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

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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 t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f the r e q u i r e m e n t s f o r an advanced degree at the U n i v e r s i t y o f B r i t i s h C o l u m b i a , I a g r e e t h a t t h e L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and s t u d y . I f u r t h e r a g r e e t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y p u r p o s e s may be g r a n t e d by the Head o f my Department o r by h i s r e p r e s e n t a t i v e s . I t i s u n d e r s t o o d t h a t c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l not be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . Department The U n i v e r s i t y o f B r i t i s h Columbia V a n c o u v e r 8, Canada Date ^ , 1-, \c\rW i ABSTRACT Several aspects of L-threonine dehydratase from seven species of unicellular marine planktonic algae were investigated; (1) the disulfide group requirement for activity of the enzymes from two cryptomonads, (2) monovalent inorganic cation requirement for enzyme activity, (3) substrate specificity and substrate analog inhibitions, (4) allosteric activation and inhibition 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 activity as exemplified by the specific inhi- bition exerted by a l l t h i o l reagents tested, which inhibition could be pa r t i a l l y reversed or prevented by the appropriate treatments. Sulfhydryl group requirement: for enzyme activity was confirmed and i t was demonstrated that these^ groups are essential for feedback inhibition from L-isoleucine. A l l algal enzymes appear to require monovalent alkali-metal cations for f u l l expression of activity, more specifically K + and NH*. Anacystis marina was exceptional i n showing maximal stimulation from L i + . Organic cations were 2+ 2+ without effect whereas some inhibition from certain divalent cations (Zn , Cu ) - 2 - 2 -and anions (N0 3, I , C10 3) were observed, whilst HPĈ  and SÔ . were stimulatory. Aside from L-threonine, the algal enzymes extended substrate activity 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 for the relatively high substrate - activity observed toward L -serine with this species. Inhibition from substrate analogs was limited to L-homoserine and L-serine , despite the substrate activity of the latter. The mechanism for the peculiar i i 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, all the algal enzymes were subject to feedback inhibition from L- isoleucine, 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, it displayed two pH-activity optima. The investigation of this pheno- menon 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. i i i 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 i i i LIST OF TABLES v i i LIST OF FIGURES v i i i 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 10 PART I. 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 15 DISCUSSION 17 PART II. 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 in 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 46 DISCUSSION 47 LITERATURE CITED 55 APPENDIX A. The Common Locus of Threonine Dehydratase in Branched-chain Amino Acid Biosynthesis 63 B. Schematic Representation of Threonine Biosynthesis and the Major Peripheral Regulatory Circuits 64 * C. 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" in two cryptophytes 66-94 II. L-Threonine Deaminase in Marine Planktonic Algae. Stimula- tion of activity by monovalent inorganic cations and diverse effects from other ions 95-129 v i i LIST OF TABLES TABLE Page . 1 Culture and standard assay conditions of the algal species used 11 2 Algal deaminase activity observed from structural analogs of L- threonine tested as substrate 20 3 Sensitivity of algal deaminases to L-isoleucine inhibition in relation to substrate 21 4 Substrate-related response of C. salina deaminase activity to various reagents and effectors 22 5 Effects of substrate analogs on algal TDH activity towards L- threonine 23 6 Effects of various amino acids on algal threonine dehydratase activity 37 7 Effects of certain nucleotides and amino acids on enzyme activity of H. virescens at pH 8.5 51 v i i i LIST OF FIGURES FIGURE Page 1 pH-activity profile of the deaminase reaction in relation to substrate 24 2 A. Effects of L-serine and L-homoserine concentration on threonine dehydratase activity of A. marina and A. quadruplicatum.. 25 B. Effects of L-homoserine and L-serine on the substrate saturation kinetics of the A. marina enzyme 25 3 Effect of L-homoserine and L-serine on the rate of L-threonine deamination by the enzymes from T. maculata and C_. salina 26 4 Effect of L-isoleucine concentration on algal threonine dehydratase activity 38 5 Influence of pH on the effects from L-isoleucine and L-valine on threonine dehydratase activity =. 39 6 Effect of L-valine concentration on algal threonine dehydratase activity 40 7 Reversal of L-isoleucine inhibition of the algal enzymes by graded concentrations of L-valine 41 8 Effects of L-valine and L-isoleucine on the substrate saturation kinetics of the enzymes from T. maculata, C. nana and A. marina 42 i x FIGURE Page 9 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 53 12 pH-activity profiles in the presence of L-isoleucine for the enzymes from three nutritionally different cultures of H. virescens 53 13 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 in 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 in 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 its 2,4,-dinitro- phenylhydrazbne. -The algal enzymes showed pH optima in 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, its scope was intended to include the study of the following features of the algal enzymes 1) the parti- cular 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 substrate- analog 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 first two features yielded such fruitful 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 will be summarized in the next two paragraphs. Disulfide and sulfhydryl group requirements of enzyme activity in two cryptophytes (see Supplement I). A systematic investigation of the cryptophyte1s "disulfide 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 in 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 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 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 HgCl2 or 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 mono- valent inorganic cations. The activation was generally the strongest (3-5 fold) with K+ and NH*, whilst L i + , Rb+, and Cs + showed intermediate orders varying with algal species. Anacystis marina was exceptional in 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 in response to ion concentration and specific for monovalent inorganic cations with indications of a coenzyme type of role. Organic cations 2*f* 24- 2-t* 2-f- were inert and the divalent cations Mg , Ca , Zn , Cu were either inhibitory or without effect. 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 is 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 8.0- 9.5 whereas the TDH (BD) range widely in their optima from pH 6.2-10.5. Mole- cular weights for these enzymes have been reported as low as 147,000 (72) although they have generally been established in the vicinity 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 sulfhy- dryl 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 is 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 conden- sation 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 coli (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 vicinity 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 ability 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 ability 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 is 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 its 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 is 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 in 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 concen- tration 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, particu- larly 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 is 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 low- ered reactivity are L-serine (26,60,69) and for a rose tissue culture enzyme, L- allocystathionine (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 lists 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 in this study were grown photoautotro- phically 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 in 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 (4mM) as N-source, ( i i i ) with glycine (4mM) and glycerol (2,17). Where present, the glycerol concentration was 0.25 M. Unless otherwise stated, the enzyme tests concerning 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 latter. Algal extracts Suspensions of algal powder in appropriate buffer were subjected to ultra- sonic 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 in 0.2 M potassium N-tris(hydroxymethyl) methylglycine (K-Tricine), pH 8.5), pyridoxal phosphate (0.1 jimole), and L- threonine (80 umoles) in 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-amino- propanol (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 finally 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. A l l the tests included.both unincubated and fully incubated controls. 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 % in 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.). All 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 Light Added organic nutrients Assay conditions Dry alga Incubation (mg) (min Q 37° C) CHLOROPHYTA Tetraselmis maculata (Te.ma.) BACILLARIOPHYTA + Cyclotella nana (Cy.na.) CRYPTOPHYTA Chroomonas salina(Ch.sa.(NA)) (Ch.sa.) ( C h . s a . C G l y . 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.) + + + nil nil nil glycerol glycerol glycine Jglycine jglycerol urea nil nil nil 4 2 2 2 4 4 2 2 40 30 20 IS 10 15 15 20 30 10 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 contamina- tion 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 (SDH) in addition to its known TDH. 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 L- serine which showed activities of 7 % and 236 % respectively when compared to equimolar concentrations of L-threonine. Evidently, the ability 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. 1A), 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 (18) . 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 in 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 is apparent that this estimated ability of the C. salina TDH to deaminate L- serine is in agreement with the values obtained for the other algal enzymes (17- 26 % ) . 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 first 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 deamina- tion 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 L- threonine functioned as the substrate. These observations confirm the occurrence of SDH in 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 in 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 antago- nism) 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 in 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-homo- serine 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 in Figs. 2A and 2B, which profiles were closely comparable for a l l the algal species examined. These results showed a distinct difference in 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 its 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 in 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 L- threonine 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 + L- threonine 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 gener- ally 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 allo- threonine 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 liver, 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, L- isoleucine 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 ability 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 inacti- vation involved although it 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), it 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 L-allothreonine 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 *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. Substrate (50 mM) (5 mM) L-Serine L-Threonine Ch.sa. - 100 100 + 88 4 Ch.sa.(NA) - 100 100 + 84 6 He.vi. - 100 100 + 48 46 Ag.qu. - 100 100 + 8 10 An.ma. - 100 100 + 6 8 Po.cr. - 100 100 + 5 4 Te.ma. - 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 n i l - 100 100 L-isoleucine 0.01 99 88 0.10 92 36 1.0 88 5 10 86 4 L-valine 0.10 101 112 1.0 100 100 10 96 48 100 89 11 dithiothreitol 1.0 93 26 10 92 25 EDTA 100 96 102 KCl f 100 428 310 NH.C1+ 100 345 400 * 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 concen- tration of L-threonine was substituted for that normally used. '.CC c o U o NO >- > < ^ 20 >- N z LU An.ma. Ag.qu. - a - •. 5 25 50 SUBSTRATE ANALOG (mM) c r_ v g . ~§ 200 D T O a o •4— '.CC - i i ! ! 5 i " B An.ma. — + /-homoserine ( 3 7 m M ) ^ ^ ^ " ^ +/-serine ( 7 3 m M ) 6 1 ^C 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 i i \ nr Te. m a . TIME (min) 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 deamina- ting 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. T. maculata was the most sensitive to L-isoleucine. 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 col 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 its 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 in 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 concen- tration giving 50 % inhibition. However, the C. nana enzyme gave no evidence of any physiologically significant increase in 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 9.5- 10.0 (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 (20). 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 sensitivity difference (100-fold) observed between the two amino acids. 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 concentra- tion must be sufficiently low to be non-inhibitory, b) the L-isoleucine concentra- tion 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 L- valine 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), ( i i i ) C. nana ( l i t t le L-isoleucine inhibition, greatest L-valine stimulation). The results depicted in Fig. 8 appear to elucidate the mode of stimulation from L- valine. 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 its 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 fu 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 Fig. 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 L- valine 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. D-Isoleucine, L-alanine and L-glycine exerted no effects whatso- ever. 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 virtually indistingui- shable 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 L- valine. Substrate saturation curves in 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 in the absence or presence of either L- norvaline, 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 criteria for the classification of a threonine dehydratase as biosynthetic is its sensitivity to feedback inhibition from isoleucine accor- ding 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 in the former species and likens i t to the desensitized bio- synthetic TDH of Rhodospirillum rub rum (28). In the case of the latter algal species, the situation is 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 is 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, (ii) 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 ability 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 L- threonine for the substrate site, which mode of inhibition has been ascribed to this amino acid in certain biodegradative threonine dehydratases (63,64). Few investigation have been performed with respect to the absolute stereo- specific nature of isoleucine inhibition. Certain compounds such as thiaisoleucine (4), L-O-methyl threonine (75), L-0-ethyl.threonine (71) and isoleucine hydroxa- mate (51) have been examined for their effect upon the growth of isoleucine- requiring autotrophs and concomitant biosynthesis of TDH following the incorporation of these antimetabolites into the growth media. However, excepting the early work of Changeux designed to reveal the specificity of substrate and inhibitor sites (15), the ability 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 criteria determining the ability 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 L- norvaline. 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. col 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 isoleucine- sensitive enzymes is likely the result of interaction from excess L-valine at the isoleucine site (39). Furthermore, the partial ability 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 in 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 in 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 10 PH 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 commen- cing 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 concen- trations 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 1 I 1 I L -5 —-.• -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), it 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, it 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, all 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 obser- vation 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 el icit ing 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 investigation of L-isoleucine inhibition at the two pH optima. 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 irst 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 vicinity 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 vicinity 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, (ii) these enzymes may occur in different proportions in the three cultures, ( i i i ) the isoleucine- insensitive enzyme (pH 9 optimum) may be present in the greatest proportion in 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 differ- ential 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 combi- nation 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. Fig. 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 dehy- dratases 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 vicinity 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 ikely 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 L- isoleucine. 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 in 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 is summarized below: . 1) In contrast to the other algal enzymes, L-isoleucine, after ensuring its 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 satura- tion 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 wi 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 in 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 DTT L-Threonine Enzyme activity Concn (mM) (0.01 mM) (mM) (% of control) n i l - - 50 100 - - 5 100 - + 50 80 - + 5 74 AMP 5 + 50 80 5 + 5 81 ADP 5 + 50 83 5 + 5 72 ATP 5 + 50 78 5 + 5 83 cyclic-AMP 5 + 50 81 5 + 5 74 L-methionine 50 - 50 . 99 50 - 5 95 5 - 50 102 5 - 5 91 L-cystathionine 50 - 50 104 50 - 5 96 5 - 50 99 5 - 5 103 L-aspartate 5 - 50 110 5 - 5 114 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. Appropriate L-threonine concentration was .substituted for that normally used. Dithiothreitol 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 appro- priate buffer (see methods) prior to the regular assay procedure commencing with the addition of pyridoxal phosphate.  54 Fig. 13. 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L-Threonine dehydratase of Azotobacter vinelandii. Doklady Akademii Nauk SSSR, 181: 997-1000. 59. MAEBA, P. § SANWAL, B.D. 1966. The allosteric threonine deaminase of Salmonella. Kinetic model for the native enzyme. Biochem. 5: 525-36. 60. MODI, S.R. $ MAZUMDER, R. 1966. A biosynthetic L-threonine dehydratase from spinach. Ind. J . Biochem. 3: 215-8. 61. MONOD, J . , CHANGEUX, J . - P . § JACOB, F. 1963. Allosteric proteins and cellular control systems. J . Mol. Biol . 6: 306-29. 62. MUDD, S.H., FINKELSTEIN, J . D . , IRREVERRE, F. § LASTER, L. 1965. Threonine dehydratase activity in humans lacking cystathionine synthase. Biochem. Biophys. Res. Comm. 19: 665-70. 63. NAKAZAWA, A. $ HAYAISHI, 0. 1967. On the mechanism of activation of L-threonine deaminase from Clostridium tetanomorphum by adenosine diphosphate. J . Biol . Chem. 242: 1146-54. 64. NIEDERMAN, R.A., RABINOWITZ, K.W. $ WOOD, W.A. 1969. 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Studies on L-threonine dehydratase from spinach (Spinacia oleracea). Ind. J . Biochem. 4: 61-4. 71. SHIGEURA, H.Y., HEN, A . C . , HIREMATH, C.B. § MAAG, T.A. 1969. L-O- Ethylthreonine: An antagonist of L-isoleucine. Arch. Biochem. Biophys. 135: 90-6. 72. 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'-monophosphate- dependent threonine deaminase from Escherichia co l i . J . Biol . Chem. 244: 1883-9. 73. THOMAS, D.A. 5 KURAMITSU, H.K. 1971. Biosynthetic L-threonine deaminase from Bacillus stearothermophilus. I. Catalytic and regulatory properties. Arch. Biochem. Biophys. 145: 96-104. 74. TCMOVA, V .S . , KAGAN, Z.S. $ KRETOVICH, V . L . 1968. L-Threonine dehydratase from pea seedlings. Biokhimiya, 33: 244-54. 75. TWAROG, R. 1972. Enzymes of the isoleucine-valine pathway in Acinetobacter. J . Bacteriol. I l l : 37-46. 62 76. UMBARGER, H.E. § BROWN, B. 1956. Threonine deamination in Escherichia co l i . I. D- and L-threonine deaminase activities of cell-free extracts. J . Bacteriol. 71: 443-9. 77. UMBARGER, H.E. § BROWN, B. 1957. Threonine deamination in Escherichia co l i . II. Evidence for two L-threonine deaminases. J . Bacteriol. 73: 105-12. 78. WHITELEY, H.R. § TAHARA, M. 1966. Threonine deaminase of Clostridium tetanomorphum. I. Purification and properties. J . Biol . 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. Acta Chem. Scand. 25: 443-50. 80. YON, R.J . 1972. 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, C=0 I COOH CH, c=o I AHA v C H -C-OH COOH Rl PYRUVATE a-ACETOLACTATE (ACTIVE ACETALDEHYDE) CH, I 3 CH, I ^ -C=0 I COOH a-KETOBUTYRATE T TDH CH, i -> c=o I AHA' CH3-CH2-C-OH COOH a-ACETO a-HYDROXY- BUTYRATE Rl CH, CH.-C-OH 3 I v H-C-OH ~? I COOH DAD a,3-DIHYDROXY- ISOVALERATE CH, I 3 CH,-CH,-C-OH H-C-OH I COOH CH, I 3 CH.-C-H 3 I v C=0 * I COOH a-KETOISO- VALERATE CH, I 3 CH.-CH.-C-H > 3 1 I DAD C=0 I COOH JA_ TA a,3-DIHYDROXY- 3-METHYLVALERATE a-KETO 3-METHYL- VALERATE CH, CH3-C-H v H-C-NH, COOH VALINE CH, I 3 CH,-CH,-C-H > 2 I 7 H-C-NH, I 1 COOH ISOLEUCINE CH 1 3 HO-C-H C-NH, I Z COOH THREONINE Abbreviations for enzymes TDH THREONINE DEHYDRATASE AHA ACETOHYDROXY ACID SYNTHETASE Rl REDUCTOISOMERASE DAD DEHYDROXY ACID DEHYDRATASE TA TRANSAMINASE Adapted from: ALLAUDEEN, H.S. $ RAMAKRISHNAN, T. 1968. Arch. Biochem. Biophys. 125: 199-209. APPENDIX B. Schematic Representation of Threonine Biosynthesis and the Major Peripheral Regulatory Circuits ********************************************** * * * * * * * * * ** ASPARTYL 8- PHOSPHATE ******* LYSINE ********* * * * * * *. ' * * i * * ' * ASPARTATE * DIHYDRODIPICOLINATE * X * * t * * * *. * £ * <****** <*********** * * * * * * * * * * * * * * * * * * * * * * ***** ISOLEUCINE * A. * * * * * a-KETOBUTYRATE * * * * * * ********.[>: % ************************* ASPARTATE 8- SEMIALDEHYDE * V * -> HOMOSERINE- •> THREONINE 7! ' * O-SUCCINYL HOMOSERINE * CYSTEINE- <•«:***** *********** * * * * * * * * * * * * * * * * * CYSTATHIONINE -> METHIONINE ( ) single enzymatic step ( ) more than one enzymatic step (****) major regulatory sequences Adapted from: DATTA,P. 1969. Science, 165: 556-62. 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 sensi- tive 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 in 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 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 for sulfhydryl groups (which requirement was common to a l l the algal enzymes) was confirmed a) by the study of their sensitivity to inhib- ition 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 divisions were reported in the previous communication of this series (13). In general, the algal enzymes showed broad resemblance in most proper- ties (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 resem- blance to the threonine dehydratases of yeast and higher plants (1). However, the algal enzymes themselves showed certain individual differ- ences, 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 all 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 unex- pected behaviour of the cryptophyte enzymes suggested that they may require both sulfhydryl [ - S H ] and disulfide [ - S - S - ] groupings for the manifestation of activity. 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). Excepting the 2 cryptophytes a l l the algae used in this study were grown photoautotrophically with added vitamins. C. salina was cultured under 3 sets of conditions, (i) photoautotrophic (vitamins, l ight) , (ii) photoheterotrophic (gly- cerol, vitamins, l ight), ( i i i ) chemoheterotrophic (glycerol, vitamins, darkness) (9). Being unable to use nitrate as N-source or to grow in darkness on organic substrates hitherto tested, H. virescens was cul- tured 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 latter. 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 in 0.2 M potassium N-tris(hydroxymethy1)methyl glycine buffer, pH 8.5), pyridoxal phosphate (0.1 umole), and L-threo- nine (80 ymoles) in a final volume of 1 ml. The mixtures were normally preincubated f irst with the test reagent for 15 min at 22°C, then with pyridoxal phosphate for 5 min at 37°C, and finally 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 fully incubated controls. A l l reagents used were of the highest purity grade commercially avail- 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. able. RESULTS Effects of thiol and reducing reagents 7 1 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: DTT = DTE = BAL > L i p f S r f ^ > GSH > Cys. This order of diminishing reactivity of the thiol 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 thiol 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 partially inactivated enzyme was reoxidized by prolonged incubation in presence of a ir . This mild reversal treatment was considered necessary for valid interpreta- tion of the tests, since the algal enzymes were known to possess -SH groups also required for activity (13), which should be left un- damaged under the conditions effecting the overall transformation ^-S-S-R^ R 1-SH + R2-SH 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 in 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 ir . 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 air stream) made no difference to the reversal. Xo attempt was made to obtain complete reversal of the thiol 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 inhi- bitions and their relationship to the cryptophyte enzyme substrate and allosteric effector, which might shed light on the nature of involve- ment of the essential disulfide groups in the mechanism of enzyme action. In the f irst place, a l l the tested thiol 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 (Figs. 1 and 2). 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 ikely 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 ob- tained. 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 phos- phate in the algal enzymes (13). An overall view of these observations suggested that the sensitive disulfide groups are not directly invol- ved in the mechanism of enzyme action but appear to be in close proxi- mity to the sites binding the substrate and allosteric effector, with the implication that such location enables them to favour enzyme action by facil itating 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 cul- tures 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 cryp- tophyte enzymes could not be satisfactorily verified at that time because of unforeseen difficulties created by the unexpected finding of thiol-reagent inhibition of these enzymes (13). However, the in- teresting observation was then made that the organomercurial p_-chlo- romercuriphenyl 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 in 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 thiol 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. Since HgC^ was uniformly totally inhibitory towards both enzymes, this 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 in these inhibitions was further verified by tests designed to reverse the observed inactivations by subsequent treatment with DTT. The enzymes were f irst inactivated 40-60 % by PCMPS or DTDP and then exposed to minimal excess of the reversal reagent, the concen- tration 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 partially inacti- vated 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 site. Otherwise, the cryptophyte enzymes resemble their bact- erial and plant counterparts, 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 in 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. The nature of this require- ment deserves a few comments. 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 facil itating the enzyme binding to the substrate. Such an influence may be exercised by assistance in maintaining the enzyme molecule in 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 ight, i t is tempting to specu- late that these disulfide groups, with their high potential for rever- sible 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 in bridging polypeptide chains of the enzyme molecule shown to be present in some purified bacterial threonine dehydratases (21,22) for which no disulfide group require- ment 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 poly- peptide chain of insulin (6,14,23,28). The close proximity of such sensitive disulfide groups to the cryptophyte enzyme sites binding threonine and isoleucine is suggested by our observations of the pro- tection 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 modi- fying such groups usually cause extremely sensitive and total inacti- 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 study- certainly 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) DTE GSH Cys Asc(OH)2 NADrt, c. 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 A. quadruplicatum 98 103 102 99 98 A. marina 102 99 100 100 98 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 NaAs02 Preincubation Enzyme activity concn concn with NaAsC^ C. salina H. virescens (mM) (mM) Temp. (°C) Period (min) % inhib. % reversal 8- S-•a i> 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) Enzyme activity {% of control) DTT Isoleucine C. salina H. virescens 1.0 - 23 31 1.0 4 48 1.0 1.0 1 30 * Method involved treatment with DTT (15 min, 22°C) followed by preincu- bation 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) Enzyme activity (% of control) Pyridoxal Threonine Isoleucine DTT C. salina H. virescens phosphate - _ 0.1 27 33 0.1 - 0.1 28 33 - 20 0.1 41 41 - 0.01 0.1 62 61 0.1 20 0.1 41 42 0.1 0.01 0.1 64 62 - 20 0.01 0.1 73 72 0.1 20 0.01 0.1 73 70 Method involved pretreatment with pyridoxal phosphate, threonine, isoleu- cine, 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 in 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 DTT concn Enzyme activity * Light Nutrients T (mM) (% of control) H. virescens + glycine 1 35 10 34 + "glycine ") 1 36 < glycerol \ 10 35 + urea 1 38 10 35 C. salina + n i l 1 28 10 26 + glycerol 1 26 10 25 - glycerol 1 24 10 25 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) in 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 DTT concn Reversal of inhibition (%) concn (mM) (mM) C. salina H. virescens DTDP 0.02 0.02 13 0 0.02 0.05 67 33 0.02 0.10 58 31 PCMPS 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 Isoleucine concn Enzyme activity (I of control) concn (mM) (mM) C. salina H. virescens n i l - 1.0 2 48 PCMPS 0.005 1.0 6 51 0.015 1.0 - 72 0.050 1.0 9 98 HgCl 2 0.001 1.0 10 60 0.005 1.0 67 84 0.010 1.0 99 101 DTDP 0.005 1.0 27 52 0.010 1.0 65 68 0.020 1.0 87 89 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. TXote that 100 % control in these tests is represented by the activ- ities obtained from a l l corresponding pretreatments effected without isoleucine. 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 thiol and reducing reagents on L-threonine dehydratase activity of Hemiselmis virescens under standard assay conditions. Reagent abbreviations are indicated in 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. Reagent abbreviations are indicated in the text. C. salina Concn: 50%inhib. (mM) Cys 1-20 GSH 0-76 Lip(SH)2 0-6 3 DTE 0-48 DTT 0-44 BAL 0-43 Asc(OH)2 H. virescens Concn: 50% (mM) Cys 2-50 GSH 1-30 Lip(SH)2 0-94 DTE 0-42 DTT 0-46 BAL 0-41 n " 1 *> 10 5 Reagent Concentration (mM) 1 0 0 9 0« o v -4— c 8 0 0 u M— 7 0 O 6 0 X 5 0 > u 4 0 < m e 3 0 N C 2 0 LU 1 0 - Concn.-50% inhib. (mM) PCMPS - 0 - 1 2 5 DTDNA 0-021 DTDP 0-017 H g C I 2 0-008 Fig. 3 Reagent Concentration (mM) c oncn: 50% inhib. (mM) PCMPS 0 021 DTDNA 0024 DTDP 0-022 HgCI2 0-006 Fig. 4 Reagent Concentration (mM) o REFERENCES ANTLA, N . J . , KRIPPS, R.S. § DESAI, I.D. 1972. L-threonine deaminase in marine planktonic algae. I I I . 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: 179-84. ANT LA, N.J. a CHORNEY, V. 1968. Nature of the nitrogen compounds supporting phototrophic growth of the marine cryptomonad Hemiselmis virescens. J . Protozool. 15: 198-201. BAREL, A .O. , DOLMANS, M. $ LEONIS, J . 1971. Spectroscopic studies on the reduction and reformation of the disulfide bonds of papaya lysozyme. Europe. J . Biochem. 19: 488-95. BLANKENSHIP, L .C. § MENCHER, J.R. 1971. 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DE ROBICHON-SZULNLAJSTER, H. § HAGEE, P.T. 1968. The regulation of isoleucine-valine biosynthesis in Saccharomyces cerevisiae. 1. Threonine deaminase. Europe. J . Biochem. 3: 492-501. 13. DESAI, I .D. , LAUB, D. § ANTIA, N.J . 1972. Comparative characte- rization of L-threonine dehydratase in 7 species of unicellu- lar marine algae. Phytochem. 11: 277-87. 14. DU VIGXEAUD, V . , FITCH, A . , PEKAREK, E. $ LOCKWOOD, W.W. 1931-32. The inactivation of crystalline insulin by cysteine and glu- tathione. J . Biol . Chem. 94: 233-42. 15. FRIEDEMANN, T .E . 1957. 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. Arch. Biochem. Biophys. 119: 41-9. 17. 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 cells . Biochem. Pharmacol. 18: 603-11. 18. GUILLARD, R.R.L. $ RYTHER, J .H. 1962. Studies of marine planktonic diatoms. I. Cvclotella nana Hustedt, and Detonula confervaceae t ——— ' (Cleve) Gran. 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. 49: 742-51. 20. HASLE, G.R. § HEIMDAL, B.R. 1970. Some species of the centric diatom genus Thalassiosira studied in the light and electron microscopes. Nova Hedwigia^31: 559-81. 21. HATFIELD, G.W. § BURNS, R.O. 1970. Threonine deaminase from Salmonella t>phimurium III. The intermediate substructure. J . Biol . Chem. 245: 787-91. 22. HATFIELD, G.W. § UMBARGER, H.E. 1970. Threonine deaminase from Bacillus subtilis I. Purification of the enzyme. J . Bio l . Chem. 245: 1736-41. 23. HEATON, G.S. , RYDON, H.N. $ SCHOFIELD, J .A. 1956. Polypeptides. Part III. The oxidation of some peptides of cysteine and gly- cine. J . Chem. Soc. 1956: 3157-68. 24. LEITZMANN, C. S BERNLOHR, R.W. 1968. Threonine dehydratase of Bacillus licheniformis I. Purification and properties. Biochim. Biophys. Acta, 151: 449-60. 25. MAEBA, P. £ SANWAL, B.D. 1966. The allosteric threonine deaminase of Salmonella. Kinetic model for the native enzyme. Biochem. 5: 525-36. 26. MONOD, J . , CHANGEUX, J . - P . $ JACOB, F. 1963. Allosteric proteins and cellular control systems. J . Mol. Biol . 6: 306-29. 27. MONOD, J . , WYMAN, J . 5 CHANGEUX, J . - P . 1965. On the nature of allosteric transitions: a plausible model. J . Mol. Biol . 12: 88-118. 94 28. RYLE, A . P . , SANGER, F . , SMITH, L . F . t\ KITAI, R. 1955. The disulphide s bond^of insulin. Biochem. J . 60: 541-56. 29. SHARMA, R.K. § MAZUMDER, R. 1970. Purification, properties, and feedback control of L-threonine dehydratase from spinach. J . Biol . Chem. 245: 3008-14. 30. TOMOVA, V . S . , KAGAN, Z.S. $ KRETOVICH, V . L . 1968. Kinetic proper- ties of desensitized "biosynthetic" L-threonine dehydratase of pea seedlings. Doklady Akademii Nauk SSSR, 180: 237-40. 31. ZAHLER, W.L. § CLELAND, W.W. 1968. A specific and sensitive assay for disulfides. J . Biol . Chem. 243: 716-9. 95 SUPPLEMENT II 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 1. The ' b i o s y n t h e t i c ' L-threonine (deaminat- ing) dehydratase of 7 marine p lanktonic species from 5 c lasses of algae showed severa l degrees of a c t i v a - t i o n from monovalent inorganic c a t i o n s . The a c t i v a - t i o n was'general ly the strongest (3 to 5-fo ld) with K + and NH^, and the weakest (1 to 2- fo ld) with X a + and T l + , whi l s t L i + , R b + , and C s + showed intermediate orders varying with a l g a l spec ies . One blue-green alga was except ional in showing strongest s t i m u l a t i o n (5-fold) from L i + and more pronounced a c t i v a t i o n from Na + than C s + , wh i l s t a green a lga showed another type of response with the l eas t e f fec t from L i + and markedly greater a c t i v a t i o n from Rb + than NH^. 2. The ca t ion a c t i v a t i o n showed ( i ) 'hyperbo l i c k i n e t i c response to ion concentra t ion , and ( i i ) high s p e c i f i c i t y for monovalent inorganic c a t i o n s , with i n d i c a t i o n s of a coenzyme type of r o l e for the a l k a l i metal type of ions . 3. Organic cat ions were i n e r t and the d i v a l e n t cat ions M g ^ + , C a ^ + , Z n ^ + , C u ^ + were e i t h e r i n h i b i t o r y or without e f f e c t . 4. .Among the anions t e s ted , c h l o r i d e , bromide, f l u o r i d e , bicarbonate showed no e f f e c t , i o d i d e , n i t r a t e , ch lorate were i n h i b i t o r y , whi l s t phosphate and su l fa te were s l i g h t l y s t i m u l a t o r y . 97 5 . It was concluded that the a l g a l enzymes may have an absolute K+ or N'H* requirement for i n vivo 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 and c h a r a c t e r i z a t i o n of the ' b i o s y n t h e t i c 1 type of L-threonine dehydratase (L-threonine hydro- lyase , deaminating; EC 4.2. 1.16) i n 7 marine p lanktonic species from 5 c lasses of algae was reported i n the f i r s t paper of th i s s er i e s (Desai e_t a l . , 1972). Apart from c e r t a i n i n d i v i d u a l d i f f erences (Antia et a l . , 1972), the a l g a l enzymes showed broad resemblance i n proper t i e s (pH optima, substrate sa turat ion k i n e t i c s , pyridoxal-phosphate and su l fhydry l -group requirements, i s o l e u c i n e - s e n s i t i v e a l lo s t er i sm) to the corresponding enzymes from b a c t e r i a , f u n g i , and higher p l a n t s . During t h i s study (Desai e_t a l . , 1972) , the determinat ion of the pH optima of the a l g a l enzymes was beset by problems of i n h i b i t i o n of a c t i v i t y from T r i s - H C l (pH 8-9) and sodium bicarbonate (pH 9.5-10.5) b u f f e r s ; the i n h i b i t i o n was c l e a r l y evident from comparison with potassium phosphate and T r i c i n e b u f f e r s , which i n turn showed smal ler but d i s t u r b i n g l y s i g n i f i c a n t d e v i a t i o n s . The nature of the i n h i b i t i o n s suggested undefined ef fects from the i o n i c composit ion and concentrat ion of the buffers used, which needed to be e f f e c t i v e l y c o n t r o l l e d for r e l i a b l e study of the a l g a l enzymes. The systematic i n v e s t i g a t i o n of these e f f e c t s , reported i n th i s communication, has revealed that (i) the algal enzymes require c e r t a i n monovalent inorganic cations for the f u l l expression of a c t i v - i t y , ( i i ) the apparent i n h i b i t i o n obtained from T r i s - HC1 was due to the absence of these ions i n the buffer used, ( i i i ) the apparent i n h i b i t i o n observed i n the sodium bicarbonate buffers was due to markedly lower a c t i v a t i o n of the enzymes by Na + r e l a t i v e to K +, and (iv) the divergences observed between potassium phosphate and T r i c i n e buffers of comparable molarity were due to differences of K + concentration i n these buffers (apart from s l i g h t stimulation by phosphate anion), which differences are normally ignored but assume importance i n the concentration-sensitive response of the a l g a l enzymes. Materials and Methods A l g a l species CHLOROPHYTA (green algae): Tetraselmis maculata (Te.ma.). BACILLARIOPHYTA (diatoms): C y c l o t e l l a nana (Cy.na.). CRYPTOPHYTA: Chroomonas s a l i n a (Ch.sa.); Hemiselmis virescens (He.vi.). RHODOPHYTA (red algae): Porphyridium cruentum (Po.cr.). CYANOPHYTA (blue-green algae): Agmenellum quadrupli- catum (Ag. qu. ) ; Anacystis -marina (An.ma. ) . Abbreviat ions shown af ter species names are used to denote these species in Figures and Tab les . D e t a i l s of the a l g a l s t r a i n s used, t h e i r source and mainten- ance have been reported (Antia and Cheng, 1970). The algae were mass-cul tured, harvested, and f r e e z e - d r i e d as p r e v i o u s l y described (Desai et a l . , 1972). Except- ing C. s a l i n a and H. v i r e s c e n s , a l l the cu l tures used i n th i s study were grown photoauto troph ica l ly with added v i tamins . C_. s a l i n a and H. v irescens were grown p h o t o t r o p h i c a l l y with enrichments of g l y c e r o l and g l y c i n e , r e s p e c t i v e l y , in a d d i t i o n to the v i tamins . Enzyme assay Suspensions of a l g a l powder i n appropriate buf fer were submitted to u l t r a s o n i c o s c i l l a t i o n and whole sonicates were assayed for enzyme a c t i v i t y as p r e - v i o u s l y described (Desai e_t al_. , 1972), the assay method being based on that of Friedemann (1957) for the c o l o r i m e t r i c determination of keto a c i d produced. A l l enzyme incubat ion mixtures contained a l g a l sonicate (0.5 ml i n buf fer of s trength double the f i n a l molar i ty r e q u i r e d ) , p y r i d o x a l phosphate (0.1 umole), and L - threonine (80 umoles) i n a f i n a l volume of 1 ml . The mixtures were preincubated with p y r i d o x a l phosphate for 5 min at 3 7 ° C , then incubated with threonine at the same temperature for periods varying with the a l g a l species (10-40 min as ind ica ted by Desai e_t a l . , 1972). When t e s ted , the s a l t s of cat ions or anions were incorporated into the mixtures at the same time as the buffered a l g a l son icate . A l l such tests inc luded unincubated and f u l l y incubated contro l s made without the s a l t s . Chromatographical ly homogeneous L-threonine was purchased from Calbiochem (Los Angeles , C a l i f . ) . The s a l t s , buf fer and other reagents used were of the highest p u r i t y grade commercially a v a i l a b l e . Results A l l the a l g a l enzymes showed very low a c t i v i t y M i n 0. lJ^Tris-HC1 b u f f e r , which was enhanced two to f i v e - f o l d by the add i t i on of severa l monovalent i n - organic cat ions as t h e i r c h l o r i d e s a l t s ( F i g . 1) . At a concentrat ion l e v e l of 0.1 M, NH^ and K + were genera l ly the most s t imulatory causing 220-390% increase i n a c t i v i t y , L i + , R b + , C s + showed intermediate degrees of a c t i v a t i o n with t h e i r order of e f fec t iveness vary- ing with the a l g a l spec ies , while Na + was u s u a l l y the l eas t e f f e c t i v e with 3-1151 increase in a c t i v i t y . The cyanophyte A. marina was except ional in showing the highest a c t i v i t y enhancement (3845) from L i + among the cations as we l l as in obta in ing greater degree of a c t i v a t i o n from Na + (1891) than C s + (166%). By c o n t r a s t , the chlorophyte T . maculata showed the l eas t e f fec t from L i + (96%), the strongest s t imula - t ion from K + (390%), and cons iderably greater a c t i v a t i o n from Rb + (285%) than NH* (160%). The diatom C. nana showed the most s p e c i f i c requirement + + + + for K , with l i t t l e or no e f f ec t from Rb , L i , Cs , + + Na , and the lowest a c t i v a t i o n from NH^ observed among the algae. T l + could not be compared d i r e c t l y with the other cat ions as the c h l o r i d e s a l t , because of i t s extremely low s o l u b i l i t y . The more so luble TINO^ of fered a means of comparison with KNO-j and NH^NO^ at a concentrat ion l e v e l of 0.05 M. The r e s u l t s (Table 1) showed no e f fec t from T l + 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 (26-61%) of the other a l g a l enzymes. It was i n t e r e s t i n g to note that the strongest a c t i v a t i o n from T l + was observed with the alga (A. marina) which had shown anomalous order of response to the other c a t i o n s . The p o s s i b i l i t y was considered that the l e s ser degree of a c t i v a t i o n obtained from some of the cat ions may be due to net i n h i b i t i o n from excess concentrat ion 103 of these ions at the l e v e l used for comparison i n F i g . 1. This was disproved by studying the e f fec t s of ca t ion concentrat ion gradations 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 hyperbo l i c increases with i o n i c concentrat ion . C e r t a i n a l g a l s p e c i e s - c a t i o n combinations tended to show ion s a t u r a t i o n at the upper concentrat ion l eve l s tested but there was no evidence of i n h i b i t i o n . Estimates of the Km values for the cat ions showing the strongest e f fec t s (see F i g . 2) ind icate high a f f i n i t y of the a l g a l enzymes for these ions . An i n t e r e s t i n g outcome of the concentrat ion gradient study was the explanat ion i t o f fered for the minor but s i g n i f i c a n t divergences obtained i n enzyme a c t i v i t y values determined with 0.1 M K- phosphate, K - T r i c i n e , K-Hepes, and T r i s - H C l (conta in- ing 0.1 M KC1) buffers at comparable pH (Table 2) . Although the molar i ty of the f i r s t 3 buffers i s the same with respect to t h e i r buf f er ing anions , they d i f f e r s u f f i c i e n t l y i n t h e i r K + concentrat ion to produce the observed divergences in response of the more s e n s i t i v e a l g a l enzymes. Excepting the case of K-phosphate, these divergences were v i r t u a l l y e l i m i n - ated whenever the KC1 concentra t ion , taken i n conjunct ion with T r i s - H C l b u f f e r , was adjusted to correspond c l o s e l y with the K + concentrat ion of the other buffers (Table 2) . K-phosphate s t i l l showed a small dev ia t ion because of the s t i m u l a t i o n obtained from the phosphate anion (see below). The correspond- ing comparison of Na-bicarbonate and K-bicarbonate buffers (Table 2) showed that the apparent i n h i b i t i o n of the a l g a l enzymes prev ious ly obtained (Desai e_t a l . , 1972) from the former buf fer was due to the l e s ser a c t i v a t i o n of the enzymes by Na + than K + . When these buffers were compared with t h e i r Na + or K + - c o n t r o l l e d counterparts i n Map-HCl b u f f e r , exce l - lent agreement was observed (Table 2) . 2+ 2+ 2+ 2 + The d iva l en t cat ions Ca , Mg , Zn , Cu showed no a c t i v a t i o n of the a l g a l enzymes when tested with T r i s - H C l and K - T r i c i n e b u f f e r s . With the former b u f f e r , the a c t i v i t y observed was too low to assess other e f fec ts from these ions . But the l a t t e r b u f f e r , due to i t s K + content , e levated a c t i v i t y l e v e l s s u f f i c i e n t l y to ind i ca te d e f i n i t e patterns of i n h i b i - t i o n from the d iva l en t cat ions (Table 3). Z n ^ + and C u 2 + were s trongly i n h i b i t o r y (50-901), while C a 2 + and M g 2 + showed l i t t l e e f fec t excepting the case of A. quadruplicatum. This cyanophyte was c o n s i s t e n t l y s e n s i t i v e to a l l the 4 d iva l en t c a t i o n s , being i n h i b i t e d about 30% by C a 2 + or M g 2 + and about 90% . - 2+ - 2 + by Zn or Cu A l l the a l g a l enzymes showed i n s i g n i f i c a n t l y low a c t i v i t i e s in T r i s - H C l and Map-HCl buffers ( i r r e s p e c t i v e of pH) i n the absence of a l k a l i - m e t a l c a t i o n s . It was necessary to e s t a b l i s h whether t h i s 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 organic cat ion strength of these b u f f e r s . The e f fects of a range of concentrat ions (10, 25, 50, 100 mM) of T r i s - H C l (pH 8.5) and Map-HCl (pH 9 .5 ) , both with and without added KC1 (5 mM), were tes ted on 2 a l g a l species (C. s a l i n a and P. cruentum). The observed a c t i v i t i e s showed no s i g n i f i c a n t e f f ec t from buf fer concentrat ion i n a l l the cases t e s t ed . S i m i l a r tests were a lso made with 2 concentrat ions (10, 50 mM) of methylamine-HCl or tr imethylamine- HCl incorporated into T r i s - H C l (0.1M, pH 8 . 5 ; ± 5 mM KC1) and again no e f fec ts were obtained from these a d d i t i o n a l organic c a t i o n s . The evidence i n d i c a t e d an o v e r a l l ind i f f erence of organic cat ions to the a l g a l enzymes. Tests of the e f f e c t s of inorganic anions on the a l g a l enzyme a c t i v i t y presented d i f f i c u l t i e s i n maintaining simultaneous control over the pH and accompanying cation, since the usual buffers con- tained unequivalent concentrations of eithe r or both ions at any chosen pH. T r i s - H + was the obvious choice of enzymically i n e r t but b u f f e r - e f f e c t i v e cation, but this required buffers of the chosen pH to be made up with acids of the anion tested (HBr, r^SO^, HNOj), where s t r i c t control over the anion concentration was not possible. However, tests with such buffers showed poor enzyme a c t i v i t y of lev e l s comparable to those obtained with Tris-HCl and indicated that any e f f e c t s from these anions were too low for s a t i s f a c t o r y evaluation i n the absence of an a c t i v a t i n g monovalent cation. Eventually, the Tris-HCl buffer containing neglig- i b l e chloride ion was chosen for the tests with K + s a l t s of the anions at a conveniently c o n t r o l l a b l e pH and the anion e f f e c t s were evaluated against KC1 c o n t r o l s . The bicarbonate e f f e c t s were obtained from d i r e c t comparison of K-bicarbonate buffer with the corresponding KCl-enriched Map-HCl buffer. The re s u l t s (Table 4) showed that f l u o r i d e , bromide, bicarbonate had no e f f e c t on the a l g a l enzymes, iodide was s trongly (81-88%) i n h i b i t o r y , n i t r a t e and ch lorate were moderately (18-45%) i n h i b i t o r y , whi l s t phosphate and su l fa t e were s l i g h t l y (8-25%) s t imula tory . Whereas the ch lorate i n h i b i t i o n was r e l a t i v e l y uniform, the n i t r a t e i n h i b i t i o n showed taxonomica l ly -re la ted d i f f e r e n t i a l response from the algae tes ted , being most pronounced with the 2 cyanophytes and r e l a t i v e l y small with the 2 crypto - phytes. Anion concentrat ion increase i n the range 0.05-0.20 M produced l i t t l e change i n the i n h i b i t i o n s or s t imulat ions obta ined, i n d i c a t i n g that these con- centrat ions were sa tura t ing i n t h e i r e f f e c t s . When n i t r i t e was tested analogously to n i t r a t e , i t showed v i r t u a l l y t o t a l a b o l i t i o n of a l g a l enzyme a c t i v i t y , but th i s was traced to i t s in ter ference i n the assay method when c o n t r o l determinations made with t ^-ketobuyric ac id showed s i m i l a r i n h i b i t i o n from n i t r i t e of the co lour normally developed. Discuss ion The resu l t s of th i s i n v e s t i g a t i o n lead to the i n e v i t a b l e conclus ion that monovalent inorganic cat ions (more s p e c i f i c a l l y a l k a l i - m e t a l type) are abso lute ly required for the expression of a c t i v i t y by a l g a l threonine deaminases. Organic cations ranging from T r i s - H + to mono- and tri-methylated ammonium ions have shown no effects whatsoever and may be considered inert towards these enzymes. Tr i s has been generally reported to i n h i b i t several enzyme systems (other than threonine deaminase) activated by monovalent cations (Betts and Evans, 1968); i n one rare instance i t proved stimulatory at low concentration while being i n h i b i t o r y at high concentration (Bewley and Marcus, 1970). These effects of Tris have been a t t r i b u t e d to i t s antagon- i s t i c competition with the required cation (generally K + ) or i t s non-competitive contribution 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 of the a l g a l enzymes to tests designed to elucidate such eff e c t s of Tris and Map buffers compels us to i n f e r that a) these buffers are i n d i f f e r e n t to the enzymes, and 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 buffers i s not due to i n h i b i t i o n from them but rather a consequence of contaminating a l k a l i - m e t a l type cations 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 buffer ca t ions . The low magnitude of the Michae l i s constants obtained for the more potent a l k a l i - m e t a l type cat ions r e f l e c t s t h e i r high s p e c i f i c i t y of enzyme "binding" comparable to that obtained from a n a t u r a l substrate or c o f a c t o r , and fur ther confirms our inference that the a l g a l enzymes have an absolute requirement for these ca t ions . Viewed in the p h y s i o l o g i c a l context , the s p e c i f i c i t y of th i s a l k a l i - m e t a l type c a t i o n requirement i s fur ther narrowed down to the ions K + and NH^, e i ther of which only may be expected to play a s i g n i f i c a n t ro le i n governing in v ivo a c t i v i t y of the a l g a l enzymes. Whether th i s ro l e involves enzyme regu la t ion by product a c t i v a t i o n i n the case of NH^ (as suggested for yeast threonine deaminase (Holzer e_t al_. , 1964)) or p a r t i c i p a t i o n i n enzyme- substrate complex formation i n the case of K + (as suggested for whole categories of enzymes c a t a l y z - ing e l i m i n a t i o n react ions (Sue l ter , 1970)) remains a quest ion for conjec ture . The degree of a c t i v a t i o n obtained from the non- p h y s i o l o g i c a l cat ions L i + , R b + , C s + , T l + may be mechan i s t i ca l ly s i g n i f i c a n t to the comparative enzymology of several types of enzyme react ions promoted by monovalent cat ions (Sue l ter , 1970), since some inves t igators have managed to c o r r e l a t e 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 ) of c e r t a i n exemplary enzymes with the c r y s t a l r a d i i of such ions (Bothwell and Datta , 1971). Prompted by the c lose s i m i l a r i t y of i o n i c r a d i i of T l + and K + , Kayne (1971) demonstrated that the former ion could indeed replace the l a t t e r in monovalent ca t ion a c t i v a t i o n of pyruvate kinase and that the consequent k i n e t i c s from T l + were c loses t to those from the cat ion Rb + with i d e n t i c a l i o n i c r a d i u s . The evidence from the present study ind ica tes that the a l g a l enzymes show no such c o r r e l a t i o n s h i p of c a t i o n a c t i v a t i o n to i o n i c rad ius ; th i s i s p a r t i c u l a r l y seen i n t h e i r genera l ly poor response to T l + and i n the case of A. marina, where the order of a c t i v a t i o n was Li + >K + >NH*>Rb + >Na + >Cs + >Tl + . The probable monovalent ca t ion requirement of prev ious ly studied 1 b i o s y n t h e t i c ' threonine deaminases from b a c t e r i a , yeast and plants may have escaped the not ice of many e a r l i e r inves t iga tors (see Desai et al . , 1972, for l i t e r a t u r e c i t a t i o n s ) , who have genera l ly used enzyme extracts prepared in phosphate buffers or (NH^^SO^ suspensions; however, the inf luence (re lated to ion ic strength) of these cat ions in s t a b i l i z i n g or p r o t e c t i n g the enzymes from i n a c t i v a t i o n by d i l u t i o n has been frequent ly noted (Changeux, 1964 ; Cennamo e_t al_. , 1964; Leitzmann and Bern lohr , 1968; Less ie and K h i t e l e y , 1969). In the case of the yeast enzyme the s t a b i l i z i n g inf luence of the monovalent cations was found to be r e l a t e d to t h e i r order of s p e c i f i c enzyme a c t i v a t i o n s subsequently observed (Holzer e_t al_. , 1964), whi l s t the enzyme of a pseudomonad (Lessie and White ley , 1969) was s t a b i l i z e d and ac t iva ted from mere increase of t o t a l i o n i c s trength , i r r e s p e c t i v e of monovalent (Na + , K + ) , d iva lent ( M g 2 + , C a 2 + , M n 2 + ) , or organic ( T r i S ' H + ) cations present . A c a r e f u l s c r u t i n y of the scanty i o n - a c t i v a t i o n observations reported i n the l i t e r a t u r e indicates that none of the b a c t e r i a l enzymes studied may have an e s s e n t i a l requirement for monovalent c a t i o n s . The threonine deaminase of Hydrogenomonas was unaffected by NH^ (Reh and Sch lege l , 1969), that of B a c i l l u s stearothermophilus was i n h i b i t e d (15%) by KH* but s t imulated (18-751) i n increas ing order by K + , N a + , L i + (Thomas and Kuramitsu, 1971), whereas the Salmonella typhimurium enzyme was only s l i g h t l y s t imulated (201) by XH^, K + , N a + , L i + (Burns and Zarlengo, 1968). On the other hand, the reported proper t i e s of the p lant and animal enzymes suggest that they may have an es sen t ia l requirement, s i m i l a r to the a l g a l enzymes, for monovalent ca t ions . The enzymes from a rose t i s sue cu l ture (Dougall, 1970) and spinach l e a f (Sharma and Mazumder, 1970) ind ica ted such a r e q u i r e - ment, being st imulated 5- to 10- fo ld by K + , with l e s ser a c t i v a t i o n from N'H^, R b + , or L i + , and l eas t or no e f fec t from N a + . The enzyme of a yeast (Holzer e_t al_. , 1964) was ac t iva ted 6 - f o l d by NH^, with other cat ion e f fec ts i n the order NH^>K+>Rb+> L i + > C s + > N a + ; the Michae l i s constants reported for NH^ and K + are of s i m i l a r magnitude to those of the a l g a l enzymes. Among animals , i t i s i n t e r e s t i n g to note that the 'b iodegradat ive ' threonine deaminase of sheep l i v e r (Nishimura and Greenberg, 1961) was s trong ly ac t ivated by the cat ions i n a manner (K+>NH^>Rb+>LI+>Na+) s i m i l a r to the h igher -p lant ' b i o s y n t h e t i c ' enzymes and with Michae l i s constants of magnitude comparable to those reported for the yeast enzyme. It thus appears that the a l g a l enzymes resemble more c l o s e l y those of the eucaryot ic p lants and animals in t h e i r monovalent ca t ion a c t i v a t i o n / requirement propert ies than those of the p r o c a r y o t i c b a c t e r i a . Since the algae examined include both procaryo t i c (blue-green algae) and eucaryot ic (other c lasses ) organisms, i t i s tempting to speculate that the monovalent cation 'promotion' of threonine deaminase was developed from a pr i m i t i v e non- s 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 procaryotic evolution 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 retained as a conservative c h a r a c t e r i s t i c during further evolution, despite subsequent modifica- tions of the enzyme function ('biosynthetic* versus 'biodegradative') i n conjunction with i t s a l l o s t e r i c properties. That the algal deaminases do not have a divalent-metal cation requirement was indicated 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 tetraacetate on t h e i r a c t i v i t y and i s confirmed by the absence of any stimulatory e f f e c t from such cations tested in the present study. In t h i s respect, the al g a l enzymes resemble a l l previously reported threonine deaminases. The rare case of ac t i v a t i o n of a pseudomonad enzyme (Lessie and Khiteley, 1969) by s a l t s of Mg 2 +, C a 2 + , Mn 2 + i s att 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 non- s p e c i f i c enhancement of the t o t a l i o n i c strength. 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 frequent ly reported (Leitzmann and Bernlohr , 1968; Sharma and Mazumder, 1970; Maeba and Sanwal, 1966; Nakazawa and H a y a i s h i , 1967 ) , which i n h i b i t i o n has been genera l ly a t t r i - buted to'mercaptide-producing modi f i ca t ion of e s s e n t i a l s u l f h y d r y l groups; th i s mechanism of H g 2 + - 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 with a b a c t e r i a l enzyme (Datta, 1966) by regenerat ion of a c t i v i t y from subsequent treatment with 2-mercaptoethan- l - o l . It appears l i k e l y that the i n h i b i t i o n s of 2+ 2 + the a l g a l enzymes from Cu and Zn may be due to s i m i l a r inter ference 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 for a c t i v i t y (Desai et a l . , 1972). The general absence of e f fec t s from 2+ 2 + Ca and Mg on the a l g a l enzymes f inds a p a r a l l e l in the s i m i l a r behaviour of the yeast enzyme (Holzer et a l . , 1964). The extreme s e n s i t i v i t y of the enzyme of A. quadruplicatum to a l l the 4 d i v a l e n t cat ions may be r e l a t e d to s i m i l a r extreme i n h i b i t o r y response cons i s t en t ly obtained from th i s enzyme on exposure to a l l the -SH group modifying agents and pyr idoxa l phosphate antagonists prev ious ly tested (Desai e_t al_. , 1972) . Apart from the enzyme of rose t i s sue c u l t u r e (Dougal l , 1970), the e f fec ts of anions have been ignored i n the l i t e r a t u r e reports on threonine deaminases. The former enzyme was found to be strongly i n h i b i t e d by n i t r a t e , n i t r i t e , iodide, and s l i g h t l y stimulated by sulf a t e and phosphate, with the halides showing a pattern of increasing i n h i b i - tory action 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 generally s i m i l a r l y affected by these anions, excepting the cases of n i t r i t e and the halide pattern. Unlike the rose culture enzyme, the algae have shown no s i g n i f i c a n t difference between f l u o r i d e , c h l o r i d e , bromide, while being strongly i n h i b i t e d by iodide. In the case of n i t r i t e , the i n h i b i t i o n reported for rose culture appears to be an erroneous i n t e r p r e t a t i o n of interference from t h i s anion i n the enzyme assay method, which interference was unequivocally established during our tests on the a l g a l enzymes. An o v e r a l l view of the anion effects on the a l g a l enzymes indicates a pattern 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 oxidation p o t e n t i a l and suggests that t h e i r i n h i b i - tory action may be due to reductive or oxidative processes rather than ion i n t e r a c t i o n . Viewed i n the physiological context, these effects are expected to be manifested only from unnaturally high concentrations of such anions , and there was no i n d i c a t i o n of a regulatory ro le of anions compar- able to that recent ly reported on the a c t i v a t i o n by b icarbonate , phosphate, and su l fa te of glucose dehydrogenase (Home and N o r d l i e , 1971). It i s concluded that anions may have no s i g n i f i c a n t inf luence on i_n vivo a c t i v i t y of a l g a l threonine deaminases, excepting i n environments p o l l u t e d with reducing or o x i d i z i n g reagents . Acknowledgement One of us ( I . D . D . ) acknowledges f i n a n c i a l support from the F i s h e r i e s Research Board of Canada and the Research Committee of the U n i v e r s i t y of B r i t i s h Columbia. TABLE 1. St imulat ion of a l g a l threonine deaminase from T l + r e l a t i v e to K + and N H t . Cat ion Enzyme a c t i v i t y (% of contro l ) ( 0 . 0 5 M ; n i t r a t e s a l t ) C h . s a . H e . v i . Ag .qu . An.ma. P o . c r . Te.ma. Cy .na K + 280 260 279 288 346 446 281 N H j 380 377 378 269 290 228 179 T l + 126 127 147 161 139 128 100 Standard enzyme incubations made with T r i s - H C l buf fer ( 0 . 1 M, pH 8 . 5 ) . The a c t i v i t i e s obtained without added ca t ion were taken as 100% c o n t r o l . TABLE 2. Enzyme a c t i v i t y of C. s a l i n a obtained with d i f f e r e n t buffers of same molar strength at comparable pH. Buffer Added Enzyme a c t i v i t y Type pH Estimated KC1 Keto a c i d % of c o r r e - K + ( o r Na + ) (M) measured sponding content (mumoles) control* 1 (M) K - T r i c i n e 8.0 0.07 - 232 102 K-Hepes 8.0 0.13 - 241 101 K-Phosphate 8.0 0.19 - 297 115 T r i s - H C l 8.0 \ - 0.07 227 - 8.0 - 0.13 238 - 8.0 - 0.19 258 - K-Bicarbon- 9.5 0.12 - 150 99 ate Na-Bicarbon- 9.5 0.13 - 68 102 ate Map-HCl 9.5 0.12 152 - 9.5 - 0.13 C 67 - a 0 . 1 M. b Above T r i s - H C l or Map-HCl buffers with corresponding pH and added KC1. C KC1 replaced by NaCl . TABLE 3. Effects of divalent cations on a l g a l threonine deaminase a c t i v i t y . Cation Enzyme a c t i v i t y (% of control) a (0.01 M d i c h l o r i d e s a l t ) Ch. sa . He.vi . Ag.qu. An.ma. Po.cr. Te. ma Cy.na. M g 2 * 105 92 72 98 102 93 95 C a 2 + 103 94 67 97 100 85 97 In1* 43 46 13 43 40 52 31 C u 2 + 20 34 11 38 27 27 25 aStandard enzyme incubations made with K-Tricine buffer (0.1 M, pH 8 .5) . The a c t i v i t i e s obtained without added cation were taken as 100% c o n t r o l . TABLE 4 . Effects of inorganic anions on a l g a l threonine deaminase a c t i v i t y . Anion (K + s a l t ) Cone. (M) u a pH Enzyme a c t i v i t y (% of control)* 5 C h . s a . H e . v i . Ag.qu . An.ma. P o . c r . Br" 0 .10 8.7 101 100 98 97 F" 0 . 10 11 101 98 101 96 I" 0.05 it 15 15 17 22 0 . 10 11 IS 10 18 18 0. 20 11 12 17 14 19 N 0 3 - 0.05 11 90 88 56 60 0 . 1 0 tt 87 87 55 61 0 . 20 n 82 84 55 55 CIO3- 0.05 it 78 74 70 79 0 . 1 0 11 76 74 72 78 s o 4 2 " 0.05 11 111 108 116 113 0 . 10 11 117 108 117 110 H P 0 4 2 " 0.05 it 119 121 127 122 0 .10 tt 121 122 125 118 HCO3- 0.07 9.5 C 99 - 95 97 a 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 obtained from standard incubations were expressed as percent of contro l s conta in ing corresponding K + concentrat ions as KC1 in place of the anion s a l t . The anion was part of 0.1 M K-bicarbonate buf fer at th i s pH; in th i s case the c o n t r o l was made with Map-HCl buffer (0.1 M, pH 9.5) conta in ing correspond- ing K + concentrat ion (0.12 M) as KC1. 122 Figure Captions F i g . 1. Stimulation of a l g a l threonine deaminase a c t i v i t y by monovalent cations. The stimulation i s shown as percent increase i n a c t i v i t y from incorporation of 0.1 M chloride s a l t s of the cations into standard incubation mixtures buffered with Tr i s - H C l (0.1 M, pH 8.5). The a c t i v i t y obtained from controls without the s a l t s was equated to zero % stimulation. F i g . 2. E f f e c t of cation concentration on threonine deaminase a c t i v i t y of (A) Chroomonas s a l i n a , (B) Porphyridium cruentum, (C) Anacystis marina, (D) Tetraselmis maculata. Standard enzyme incubations made with Tr i s - H C l buffer (0.1 M, pH 8.5). Note that the a c t i v i t i e s are shown for the t o t a l incuba- t i o n periods used (10-40 min varying with the a l g a l species). The Km values were estimated from double r e c i p r o c a l p l o t s of reaction v e l o c i t y (mumoles keto acid pro- duced per min per mg protein) versus cation concentration (moles per l i t e r ) . In view of the l i k e l i h o o d of contaminating cations i n the enzyme extracts used, these Km values cannot be considered absolute but r e f l e c t the magnitude of cation concentration giving half-maximum v e l o c i t y . Li Na K Rb Cs NH 50 100 150 200 250 300 350 i i i i r 1 T •\ Ch.sa. Li Na K Rb Cs NH, He.vi. Li Na K Rb Cs NH. Ag.qu. Li Na K Rb Cs NH. An. ma. Li Na K Rb Cs NH. Po.cr. Li Na K Rb Cs NH. Te.ma. Li Na K Rb Cs NH 4 H •I 1 1 1 1 Cy. na J i 50 100 150 200 250 300 350 125 NhLCI CD U D 4 0 0 h 3 0 0 h O CL z. 2 0 0 h D 1 0 0 IS O £ 3.. E > » MM u K 1 I M V 3 Km M N H 4 + 4 08 x l O " 4 K + 5-50 x l O " 4 1 I . 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