activity (I of control) C. salina  H. virescens  1.0  2  48  0.005  1.0  6  51  0.015  1.0  -  72  0.050  1.0  9  98  0.001  1.0  10  60  0.005  1.0  67  84  0.010  1.0  99  101  0.005  1.0  27  52  0.010  1.0  65  68  0.020  1.0  87  89  -  nil PCMPS  Isoleucine concn  Method involved pretreatment with -SH binding agent (15 min, 22°C) followed by preincubation with isoleucine (1 min, 22°C) prior to the regular assay procedure commencing with pyridoxal phosphate addition. T  Xote that 100 % control in these tests is represented by the activi t i e s obtained from a l l corresponding isoleucine.  pretreatments effected without  86  Figure Legends  FIG. 1. Effects of thiol and reducing reagents on L-threonine dehydratase activity of Giroomonas salina under standard assay conditions.  Reagent abbreviations are indicated in  the text. FIG. 2.  Effects of t h i o l and reducing reagents on L-threonine dehydratase activity of Hemiselmis virescens under standard assay conditions.  Reagent abbreviations are indicated i n  the text. FIG. 3.  Effects of -SH binding or modifying agents on L-threonine dehydratase activity of Chroomonas salina under standard assay conditions.  Reagent abbreviations are indicated  in the text. FIG. 4.  Effects of -SH binding or modifying agents on L-threonine dehydratase activity of Hemiselmis virescens under standard assay conditions. the text.  Reagent abbreviations are indicated i n  Concn: 50%inhib. (mM) C. salina  Cys GSH Lip(SH) DTE DTT BAL  2  1-20 0-76 0-6 3 0-48 0-44 0-43  Asc(OH)  2  Concn: 50% (mM) Cys GSH Lip(SH) DTE DTT BAL  H. virescens  2  n 10  "  1  5  Reagent Concentration (mM)  2-50 1-30 0-94 0-42 0-46 0-41  *>  Concn.-50% inhib. (mM)  100  9 0«  o  -4—  v  80  M—  70  c 0 u  O  PCMPS DTDNA DTDP HgCI 2  -0-125 0-021 0-017 0-008  60  X >  50  <  40  me  u  30  N C LU  20  1 0 -  Fig. 3  Reagent Concentration (mM)  c oncn: 50% inhib. (mM) PCMPS DTDNA DTDP HgCI 2  Fig. 4  0 021 0024 0-022 0-006  Reagent Concentration (mM)  o  REFERENCES  ANTLA, N . J . , KRIPPS, R.S. § DESAI, I.D. 1972. in marine planktonic algae. I I I .  L-threonine deaminase  Stimulation of activity by  monovalent inorganic cations and diverse effects from other ions.  Iii preparation.  ANT LA, N . J . $ CHENG, J . Y . 1970.  The survival of axenic cultures  of marine planktonic algae from prolonged exposure to darkness at 20°C.  Phycologia, 9:  ANT LA, N . J . a CHORNEY, V. 1968.  179-84. Nature of the nitrogen compounds  supporting phototrophic growth of the marine cryptomonad Hemiselmis virescens.  J . Protozool.  15:  BAREL, A . O . , DOLMANS, M. $ LEONIS, J . 1971.  198-201.  Spectroscopic studies  on the reduction and reformation of the disulfide bonds of papaya lysozyme.  Europe. J . Biochem. 19:  BLANKENSHIP, L . C . § MENCHER, J.R. 1971.  488-95.  A disulfide reductase  in spores of Bacillus cereus T. Can. J . Microbiol.  17:  1273-7. BROWN, H . , SANGER, F. $ KITAI, R. 1955. and sheep insulins.  Biochem. J .  The structure of pig 60:  CENNA^D, C , BOLL, M. $ HOLZER, H. 1964. ratase aus Saccharomyces cerevisiae.  556-65. Uber ThreonindehydBiochem. Z.  340:  125-  45. CRANGEUX, J . - P . 1961.  The feedback control mechanism of biosyn-  thetic L-threonine deaminase by L-isoleucine. Harbor Symp.  Quant. B i o l .  CHENG, J . Y . $ ANT LA, N . J . 1970.  26:  Cold Spring  313-8.  Enhancement by glycerol of photo-  trophic growth of marine planktonic algae and its cance to the ecology of glycerol pollution.  signifi-  J . Fisheries  92  Res. Board Can. 10.  CLELAND, W.K. 1964. for SH groups.  11.  DATTA, P. 1966.  27:  335-46.  Dithiothreitol, a new protective reagent Biochem.  3:  480-2.  Purification and feedback control of threonine  deaminase activity of Rhodopseudomonas spheroides. Chem. 12.  241:  J . Biol.  5836-44.  DE ROBICHON-SZULNLAJSTER, H. § HAGEE, P.T. 1968.  The regulation  of isoleucine-valine biosynthesis in Saccharomyces cerevisiae. 1. 13.  Threonine deaminase.  Europe. J . Biochem.  DESAI, I . D . , LAUB, D. § ANTIA, N . J . 1972.  3:  492-501.  Comparative characte-  rization of L-threonine dehydratase i n 7 species of unicellular marine algae. 14.  Phytochem.  11:  277-87.  DU VIGXEAUD, V . , FITCH, A . , PEKAREK, E . $ LOCKWOOD, W.W. 1931-32. The inactivation of crystalline insulin by cysteine and glutathione.  15.  J . B i o l . Chem.  FRIEDEMANN, T . E . 1957.  94:  233-42.  Determination of cC-keto acids.  In  Colowick, S.P. § Kaplan, N.O. [ed.] , Methods in Enzymology, Vol. 3, Academic Press, N . Y . , 414-8. 16.  GRASSETTI, D.R. § MURRAY, J . F . , JR. 1967.  Determination of sulf-  hydryl groups with 2,2'- or 4,4'-dithiodipyridine. Biochem. Biophys. 17.  119:  Arch.  41-9.  GRASSETTI, D.R., MURRAY, J . F . , JR. $ RUAN, H.T. 1969.  The inter-  action of 6,6'-dithiodinicotinic acid with thiols and with Ehrlich ascites tumor c e l l s . 18.  Biochem. Pharmacol.  GUILLARD, R.R.L. $ RYTHER, J . H . 1962. diatoms.  I.  (Cleve) Gran.  18:  603-11.  Studies of marine planktonic  Cvclotella nana Hustedt,' and Detonula confervaceae t ——— Can. J . Microbiol.  8:  229-39.  9.3 19.  RASINOFF, B . B . , MADSEN, N.B. $ AVTRAMOVIC-ZIKIC, 0. 1971.  Kinetics  of the reaction of p_-chloromercuribenzoate with the sulfhydryl groups of glutathione, 2-mercaptoethanol, and phosphorylase b. Can. J . Biochem. 20.  49:  742-51.  HASLE, G.R. § HEIMDAL, B.R. 1970.  Some species of the centric  diatom genus Thalassiosira studied i n the light and electron microscopes. 21.  Nova Hedwigia^31:  HATFIELD, G.W. § BURNS, R.O. 1970. Salmonella t>phimurium J . B i o l . Chem. 245:  22.  Chem.  245:  The intermediate substructure.  787-91.  cine.  Purification of the enzyme.  J . Chem. Soc. 1956:  3157-68.  MAEBA, P. £ SANWAL, B.D. 1966. of Salmonella.  449-60.  The allosteric threonine deaminase  Kinetic model for the native enzyme.  MONOD, J . , CHANGEUX, J . - P . $ JACOB, F. 1963.  allosteric transitions: 88-118.  A l l o s t e r i c proteins  J . Mol. B i o l .  MONOD, J . , WYMAN, J . 5 CHANGEUX, J . - P . 1965.  12:  Biochem.  525-36.  and cellular control systems. 27.  Threonine dehydratase of  Purification and properties.  Biochim. Biophys. Acta, 151:  26.  Polypeptides.  The oxidation of some peptides of cysteine and gly-  LEITZMANN, C. S BERNLOHR, R.W. 1968.  5:  J . Biol.  1736-41.  Bacillus licheniformis I.  25.  Threonine deaminase from  HEATON, G . S . , RYDON, H.N. $ SCHOFIELD, J . A . 1956. Part III.  24.  Threonine deaminase from  HATFIELD, G.W. § UMBARGER, H.E. 1970. Bacillus subtilis I.  23.  III.  559-81.  6:  306-29.  On the nature of  a plausible model.  J . Mol. B i o l .  94  28.  RYLE, A . P . , SANGER, F . , SMITH, L . F . t\ KITAI, R. 1955. s bond^of insulin.  29.  Biochem. J .  SHARMA, R.K. § MAZUMDER, R. 1970.  60:  The disulphide  541-56.  Purification, properties, and  feedback control of L-threonine dehydratase from spinach. J . B i o l . Chem. 30.  245:  3008-14.  TOMOVA, V . S . , KAGAN, Z.S. $ KRETOVICH, V . L . 1968.  Kinetic proper-  ties of desensitized "biosynthetic" L-threonine dehydratase of pea seedlings. 31.  Doklady Akademii Nauk SSSR, 180:  ZAHLER, W.L. § CLELAND, W.W. 1968. for disulfides.  J . B i o l . Chem.  237-40.  A specific and sensitive assay 243:  716-9.  95  SUPPLEMENT I I  L-Threonine Deaminase in Marine Planktonic Algae. Stimulation of activity by monovalent inorganic cations and diverse effects from other ions  Submitted and accepted for publication in Archives fur Mikrobiologie  Summary ing)  1.  The ' b i o s y n t h e t i c '  dehydratase of  5 c l a s s e s of algae  showed s e v e r a l  tion was'generally +  +  , whilst  degrees of  inorganic cations.  the s t r o n g e s t  and NH^, and the weakest  and T l  Li ,Rb , +  +  (1 to  and C s  a l g a was e x c e p t i o n a l  from N a  from L i  +  2-fold)  stimulation  a green a l g a showed  w i t h the  least  effect +  from L i  (i)  to i o n c o n c e n t r a t i o n ,  and ( i i )  f o r monovalent  indications  of a coenzyme type of r o l e f o r the  3. cations  4. fluoride, nitrate, and  inorganic cations,  were  Mg^ ,Ca^ ,Zn^ ,Cu^  or without  and  high  with alkali  ions.  Organic c a t i o n s +  +  'hyperbolic  specificity  metal type of  another  than NH^.  The c a t i o n a c t i v a t i o n showed  k i n e t i c response  intermediate  One b l u e - g r e e n  markedly g r e a t e r a c t i v a t i o n from R b 2.  +  and more pronounced a c t i v a t i o n  +  type of response  with  with X a  i n showing s t r o n g e s t  than C s , w h i l s t  +  activa-  5-fold)  showed  +  from  The a c t i v a -  (3 to  orders varying with a l g a l s p e c i e s .  (5-fold)  (deaminat-  7 marine p l a n k t o n i c s p e c i e s  t i o n from monovalent  K  L-threonine  +  +  +  i n e r t and the  were e i t h e r  divalent  inhibitory  effect.  .Among the  anions  tested,  c h l o r i d e , bromide,  b i c a r b o n a t e showed no e f f e c t , c h l o r a t e were  sulfate  iodide,  i n h i b i t o r y , whilst  were s l i g h t l y  stimulatory.  phosphate  97  5.  I t was concluded that the a l g a l enzymes  may have an absolute K  +  or N'H* requirement f o r i n  v i v o expression of a c t i v i t y .  The i d e n t i f i c a t i o n 'biosynthetic  type  1  and c h a r a c t e r i z a t i o n of  of L - t h r e o n i n e  (L-threonine hydro-lyase,  algae was r e p o r t e d i n the (Desai  e_t a l . , 1972).  differences  ( A n t i a et  first  paper o f  al.,  1972),  the  allosterism)  t h i s study  fungi,  (Desai  to the  (pH 8-9) the  isoleucine-  c o r r e s p o n d i n g enzymes  e_t a l . , 1972) , the  i n h i b i t i o n of a c t i v i t y  w i t h potassium phosphate  determination  from T r i s - H C l  (pH 9 . 5 - 1 0 . 5 )  and T r i c i n e b u f f e r s ,  The nature of from the  the  be e f f e c t i v e l y algal  enzymes.  effects,  controlled  inhibitions  used,  suggested and  which needed  for r e l i a b l e  The s y s t e m a t i c  which  significant  i o n i c composition  c o n c e n t r a t i o n o f the b u f f e r s  buffers;  from comparison  i n t u r n showed s m a l l e r but d i s t u r b i n g l y  effects  During  a l g a l enzymes was b e s e t by  i n h i b i t i o n was c l e a r l y e v i d e n t  undefined  enzymes  pyridoxal-phosphate  and sodium b i c a r b o n a t e  deviations.  series  (pH o p t i m a ,  and h i g h e r p l a n t s .  of the pH optima of the problems o f  of  this  algal  in properties  and s u l f h y d r y l - g r o u p r e q u i r e m e n t s ,  from b a c t e r i a ,  1.16)  from 5 c l a s s e s  saturation k i n e t i c s ,  sensitive  EC 4 . 2 .  A p a r t from c e r t a i n i n d i v i d u a l  showed broad resemblance substrate  dehydratase  deaminating;  i n 7 marine p l a n k t o n i c s p e c i e s  the  study o f  investigation  r e p o r t e d i n t h i s communication, has  of  to the these  revealed  that  ( i ) the a l g a l enzymes r e q u i r e c e r t a i n monovalent  inorganic cations ity,  f o r the f u l l  expression  of activ-  ( i i ) the apparent i n h i b i t i o n o b t a i n e d  from  Tris-  HC1 was due to the absence o f these ions i n the b u f f e r used,  (iii)  the apparent i n h i b i t i o n observed i n the  sodium b i c a r b o n a t e  b u f f e r s was due t o markedly lower  a c t i v a t i o n o f the enzymes by N a  +  relative  t o K , and +  ( i v ) the d i v e r g e n c e s observed between potassium phosphate and T r i c i n e b u f f e r s o f comparable  molarity  were due t o d i f f e r e n c e s o f K  i n these  buffers  +  concentration  (apart from s l i g h t s t i m u l a t i o n by phosphate  a n i o n ) , which d i f f e r e n c e s a r e n o r m a l l y i g n o r e d b u t assume importance i n the c o n c e n t r a t i o n - s e n s i t i v e response o f the a l g a l enzymes.  Materials Algal  and Methods  species  CHLOROPHYTA (green a l g a e ) : T e t r a s e l m i s  maculata  (Te.ma.). BACILLARIOPHYTA (diatoms):  C y c l o t e l l a nana  CRYPTOPHYTA: Chroomonas s a l i n a (Ch.sa.); virescens  (Cy.na.).  Hemiselmis  (He.vi.).  RHODOPHYTA (red a l g a e ) : Porphyridium cruentum CYANOPHYTA catum  (blue-green  a l g a e ) : Agmenellum  (Po.cr.).  quadrupli-  (Ag. qu. ) ; A n a c y s t i s -marina (An.ma. ) .  Abbreviations  shown a f t e r  to denote these s p e c i e s of  the  algal  strains  i n F i g u r e s and T a b l e s .  used,  ance have been r e p o r t e d algae were m a s s - c u l t u r e d , as p r e v i o u s l y d e s c r i b e d  their  source  harvested, (Desai  et  and  al.,  all  freeze-dried 1972).  the  Except-  cultures  used  with  C_. s a l i n a and H. v i r e s c e n s were  grown p h o t o t r o p h i c a l l y w i t h enrichments and g l y c i n e ,  Details  and m a i n t e n -  study were grown p h o t o a u t o t r o p h i c a l l y  added v i t a m i n s .  used  ( A n t i a and Cheng, 1970). The  i n g C . s a l i n a and H. v i r e s c e n s , in this  s p e c i e s names are  respectively,  of  i n a d d i t i o n to  glycerol the  vitamins.  Enzyme assay Suspensions  o f a l g a l powder i n a p p r o p r i a t e  were submitted to u l t r a s o n i c  oscillation  and whole  s o n i c a t e s were assayed f o r enzyme a c t i v i t y viously  described  (Desai  e_t al_. , 1972),  method b e i n g based on t h a t the  buffer  the  as  pre-  assay  of Friedemann (1957)  for  c o l o r i m e t r i c d e t e r m i n a t i o n of keto a c i d p r o d u c e d . All  sonicate final  enzyme (0.5  i n c u b a t i o n mixtures  ml i n b u f f e r o f  contained  strength  double  m o l a r i t y r e q u i r e d ) , p y r i d o x a l phosphate  and L - t h r e o n i n e  (80 umoles)  were p r e i n c u b a t e d w i t h p y r i d o x a l  for  37°C,  then  the (0.1  i n a f i n a l volume of  The mixtures 5 min at  algal  umole),  1 ml.  phosphate  incubated with threonine  at  the same temperature f o r p e r i o d s v a r y i n g w i t h algal al.,  species 1972).  anions were  (10-40 min as When t e s t e d ,  i n d i c a t e d by D e s a i e_t  the s a l t s o f c a t i o n s  i n c o r p o r a t e d i n t o the mixtures  same time as the b u f f e r e d a l g a l s o n i c a t e . tests  i n c l u d e d u n i n c u b a t e d and f u l l y  c o n t r o l s made without homogeneous  the s a l t s .  Calif.).  used were o f  commercially  at  or the  A l l such  incubated  Chromatographically  L - t h r e o n i n e was purchased from Calbiochem  (Los A n g e l e s , reagents  the  The s a l t s , the h i g h e s t  b u f f e r and o t h e r p u r i t y grade  available.  Results All  the  a l g a l enzymes  showed v e r y low  activity  M i n 0. lJ^Tris-HC1 b u f f e r , which was enhanced two five-fold  to  by the a d d i t i o n of s e v e r a l monovalent  organic cations  as t h e i r c h l o r i d e s a l t s  At a c o n c e n t r a t i o n l e v e l  (Fig.  of 0.1 M, NH^ and K  +  1). were  g e n e r a l l y the most s t i m u l a t o r y c a u s i n g 220-390% in a c t i v i t y ,  L i  +  , Rb , Cs +  +  showed i n t e r m e d i a t e  of a c t i v a t i o n with t h e i r o r d e r of e f f e c t i v e n e s s i n g w i t h the a l g a l s p e c i e s , the  least effective  while Na  +  was  w i t h 3-1151 i n c r e a s e  The cyanophyte A . marina was e x c e p t i o n a l  in-  increase degrees vary-  usually in  activity.  i n showing  the h i g h e s t  a c t i v i t y enhancement  among the c a t i o n s degree  as w e l l  (3845) from L i  as i n o b t a i n i n g  o f a c t i v a t i o n from N a  +  greater  (1891) than C s  (166%).  +  By c o n t r a s t ,  the c h l o r o p h y t e T . maculata showed  least effect  from L i  t i o n from K  +  +  (96%), the s t r o n g e s t  (390%), and c o n s i d e r a b l y  a c t i v a t i o n from R b  +  The  requirement +  or no e f f e c t  +  +  from Rb , L i , Cs ,  +  Na  stimula-  (285%) than NH* (160%).  +  K , with l i t t l e  the  greater  diatom C . nana showed the most s p e c i f i c for  +  +  , and the  among the Tl  +  algae. c o u l d not be compared d i r e c t l y w i t h  other c a t i o n s extremely  lowest a c t i v a t i o n from NH^ o b s e r v e d  as the c h l o r i d e s a l t ,  low s o l u b i l i t y .  because  the of  The more s o l u b l e  its  TINO^  o f f e r e d a means of comparison w i t h KNO-j and NH^NO^ at a c o n c e n t r a t i o n l e v e l (Table 1)  o f 0.05  showed no e f f e c t  M.  from T l  +  The  on the diatom  enzyme and r e l a t i v e l y minor s t i m u l a t i o n the o t h e r a l g a l enzymes. that the  strongest  w i t h the  alga  (26-61%) o f  It was i n t e r e s t i n g  a c t i v a t i o n from T l  +  was  to note  observed  (A. marina) which had shown anomalous  order of response  to the other  cations.  The p o s s i b i l i t y was c o n s i d e r e d t h a t degree  results  the  of a c t i v a t i o n o b t a i n e d from some o f  may be due to net  i n h i b i t i o n from excess  lesser  the  cations  concentration  103  of these ions Fig.  1.  at the  level  used f o r comparison i n  T h i s was d i s p r o v e d by s t u d y i n g the  effects  of c a t i o n c o n c e n t r a t i o n g r a d a t i o n s on 5 a l g a l species  (examples  shown i n F i g . 2 ) .  In a l l  cases,  enzyme a c t i v i t y showed h y p e r b o l i c i n c r e a s e s ionic concentration.  Certain algal  with  species-cation  combinations tended to show i o n s a t u r a t i o n at upper c o n c e n t r a t i o n l e v e l s no evidence values (see  of i n h i b i t i o n .  f o r the c a t i o n s  F i g . 2)  enzymes  t e s t e d but there  was  E s t i m a t e s of the Km  showing the  indicate high a f f i n i t y  f o r these  the  strongest of the  effects  algal  ions.  An i n t e r e s t i n g  outcome o f the  concentration  g r a d i e n t study was the e x p l a n a t i o n i t  offered  the minor but s i g n i f i c a n t  obtained  enzyme a c t i v i t y v a l u e s phosphate, i n g 0.1  K-Tricine,  M KC1) b u f f e r s  divergences  determined w i t h 0.1 K-Hepes,  differ  M K-  and T r i s - H C l  (contain-  first  3 buffers  to t h e i r b u f f e r i n g a n i o n s ,  sufficiently  in  at comparable pH (Table  Although the m o l a r i t y of the same w i t h r e s p e c t  for  in their K  concentration  +  produce the observed d i v e r g e n c e s  i n response  2).  is  the  they to of  the  more s e n s i t i v e a l g a l enzymes.  E x c e p t i n g the case o f  K-phosphate,  were v i r t u a l l y e l i m i n -  these d i v e r g e n c e s  ated whenever the  KC1 c o n c e n t r a t i o n , taken i n  c o n j u n c t i o n with T r i s - H C l  b u f f e r , was a d j u s t e d  correspond c l o s e l y w i t h the other b u f f e r s  (Table 2 ) .  a s m a l l d e v i a t i o n because from the phosphate ing  c o n c e n t r a t i o n of  +  K-phosphate s t i l l of  the  showed  the s t i m u l a t i o n o b t a i n e d  (see  below).  The c o r r e s p o n d -  comparison of N a - b i c a r b o n a t e and K - b i c a r b o n a t e  buffers of  anion  K  to  (Table 2)  showed t h a t  the  apparent i n h i b i t i o n  the a l g a l enzymes p r e v i o u s l y o b t a i n e d  a l . , 1972)  (Desai e_t  from the former b u f f e r was due to  l e s s e r a c t i v a t i o n of the When these b u f f e r s  enzymes by N a  +  the  than K . +  were compared w i t h t h e i r N a  K - c o n t r o l l e d c o u n t e r p a r t s i n Map-HCl b u f f e r ,  excel-  +  lent  agreement was observed The  divalent cations  (Table  or  +  2).  2+ 2+ 2+ 2+ Ca , Mg , Zn , Cu  showed no a c t i v a t i o n o f the a l g a l enzymes when t e s t e d with T r i s - H C l buffer,  and K - T r i c i n e b u f f e r s .  the a c t i v i t y observed was too  other e f f e c t s due to  its  K  sufficiently  +  2 +  But the  content,  activity  elevated  to i n d i c a t e d e f i n i t e  were s t r o n g l y  and M g A.  2 +  low to  from these i o n s .  t i o n from the d i v a l e n t c a t i o n s Cu  With the  quadruplicatum.  effect  (Table 3 ) .  T h i s cyanophyte was  buffer,  levels  patterns  excepting  assess  latter  of  inhibi-  Z n ^ and +  i n h i b i t o r y (50-901), w h i l e  showed l i t t l e  former  Ca  2 +  the case o f consistently  sensitive  to a l l the  4 divalent cations,  i n h i b i t e d about 30% by C a .  - 2+  by  Zn  or M g  2 +  2 +  being  and about 90%  -2 + or Cu  All  the a l g a l enzymes  low a c t i v i t i e s  in Tris-HCl  (irrespective  of pH) i n the  cations.  It was n e c e s s a r y  showed  insignificantly  and Map-HCl absence  buffers  of a l k a l i - m e t a l  to e s t a b l i s h whether  was due to enzyme i n h i b i t i o n or i n d i f f e r e n c e  from  the o r g a n i c c a t i o n s t r e n g t h o f these b u f f e r s . effects  o f a range o f c o n c e n t r a t i o n s  100 mM) of T r i s - H C l  (pH 8.5)  both w i t h and without on  2 a l g a l species  The  (10,  and Map-HCl  25, (pH  added KC1 (5 mM), were  this  The 50, 9.5), tested  (C. s a l i n a and P. cruentum).  observed a c t i v i t i e s  showed no s i g n i f i c a n t  from b u f f e r c o n c e n t r a t i o n i n a l l the cases  effect  tested.  S i m i l a r t e s t s were a l s o made w i t h 2 c o n c e n t r a t i o n s (10,  50 mM) of methylamine-HCl or t r i m e t h y l a m i n e -  HCl i n c o r p o r a t e d i n t o T r i s - H C l KC1)  and a g a i n no e f f e c t s were o b t a i n e d from these  a d d i t i o n a l organic c a t i o n s . an o v e r a l l i n d i f f e r e n c e algal  (0.1M, pH 8 . 5 ; ± 5 mM  enzymes.  The evidence  of o r g a n i c c a t i o n s  indicated to  the  T e s t s o f the e f f e c t s the a l g a l  o f i n o r g a n i c anions on  enzyme a c t i v i t y p r e s e n t e d d i f f i c u l t i e s i n  maintaining  simultaneous  c o n t r o l over the pH and  accompanying c a t i o n , s i n c e the u s u a l b u f f e r s cont a i n e d u n e q u i v a l e n t c o n c e n t r a t i o n s o f e i t h e r o r both ions a t any chosen pH.  Tris-H  +  was the obvious  c h o i c e o f e n z y m i c a l l y i n e r t but b u f f e r - e f f e c t i v e c a t i o n , but t h i s r e q u i r e d b u f f e r s o f the chosen pH to be made up with a c i d s o f the a n i o n t e s t e d (HBr, r^SO^,  HNOj), where s t r i c t  c o n t r o l over the a n i o n  c o n c e n t r a t i o n was not p o s s i b l e .  However,  tests  w i t h such b u f f e r s showed poor enzyme a c t i v i t y o f levels and  comparable t o those o b t a i n e d w i t h  i n d i c a t e d t h a t any e f f e c t s  from  Tris-HCl  these  anions  were too low f o r s a t i s f a c t o r y e v a l u a t i o n i n the absence o f an a c t i v a t i n g monovalent  cation.  E v e n t u a l l y , the T r i s - H C l b u f f e r c o n t a i n i n g n e g l i g ible salts  c h l o r i d e i o n was chosen f o r the t e s t s w i t h o f the anions  K  +  at a c o n v e n i e n t l y c o n t r o l l a b l e  pH and the anion e f f e c t s were e v a l u a t e d a g a i n s t KC1 c o n t r o l s . from d i r e c t  The b i c a r b o n a t e e f f e c t s were o b t a i n e d  comparison  the c o r r e s p o n d i n g results  (Table 4)  of K-bicarbonate  buffer  with  K C l - e n r i c h e d Map-HCl b u f f e r .  The  showed t h a t f l u o r i d e ,  b i c a r b o n a t e had no e f f e c t  on the a l g a l  bromide, enzymes,  i o d i d e was s t r o n g l y  (81-88%) i n h i b i t o r y ,  nitrate  and c h l o r a t e were moderately  (18-45%) i n h i b i t o r y ,  whilst  were s l i g h t l y  phosphate  stimulatory. relatively  and s u l f a t e  Whereas the c h l o r a t e  u n i f o r m , the n i t r a t e  (8-25%)  i n h i b i t i o n was  i n h i b i t i o n showed  taxonomically-related d i f f e r e n t i a l  response  from  the algae t e s t e d , b e i n g most pronounced w i t h 2 cyanophytes phytes.  and r e l a t i v e l y  Anion c o n c e n t r a t i o n  0 . 0 5 - 0 . 2 0 M produced l i t t l e or s t i m u l a t i o n s centrations  obtained,  s m a l l w i t h the increase change  but t h i s  total  i n the  to n i t r a t e ,  a b o l i t i o n o f a l g a l enzyme  was t r a c e d to  its  range  inhibitions these con-  in their effects.  n i t r i t e was t e s t e d a n a l o g o u s l y virtually  2 crypto-  i n the  i n d i c a t i n g that  were s a t u r a t i n g  the  interference  method when c o n t r o l d e t e r m i n a t i o n s  it  When showed  activity,  i n the  assay  made w i t h  t ^ - k e t o b u y r i c a c i d showed s i m i l a r i n h i b i t i o n from nitrite  of the c o l o u r n o r m a l l y d e v e l o p e d . Discussion  The r e s u l t s inevitable cations  of t h i s  conclusion  investigation  t h a t monovalent  (more s p e c i f i c a l l y  absolutely  r e q u i r e d f o r the  l e a d to inorganic  a l k a l i - m e t a l type) expression  the  of  are  activity  by a l g a l threonine  deaminases.  ranging  +  from T r i s - H  Organic c a t i o n s  to mono- and t r i - m e t h y l a t e d  ammonium ions have shown no e f f e c t s whatsoever and may be considered  i n e r t towards these enzymes.  T r i s has been g e n e r a l l y reported to i n h i b i t s e v e r a l enzyme systems (other than threonine  deaminase)  a c t i v a t e d by monovalent c a t i o n s (Betts and Evans, 1968); i n one rare instance i t proved s t i m u l a t o r y at low concentration while being i n h i b i t o r y at high concentration  (Bewley and Marcus, 1970).  These  e f f e c t s of T r i s have been a t t r i b u t e d to i t s antagoni s t i c competition w i t h the r e q u i r e d c a t i o n ( g e n e r a l l y K ) +  or i t s non-competitive c o n t r i b u t i o n to the t o t a l  c a t i o n i c strength.  The e n t i r e l y negative  response o f  the a l g a l enzymes to t e s t s designed to e l u c i d a t e such e f f e c t s of T r i s and Map b u f f e r s compels us to i n f e r that and  a) these b u f f e r s are i n d i f f e r e n t to the enzymes, b) the i n s i g n i f i c a n t l y low, though measureable,  a c t i v i t y obtained  i n these b u f f e r s i s not due to  i n h i b i t i o n from them but r a t h e r a consequence of contaminating a l k a l i - m e t a l type c a t i o n s expected to be present i n the a l g a l preparations  used.  This  i n t e r p r e t a t i o n i s supported by the extreme s e n s i t i v i t y of response of the a l g a l enzymes to minute amounts of added K  +  or NHT i n the presence of large excess  of b u f f e r c a t i o n s .  The low magnitude o f  the  M i c h a e l i s c o n s t a n t s o b t a i n e d f o r the more alkali-metal specificity  type c a t i o n s of enzyme  reflects  their  and f u r t h e r confirms our i n f e r e n c e enzymes have an a b s o l u t e  the  requirement i s K  +  of this  and NH^, e i t h e r  the  cofactor,  the  requirement f o r  algal  these  context,  a l k a l i - m e t a l type  cation ions  o f which o n l y may be expected role  a l g a l enzymes.  i n governing Whether t h i s  in vivo role  enzyme r e g u l a t i o n by p r o d u c t a c t i v a t i o n of NH^ (as  to  f u r t h e r narrowed down to the  play a s i g n i f i c a n t of  or  that  Viewed i n the p h y s i o l o g i c a l  specificity  high  " b i n d i n g " comparable  t h a t o b t a i n e d from a n a t u r a l s u b s t r a t e  cations.  potent  to  activity  involves i n the  suggested f o r y e a s t t h r e o n i n e  case  deaminase  (Holzer e_t al_. , 1964)) or p a r t i c i p a t i o n i n enzymesubstrate  complex f o r m a t i o n i n the case of K  suggested f o r whole c a t e g o r i e s of enzymes ing e l i m i n a t i o n r e a c t i o n s a question  for  (Suelter,  +  (as  catalyz-  1970))  remains  conjecture.  The degree of a c t i v a t i o n o b t a i n e d from the nonp h y s i o l o g i c a l cations mechanistically  L i  +  , Rb , Cs , T l  significant  enzymology of s e v e r a l promoted by monovalent  +  +  to the  may be  comparative  types of enzyme cations  +  reactions  (Suelter,  1970),  since  some i n v e s t i g a t o r s  have managed to  the degree of a c t i v a t i o n  Cor s t a b i l i z a t i o n )  c e r t a i n exemplary enzymes with  the  such ions  1971).  (Bothwell and D a t t a ,  the c l o s e s i m i l a r i t y of Kayne  that  the  the l a t t e r  kinetics  from the c a t i o n R b The evidence the  from T l  from the p r e s e n t  +  former i o n  i n monovalent  with i d e n t i c a l  +  and K ,  +  and t h a t  were c l o s e s t  +  of  Prompted by  of T l  c a t i o n a c t i v a t i o n of pyruvate kinase consequent  of  crystal radii  ionic radii  (1971) demonstrated  c o u l d indeed r e p l a c e  correlate  ionic  study  to  the those  radius.  indicates  that  a l g a l enzymes show no such c o r r e l a t i o n s h i p o f  c a t i o n a c t i v a t i o n to  ionic radius; this  seen i n t h e i r g e n e r a l l y poor response  is p a r t i c u l a r l y  to T l  the case o f A . m a r i n a , where the o r d e r of  and i n  +  activation  was L i > K > N H * > R b > N a > C s > T l . +  +  +  +  +  The p r o b a b l e monovalent previously studied  1  +  c a t i o n requirement o f  b i o s y n t h e t i c ' threonine  deaminases  from b a c t e r i a , y e a s t and p l a n t s may have escaped notice al.,  of many e a r l i e r  1972,  for l i t e r a t u r e c i t a t i o n s ) ,  used enzyme e x t r a c t s or  investigators  to  Desai  who have  p r e p a r e d i n phosphate  (NH^^SO^ s u s p e n s i o n s ;  (related  (see  however,  ionic strength)  s t a b i l i z i n g or p r o t e c t i n g  the  the et  generally  buffers  influence  of these c a t i o n s  the enzymes from  in inactivation  by d i l u t i o n has been f r e q u e n t l y noted 1964 ; Cennamo e_t al_. , 1964; 1968;  monovalent  cations  order of s p e c i f i c observed  stabilizing  was  In the case o f influence  of  the  found to be r e l a t e d  to  their  enzyme a c t i v a t i o n s  subsequently  (Holzer e_t al_. , 1964), w h i l s t  a pseudomonad stabilized  (Lessie  ionic strength,  (Na ,  K ) , divalent +  (TriS'H ) +  and W h i t e l e y ,  and a c t i v a t e d  total +  Leitzmann and B e r n l o h r ,  L e s s i e and K h i t e l e y , 1969).  the y e a s t enzyme the  (Changeux,  cations  the  scanty  the  literature  1969)  (Mg , 2 +  present.  Ca  2 +  ,  of  Mn  2 +  ),  was i n h i b i t e d  that  (15%)  enzyme was only s l i g h t l y +  Na , L i +  +  deaminase  of  stearothermophilus  by KH* but s t i m u l a t e d  1971), whereas the  bacterial  by NH^ (Reh and  of B a c i l l u s  +  K ,  reported i n  requirement  The t h r e o n i n e  i n i n c r e a s i n g order by K , N a , L i Kuramitsu,  of  or o r g a n i c  t h a t none of the  Hydrogenomonas was u n a f f e c t e d 1969),  was  A careful scrutiny of  enzymes s t u d i e d may have an e s s e n t i a l  Schlegel,  of  monovalent  i o n - a c t i v a t i o n observations  f o r monovalent c a t i o n s .  enzyme  from mere i n c r e a s e  irrespective  indicates  the  +  +  (18-751)  (Thomas and  S a l m o n e l l a typhimurium  stimulated  (201) by X H ^ ,  (Burns and Z a r l e n g o , 1968).  other hand, the r e p o r t e d p r o p e r t i e s and animal enzymes suggest that  of the  On the plant  they may have an  essential  requirement,  s i m i l a r to the a l g a l  f o r monovalent  cations.  tissue culture  (Dougall,  The enzymes from a rose 1970)  (Sharma and Mazumder, 1970) ment,  being s t i m u l a t e d  lesser activation or no e f f e c t  5-  (Holzer e_t al_. , 1964)  +  algal  of  , and l e a s t  +  was a c t i v a t e d  6-fold  by NH^,  i n the o r d e r NH^>K >Rb > +  the  Among a n i m a l s , 'biodegradative'  sheep l i v e r  it  is  threonine  cations  +  +  'biosynthetic'  deaminase  +  enzymes and w i t h M i c h a e l i s c o n s t a n t s  It  thus  resemble more c l o s e l y and animals  appears t h a t  i n t h e i r monovalent  Since the  procaryotic  cation  it  the  a l g a l enzymes plants  activation/  than those of the p r o c a r y o t i c  algae examined i n c l u d e  (blue-green  organisms,  the  those of the e u c a r y o t i c  requirement p r o p e r t i e s bacteria.  was  higher-plant  of magnitude comparable to those r e p o r t e d f o r y e a s t enzyme.  to  i n a manner  (K >NH^>Rb >LI >Na ) s i m i l a r to the +  the  interesting  (Nishimura and G r e e n b e r g , 1961)  s t r o n g l y a c t i v a t e d by the  classes)  +  are of s i m i l a r magnitude to those of  +  enzymes.  note t h a t  +  the M i c h a e l i s c o n s t a n t s r e p o r t e d f o r  +  NH^ and K  1 0 - f o l d by K , w i t h  The enzyme o f a y e a s t  with other c a t i o n e f f e c t s +  leaf  i n d i c a t e d such a r e q u i r e -  +  +  Li >Cs >Na ;  to  and s p i n a c h  from N'H^, R b , or L i  from N a .  enzymes,  algae) is  both  and e u c a r y o t i c  tempting  to s p e c u l a t e  (other that  the monovalent c a t i o n 'promotion' o f threonine deaminase was developed from a p r i m i t i v e nons p e c i f i c i o n i c strength e f f e c t to a s p e c i f i c requirement during p r o c a r y o t i c e v o l u t i o n from b a c t e r i a l ancestors to blue-green algae and that t h i s 'selected" requirement was subsequently r e t a i n e d as a conservative  c h a r a c t e r i s t i c during  f u r t h e r e v o l u t i o n , despite subsequent m o d i f i c a t i o n s of the enzyme f u n c t i o n versus 'biodegradative')  ('biosynthetic*  i n conjunction with i t s  a l l o s t e r i c properties. That the a l g a l deaminases do not have a d i v a l e n t - m e t a l c a t i o n requirement was i n d i c a t e d by our e a r l i e r observation  (Desai e_t al_. , 1972)  of no s i g n i f i c a n t e f f e c t from ethylenediamine t e t r a a c e t a t e on t h e i r a c t i v i t y and i s confirmed by the absence of any s t i m u l a t o r y e f f e c t from such c a t i o n s t e s t e d i n the present study.  In t h i s  respect, the a l g a l enzymes resemble a l l p r e v i o u s l y reported  threonine  deaminases.  The rare case o f  a c t i v a t i o n of a pseudomonad enzyme (Lessie and K h i t e l e y , 1969)  by s a l t s of M g , C a , M n 2+  2 +  2+  is  a t t r i b u t e d to i t s s t a b i l i z a t i o n from t h e i r nons p e c i f i c enhancement o f the t o t a l i o n i c s t r e n g t h . On the other hand, s e n s i t i v e i n h i b i t i o n of these  enzymes from Hg  +  has been f r e q u e n t l y  (Leitzmann and B e r n l o h r , 1968; 1970; 1967  Maeba and Sanwal, 1966; ),  reported  Sharma and Mazumder, Nakazawa and H a y a i s h i ,  which i n h i b i t i o n has been g e n e r a l l y  attri-  buted t o ' m e r c a p t i d e - p r o d u c i n g m o d i f i c a t i o n o f essential  s u l f h y d r y l groups;  this  mechanism of  H g - i n d u c e d i n h i b i t i o n has been v e r i f i e d w i t h a 2 +  bacterial activity l-ol.  enzyme  (Datta,  from subsequent  It  appears  1966)  by r e g e n e r a t i o n  treatment  l i k e l y that  the  2+ the  a l g a l enzymes from Cu  similar interference  al.,  1972).  2+ Ca  2-mercaptoethan-  inhibitions 2+  and Zn  may be due  The g e n e r a l  for a c t i v i t y  to  (Desai  absence o f e f f e c t s  from  2+ and Mg  on the  a l g a l enzymes f i n d s  a parallel  i n the s i m i l a r b e h a v i o u r of the y e a s t enzyme et  of  with s e n s i t i v e s u l f h y d r y l  groups known to be e s s e n t i a l et  with  of  al.,  1964).  The extreme  sensitivity  enzyme of A . q u a d r u p l i c a t u m to a l l the cations  may be r e l a t e d  response  consistently  exposure  to a l l the  p y r i d o x a l phosphate (Desai  of  (Holzer  the  4 divalent  to s i m i l a r extreme o b t a i n e d from t h i s  inhibitory enzyme on  -SH group m o d i f y i n g agents and antagonists  previously  tested  e_t al_. , 1972) .  Apart from the enzyme of rose (Dougall,  1970),  the e f f e c t s  tissue  culture  of anions have  been  ignored  i n the l i t e r a t u r e reports on  deaminases.  threonine  The former enzyme was found to be  s t r o n g l y i n h i b i t e d by n i t r a t e , n i t r i t e , i o d i d e , and s l i g h t l y stimulated by s u l f a t e and phosphate, w i t h the h a l i d e s showing a p a t t e r n of i n c r e a s i n g  inhibi-  tory a c t i o n p a r a l l e l i n g t h e i r atomic weight.  The  a l g a l enzymes were g e n e r a l l y s i m i l a r l y a f f e c t e d by these anions, excepting halide pattern.  the cases of n i t r i t e and the  U n l i k e the rose c u l t u r e enzyme,  the algae have shown no s i g n i f i c a n t d i f f e r e n c e between f l u o r i d e , c h l o r i d e , bromide, while being s t r o n g l y i n h i b i t e d by i o d i d e . the i n h i b i t i o n reported  In the case o f n i t r i t e ,  f o r rose c u l t u r e appears  to be an erroneous i n t e r p r e t a t i o n of i n t e r f e r e n c e from t h i s anion i n the enzyme assay method, which i n t e r f e r e n c e was unequivocally  established  our t e s t s on the a l g a l enzymes.  during  An o v e r a l l view of  the anion e f f e c t s on the a l g a l enzymes i n d i c a t e s a p a t t e r n of i n h i b i t i o n a r i s i n g only from the ions (I~, ClOj,  known to possess a high reducing or  o x i d a t i o n p o t e n t i a l and suggests that t h e i r i n h i b i t o r y a c t i o n may be due to r e d u c t i v e or o x i d a t i v e processes rather than i o n i n t e r a c t i o n .  Viewed i n  the p h y s i o l o g i c a l context, these e f f e c t s are expected to be manifested only from u n n a t u r a l l y  high concentrations  of  such a n i o n s ,  and there  no i n d i c a t i o n of a r e g u l a t o r y r o l e of anions able  to t h a t  recently  influence  and s u l f a t e  anions may have no  on i_n v i v o a c t i v i t y  deaminases,  of  glucose  (Home and N o r d l i e , 1971).  concluded that  excepting  r e d u c i n g or o x i d i z i n g  compar-  r e p o r t e d on the a c t i v a t i o n by  b i c a r b o n a t e , phosphate, dehydrogenase  was  It  is  significant  of a l g a l  i n environments  threonine p o l l u t e d with  reagents.  Acknowledgement One of us  (I.D.D.)  acknowledges  support from the F i s h e r i e s  financial  Research Board o f Canada  and the Research Committee of the U n i v e r s i t y of British  Columbia.  TABLE 1 .  S t i m u l a t i o n of a l g a l t h r e o n i n e from T l  Cation  +  relative  Enzyme a c t i v i t y  M  ;  nitrate salt) Ch.sa.  He.vi.  (0.05  K  to K  +  deaminase  and N H t .  (% o f  Ag . q u . An.ma.  control)  Po.cr.  Te.ma.  Cy .na  +  280  260  279  288  346  446  281  NHj  380  377  378  269  290  228  179  Tl  126  127  147  161  139  128  100  +  Standard enzyme i n c u b a t i o n s made w i t h T r i s - H C l ( 0 . 1 M, pH 8 . 5 ) .  The a c t i v i t i e s  buffer  o b t a i n e d without  added c a t i o n were taken as 100% c o n t r o l .  TABLE 2.  Enzyme a c t i v i t y o f C . s a l i n a with d i f f e r e n t  buffers  o f same molar  strength  at comparable pH.  Buffer  Added  Type  pH E s t i m a t e d K ( o r Na ) +  +  obtained  Enzyme a c t i v i t y  KC1  Keto a c i d  (M)  measured  sponding  (mumoles)  control*  content (M)  % of  corre-  K-Tricine  8.0  0.07  -  232  102  K-Hepes  8.0  0.13  -  241  101  K-Phosphate  8.0  0.19  -  297  115  Tris-HCl  8.0  \  -  0.07  227  -  8.0  -  0.13  238  -  8.0  -  0.19  258  -  K-Bicarbonate  9.5  0.12  -  150  99  Na-Bicarbonate  9.5  0.13  -  68  102  Map-HCl  9.5  0.12  -  9.5  a  0 . 1 M.  b  Above T r i s - H C l  KC1 r e p l a c e d by  C  or Map-HCl b u f f e r s  pH and added KC1. C  0.13  NaCl.  152  -  67  -  1  with c o r r e s p o n d i n g  TABLE 3.  E f f e c t s of d i v a l e n t c a t i o n s on a l g a l threonine deaminase a c t i v i t y .  Enzyme a c t i v i t y (% of c o n t r o l ) a  Cation (0.01  M  dichloride salt)  An.ma. Po.cr. Te. ma Cy.na.  Mg *  105  92  72  98  102  93  95  Ca  103  94  67  97  100  85  97  In *  43  46  13  43  40  52  31  Cu  20  34  11  38  27  27  25  2  2  +  1  a  Ch. sa . He.vi. Ag.qu.  2 +  S t a n d a r d enzyme incubations made w i t h K - T r i c i n e b u f f e r (0.1 M, pH 8 . 5 ) .  The a c t i v i t i e s  obtained  without added c a t i o n were taken as 100% c o n t r o l .  TABLE 4 .  Effects  of  threonine  Anion (K  salt)  +  i n o r g a n i c anions deaminase  a Cone. pH  activity.  Enzyme a c t i v i t y  u  (M)  Ch.sa.  on a l g a l  (% of  control)*  H e . v i . Ag.qu . An.ma.  Br"  0.10  8.7  101  100  98  97  F"  0.10  11  101  98  101  96  I"  0.05  15  15  17  22  N0 3  CIO3-  so  2 4  "  HP0 " 2  4  HCO3-  a  it  0.10  11  IS  10  18  18  0. 20  11  12  17  14  19  0.05  11  90  88  56  60  0.10  tt  87  87  55  61  0.20  n  82  84  55  55  0.05  it  78  74  70  79  0.10  11  76  74  72  78  0.05  11  111  108  116  113  0.10  11  117  108  117  110  0.05  it  119  121  127  122  0.10  tt  121  122  125  118  0.07  9.5  C  99  -  Po.cr.  97  95  B u f f e r e d with T r i s - H C l f O . l M) . The a c t i v i t i e s  o b t a i n e d from s t a n d a r d  incubations  were expressed  as p e r c e n t  containing  of c o n t r o l s  5  corresponding of the  anion  K  +  concentrations  M K-bicarbonate  t h i s pH; i n t h i s case the  Map-HCl b u f f e r ing K  +  place  salt.  The anion was p a r t o f 0.1 at  as KC1 i n  (0.1  concentration  c o n t r o l was made w i t h  M, pH 9.5) (0.12  buffer  containing  M) as KC1.  correspond-  122  Figure F i g . 1.  Captions  Stimulation of a l g a l  t h r e o n i n e deaminase  a c t i v i t y by monovalent c a t i o n s .  The  s t i m u l a t i o n i s shown as p e r c e n t i n c r e a s e i n a c t i v i t y from  i n c o r p o r a t i o n o f 0.1  c h l o r i d e s a l t s of the c a t i o n s i n t o i n c u b a t i o n mixtures (0.1 M,  pH  8.5).  buffered with  The  activity  from c o n t r o l s without  M  standard Tris-HCl  obtained  the s a l t s was  equated  to zero % s t i m u l a t i o n .  Fig.  2.  E f f e c t o f c a t i o n c o n c e n t r a t i o n on deaminase a c t i v i t y o f (B) Porphyridium marina,  (A) Chroomonas  (D) T e t r a s e l m i s maculata.  (0.1 M,  pH  8.5).  Note t h a t the  (10-40 min  the a l g a l s p e c i e s ) . e s t i m a t e d from double reaction velocity  The  Standard  Tris-HCl  a c t i v i t i e s are shown f o r the t o t a l t i o n p e r i o d s used  salina,  cruentum, (C) A n a c y s t i s  enzyme i n c u b a t i o n s made w i t h buffer  threonine  incuba-  varying with  Km v a l u e s were  reciprocal plots  (mumoles keto a c i d  duced per min  per mg  p r o t e i n ) versus  concentration  (moles per l i t e r ) .  of  procation  In view  of the l i k e l i h o o d of contaminating c a t i o n s i n the enzyme e x t r a c t s used, these Km values cannot be considered absolute but r e f l e c t the magnitude of cation concentration giving velocity.  half-maximum  50 i  Li Na K Rb Cs NH Li Na K Rb Cs NH, Li Na K Rb Cs NH.  100 i  150 i  200 1  i  250 r  300  T  •\ Ch.sa.  He.vi.  Ag.qu.  Li Na K Rb Cs NH.  An. ma.  Li Na K Rb Cs NH. Li Na K Rb Cs NH. Li Na K Rb Cs NH  350  Po.cr.  Te.ma. H  •I  4  Cy. na 1  50  100  1  150  1  200  1  250  J  300  i  350  125  NhLCI 4 0 0 h  CD 3 0 0 h U D  O z.  CL  200 h I M V 3  1  100  D  Km NH  4  +  K  IS  I.L. 1  1  Km  M  +  4 08 x l O "  4  5-50 x l O "  4  1  1  K  1  1  l  O  £  3..  E  -  400  u  1-73 x 10" 4  •4  NH4  2-75 x 10"  Rb  4-01 x 10'  +  I  J  Km  -  LiCI  l  +  M  +  3-44  x 10"  4  Rb  +  4-72  x 10"  4  MM  K  200  k  M  Km 100'£  U  +  K+  I  c  20  Fig. 2  '  I 40  L__J  2-24 x T O "  4  2-90x 1 0 "  4  1  L_J  60  Salt  80  I 100  20  Concentration  I  K  > u  I  M  3 0 0 h  »  -4  40  (mM)  60  References Antia,  N . J . , and Cheng, J . Y . : The s u r v i v a l of  cultures  of marine p l a n k t o n i c algae from p r o -  longed exposure 9,179-184 Antia,  to darkness at  fide  R . S . , and D e s a i ,  Phycologia,  I . D . : L-threonine  i n marine p l a n k t o n i c a l g a e  II.  and s u l f h y d r y l group requirements  enzyme a c t i v i t y in press Betts,  20 C .  (1970).  N . J . , Kripps,  deaminase  axenic  i n two c r y p t o p h y t e s .  Disul-  of J . Phycol.  (1972).  G . F . , and E v a n s , H . J . : The i n h i b i t i o n of  univalent cation activated (hydroxymethyl) Biophys. A c t a , Bewley,  enzymes by t r i s -  aminomethane. 167,193-196  Biochim.  (1968).  J . D . , and M a r c u s , A . : S t i m u l a t o r y e f f e c t  tris  of  b u f f e r on a wheat embryo amino a c i d  i n c o r p o r a t i n g system.  Phytochem.  9,1031-1033  (1970) . B o t h w e l l , M . A . , and D a t t a , P . : E f f e c t s catalytic serine  and r e g u l a t o r y p r o p e r t i e s  dehydrogenase  Biochim.  of K  235,1-13  on the  o f homo-  of Pseudomonas  Biophys. A c t a ,  +  fluorescens.  (1971).  Burns, R . O . , and Z a r l e n g o , M . H . : Threonine Deaminase from S a l m o n e l l a typhimurium. J . B i o l . 178-185  (1968).  Chem.  243,  127 -  Cennamo, C ,  Boll,  hydratase Zeit.  i  •  M . , and H o l z e r , H. : Uber T h r e o n i n d e -  aus  Saccharoinyces  340,125-145  Changeux, J . - P . :  .  cerevisiae .  Biochem.  (1964).  Sur l e s  proprietes  allosteriques  de l a L - t h r e o n i n e - d e s a m i n a s e .  I.  Methodes  d'etude de l a L - t h r e o n i n e desaminase de b i o s y n t h e s e . Bull. Datta,  Soc.  Chim.  46,927-946  P . : P u r i f i c a t i o n and feedback  onine  deaminase a c t i v i t y  spheroides. Desai,  Biol.  J . Biol.  of  c o n t r o l of  Chem.  241,5836-5844  Dougall,  11,  of L - t h r e o n i n e dehydratase  277-287  (1972).  Rose t i s s u e c u l t u r e s .  9,959-964  Phytochem.  (1970).  Friedemann, T . E . : D e t e r m i n a t i o n o f « - k e t o 414-418.  Kaplan  In: S.P.  ( e d . ) : Methods i n enzymology, Inc.  H. , Cennamo, C . , and B o l l ,  activation  of y e a s t  threonine  ammonia. Biochem. B i o p h y s . 14,487-492  (1964).  acids,  Colowick and N. 0.  New York: Academic Press Holzer,  algae.  D . K . : Threonine deaminase from P a u l ' s  Scarlet  pp.  (1966).  N . J . : Comparative  seven s p e c i e s o f u n i c e l l u l a r marine Phytochem.  thre-  Rhodopseudomonas  I . D . , Laub, D . , and A n t i a ,  characterization  (1964).  V o l . 3.  1957. M . : Product dehydratase  Res. Commun.  by  in  Home,  R . N . , and X o r d l i e ,  carbonate,  R . C . : A c t i v a t i o n by b i -  orthophosphate,  l i v e r microsomal g l u c o s e Biochim.  Biophys. A c t a ,  Kayne, F . J . : T h a l l i u m kinase.  Arch.  (I)  and s u l f a t e  of r a t  dehydrogenase. 242,1-13  (1971).  a c t i v a t i o n of p y r u v a t e  Biochem. B i o p h y s .  143,232-239  (1971). Leitzmann,  C , and B e r n l o h r , R.V.'. : T h r e o n i n e  dehydratase  of B a c i l l u s l i c h e n i f o r m i s  Purification Acta, Lessie,  151,449-460  Biochim. Biophys.  (1968).  T . G . , and W h i t e l e y , H . R . : P r o p e r t i e s o f  threonine use  and p r o p e r t i e s .  I.  deaminase  threonine  Bacteriol.  from a b a c t e r i u m a b l e  as s o l e source  100,878-889  of carbon.  to J.  (1969).  Maeba, P . , and Sanwal, B . D . : The a l l o s t e r i c threonine  deaminase  of Salmonella.  model f o r the n a t i v e 536  enzyme.  Kinetic  Biochem.  5,525-  (1966).  Nakazava, A . , and H a y a i s h i , 0 . :  On the mechanism o f  a c t i v a t i o n o f L - t h r e o n i n e deaminase  from  C l o s t r i d i u m tetanomorphum by adenosine phate.  J . Biol.  Chem.  242,1146-1154  diphos-  (1967).  N i s h i m u r a , J . S . , and G r e e n b e r g , D . M . : P u r i f i c a t i o n and p r o p e r t i e s  o f L - t h r e o n i n e dehydrase o f  sheep l i v e r . J . B i o l .  Chem.  236,2684-2691  (1961).  Reh, M . , and S c h l e g e l , Isoleucin Arch.  H , G . : Die B i o s y n t h e s e  und Y a l i n  Mikrobiol.  von  i n Hydrogenomonas H 16.  67,110-127  (1969).  Sharma, R . K . , and Mazumder, R . : P u r i f i c a t i o n , properties, threonine Chem. Suelter,  and feedback  dehydratase  245,3008-3014  control of L -  from s p i n a c h . (1970).  C . H . : Enzymes a c t i v a t e d  cations.  J . Biol  by monovalent  Science, 168,789 - 795  (1970).  Thomas, D . A . , and K u r a m i t s u , H . K . : B i o s y n t h e t i c L threonine  deaminase  from B a c i l l u s  stearo-  thermophilus  I . C a t a l y t i c and r e g u l a t o r y  properties.  Arch.  96-104  (1971).  Biochem. B i o p h y s .  145,  

Cite

Citation Scheme:

    

Usage Statistics

Country Views Downloads
United States 10 0
China 4 28
India 1 0
Germany 1 0
Chile 1 0
Czech Republic 1 0
Russia 1 0
City Views Downloads
Ashburn 6 0
Unknown 5 0
Shenzhen 3 26
Redmond 1 0
Wilmington 1 0
Seattle 1 0
Washington 1 0
Beijing 1 1

{[{ mDataHeader[type] }]} {[{ month[type] }]} {[{ tData[type] }]}
Download Stats

Share

Embed

Customize your widget with the following options, then copy and paste the code below into the HTML of your page to embed this item in your website.
                        
                            <div id="ubcOpenCollectionsWidgetDisplay">
                            <script id="ubcOpenCollectionsWidget"
                            src="{[{embed.src}]}"
                            data-item="{[{embed.item}]}"
                            data-collection="{[{embed.collection}]}"
                            data-metadata="{[{embed.showMetadata}]}"
                            data-width="{[{embed.width}]}"
                            async >
                            </script>
                            </div>
                        
                    
IIIF logo Our image viewer uses the IIIF 2.0 standard. To load this item in other compatible viewers, use this url:
http://iiif.library.ubc.ca/presentation/dsp.831.1-0101499/manifest

Comment

Related Items