Medicine, Faculty of
Biochemistry and Molecular Biology, Department of
DSpace
UBCV
Lau, D.C.C.
Master of Science - MSc
University of British Columbia
The compound α-acetolactate has been prepared by the chemical method of Krampltz (1948) and by an enzymatic procedure. The products obtained by each method were characterized chromatographically and spectrophotometrically by conversion to their 2,4—dinitrophenylhydrazone derivatives as well as by conversion to acetoin. The Identity of the hydrazone of enzymatically prepared α-acetolactate with hydrazones of the chemical product was established by comparison of R[subscript: F] values, the identical absorption maxima, and by the infrared spectra of these compounds. The α-acetolactate so obtained was used as substrate in a comparison of the activity in streptomycin mutants of the reductoisomerase enzyme which catalyzes the rearrangement and reduction of α-acetolactate to α,β-dihydroxyisovaleric acid. This reaction is the second step in valine biosynthesis. Streptomycin-dependent mutants of Escherichia coli previously has been shown to be derepressed in acetohydroxy acid synthetase, the enzyme which initiates valine biosynthesis. In addition, it had been reported previously that in streptomycin-dependent E. coli K-12, threonine dehydratase, the enzyme which initiates biosynthesis of isoleucine, also is derepressed. In contrast, reductoisomerase, which is common to both the valine and isoleucine pathway, has been found in this work to be normal (i.e., not derepressed) in streptomycin mutants. An additional enzyme of the common pathway, transaminase B, was found to be about 2 to 3 times higher in streptomycin-dependent mutants than in sensitive or resistant strains. The structural genes for both transaminase B and threonine dehydratase of E. coli K-12 have been shown by genetic studies (Ramakrishnan and Adelberg, 1965b) to be coordinately regulated (i.e., on the same operon). The observations made in this study with streptomycin-dependent E. coli K-12 support the observations of these workers. That is, transaminase B of streptomycin-dependent E. coli K-12 is derepressed coordinately with threonine dehydratase. However, the degree of derepression of transaminase B (about 2 to 3 fold) was much less than that of threonine dehydratase (about 9 fold, according to Desai and Polglase, 1966). It may be concluded from these studies that the type of derepression of certain enzymes which has been observed in streptomycin-dependent E. coli has contrasting features to the type of derepression which would be expected on the basis of the Jacob and Monod model (1961) from a nonfunctional regulatory gene or product thereof.
ENZYME ACTIVITIES OF THE ISOLEUCINE-VALINE BIOSYNTHETIC PATHWAY IN STREPTOMYCIN MUTANTS OF ESCHERICHIA COLI by D.C.C. LAU B. Sc., University of B r i t i s h Columbia, 196*4-. A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n the Department of Biochemistry. We accept t h i s thesis as conforming to the required standard for the degree of MASTER OF SCIENCE. The University of B r i t i s h Columbia A p r i l , 1966. In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of ' • British Columbia, I agree that the Library shall make i t freely available for reference and study. I further agree that per- ; mission for extensive copying of this thesis for scholarly purposes may be granted by the Head of my Department or by. his representatives. It is understood that; copying or publi-cation of this thesis for financial gain shall: not be allowed without my written permission. ;- *; ,%-v,x-;.-/, •. ' Department of The University of British Columbia,: Vancouver 8, Canada. j " Date $ , : - 1 -ABSTRACT The compound oU-acetolactate has been prepared by the chemical method of Krampltz (19^8) and by an enzymatic procedure. The products obtained by each method were characterized chromatographically and spectrophotometrically by conversion to t h e i r 2 ,4—dinitrophenylhydrazone derivatives as well as by conversion to acetoin. The Identity of the hydrazone of enzymatically prepared OC-acetolactate with hydrazones of the chemical product was established by comparison of Ep values, the i d e n t i c a l absorption maxima, and by the i n f r a r e d spectra of these compounds. The a-acetolactate so obtained was used as substrate i n a comparison of the a c t i v i t y i n streptomycin mutants of the reductoisomerase enzyme which catalyzes the rearrangement and reduction of a-acetolactate to at,^-dihydroxyisovaleric a c i d . This reaction i s the second step i n valine biosynthesis. Streptomycin-dependent mutants of Escherichia c o l l previously has been shown to be derepressed i n acetohydroxy ac i d synthetase, the enzyme which i n i t i a t e s valine biosynthesis. In addition, i t had been reported previously that i n streptomycin-dependent E. c o l l K-12, threonine dehydratase, the enzyme which i n i t i a t e s biosynthesis of isoleucine, also i s derepressed. In contrast, reductoisomerase, which i s common to both the valine and isoleucine pathway, has been found i n t h i s work to be normal ( i . e . , not derepressed) i n - i i -streptomycin mutants. An a d d i t i o n a l enzyme of the common pathway, transaminase B, was found to be about 2 to 3 times higher i n streptomycin-dependent mutants than i n sensitive or r e s i s t a n t s t r a i n s . The s t r u c t u r a l genes f o r both trans-aminase B and threonine dehydratase of E. c o l l K-12 have been shown by genetic studies (Ramakrishnan and Adelberg, 1965b) to be coordinately regulated ( i . e . , on the same operon). The observations made i n t h i s study with streptomycin-dependent E. c o l i K-12 support the observations of these workers. That i s , transaminase B of streptomycin-dependent E. c o l i K-12 i s derepressed coordinately with threonine dehydratase. However, the degree of derepression of trans-aminase B (about 2 to 3 fold) was much l e s s than that of threonine dehydratase (about 9 f o l d , according to Desai and Polglase, I 9 6 6 ) . I t may be concluded from these studies that the type of derepression of cert a i n enzymes which has been observed i n streptomycin-dependent E. c o l l has contrasting features to the type of derepression which would be expected on the basis of the Jacob and Monod model ( I 9 6 I ) from a non-functional regulatory gene or product thereof. i i i -ACKNOWLEDGMENT The author wishes to express h i s sincere thanks and deep appreciation to Dr. W.J. Polglase f o r h i s guidance and encouragement during the course of t h i s research. - i v -TABLE OP CONTENTS PAGE A. INTRODUCTION I. The Metabolic Role of Ct-Acetolactate 1 (a) Formation of acetoin 1 (b) Formation of L-valine 1 I I . Interrelationships Between L-valine and L-Isoleucine Biosynthesis 3 (a) Enzymes shared by the two pathways 3 (b) Feedback i n h i b i t i o n of acetohydroxy aci d synthetase by valine i n E. c o l l K-12 3 (c) Relation between threonine dehydratase a c t i v i t y and i n h i b i t i o n of E. c o l l K-12 by valine 5 I I I . E f f e c t of Streptomycin on the Isoleucine-Valine Biosynthetic Pathway i n Streptomycin-Mutants of E. c o l l 6 IV. Isoleucine-Valine Operon 8 V. Reductoisomerase 8 VI. Transaminase B 10 - V -PAGE B. MATERIALS AND METHODS I. Synthesis of OC-Acetolaotate 12 (a) Chemical method 12 (b) Enzymatic method 15 I I . Colorlmetrlc Determination of Acetoin and a-Acetolactate 16 (a) Acetoin 16 (b) Oc-Acetolactate 17 I I I . Characterization of a-Acetolactate 18 (a) Preparation of acetolactate 2 , 4-dinitrophenylhydrazone ........ 18 (b) I d e n t i f i c a t i o n of hydrazones by paper chromatography 18 (c) U l t r a v i o l e t and i n f r a r e d spectroscopy. 18 IV. Cultures and C e l l Extracts 19 (a) Summary of E. c o l l s t r a i n s used 19 (b) Preparation of media 21 (c) Growth of cultures • 21 (d) Preparation of c e l l extracts ........ 21 V. Colorlmetrlc Determination of Proteins .... 22 VI. Enzyme Assays 23 (a) Reductoisomerase 23 (b) Transaminase B - v i -PAGE C. RESULTS I i Synthesis of a-Acetolactate 26 (a) Chemical synthesis 26 (b) Enzymatic synthesis 26 II . Colorlmetrlc Determination of Acetoin and a-Acetolactate 27 III . S t a b i l i t y of a-Acetolactate 27 IV. Characterization of a-Acetolactate ........ 30 V. Enzyme Assays 30 (a) Reductoisomerase 30 (b) Transaminase B 36 VI. Colorimetric Determination of Proteins 36 D. DISCUSSION I. Relationship Between Streptomycin-Dependency and the Elevation of Acetohydroxy Acid Synthetase 40 I I . Synthesis and Characterization of a-Acetolactate 42 (a) General properties of keto-acid 2,4-dinitrophenylhydrazones 42 (b) Hydrazones of a-Acetolactate 44 - v i i -PAGE I I I . Reductoisomerase A c t i v i t i e s i n Streptomycin-Mutants of E. p o l l IV. Transaminase B A c t i v i t i e s i n Streptomycin-Resistant, -Sensitive, and -Dependent E. c o l l K-12 48 V. Interpretation of the Results 49 E. BIBLIOGRAPHY 5k - v i i i -LIST OP FIGURES FIGURE PAGE 1. Intermediates and enzymes i n the biosynthesis of isoleucine and valine 4 2. Sequence of genes c o n t r o l l i n g the formation of enzymes i n the isoleucine-valine blosynthetic pathway 9 3. Enzymie synthesis of a-acetolactate .... 28 4. Standard curve of acetoin(acetylmethylcarbinol) 29 5. S t a b i l i t y of chemical a-acetolactate 31 6. Standard curve of protein 39 7(a). Infrared spectra of a-acetolactate 2,4-dlnitrophenylhydrazone (Spot 1) 33 7(b). Infrared spectra of a-acetolactate 2,4-dinltrophenylhydrazone (Spot 2) 34 7(c). Infrared spectra of acetoin 2 , 4 - d i n i t r ophenylhydrazone 35 8(a). Cls-trans geometrical isomers of pyruvic ac i d 2,4-dinitrophenylhydrazone ............ 45 8(b). Cis-trans geometrical isomers of ^acetolactic acid 2,4-dinitrophenylhydrazone 45 8(c). Cis-isomer of pyruvic a c i d 2,4-dinitrophenyl-hydrazone, showing s t a b i l i z e d excited state by e l e c t r o s t a t i c a t t r a c t i o n 45 - i x -LIST OP TABLES TABLE PAGE I. R F Values of 2,4-Dinitrophenylhydrazone Derivatives 32 I I . Absorption Maxima and Minima of 2,4-Dinitrophenylhydrazone Derivatives.... 32 I I I . Reductoisomerase A c t i v i t i e s Using E i t h e r Chemical or Enzymatic GK-Acetolactate as Substrate 37 IV. Transaminase B A c t i v i t i e s i n Extracts of Streptomycin-Resistant, -Dependent and -Sensitive E. c o l l K-12 38 - 1 -A. INTRODUCTION I. The Metabolic Role of tt-Acetolaotate (a) Formation of acetoin The formation of acetoin (acetylmethylcarbinol) has been observed i n a va r i e t y of b i o l o g i c a l systems: i n yeast, by Neuberg and Simon ( 1 9 2 5 ) , i n plants, by Tomiyasu ( 1 9 3 7 ) * and i n Aerobacter aerogenes by Silverman and Werkman ( 1 9 4 1 ) . Studies on pyruvic acid metabolism of A. aerogenes l e d Watt and Krampitz (1947) to suggest oC-acetolactate as an intermediate i n the formation of acetoin. In 1948, Krampitz f i r s t postulated a condensation reaction involving acetaldehyde and pyruvic a c i d to form ot-acetolactate which upon decarboxylation yielded acetoin. By the excellent work of Juni ( 1 9 5 2 ) , Juni and Heym (1956) i t was established that oC-acetolactate played the role of an intermediate i n acetoin formation both i n acetoin-producing bacteria (A. aerogenes) and i n pigeon breast muscle. (b) Formation of L-valine On the basis of is o t o p i c studies by Strassman et a l . - 2 -(1953) acetolactate was then suggested as an Intermediate i n the biosynthesis of L-valine i n Torulopsls u t l l l s . These authors proposed a scheme involving a keto condensation of pyruvate with acetaldehyde to y i e l d ' tX-acetolactate, followed by an Intramolecular migration of the oc-methyl group to the /B-carbon to y i e l d #-keto-/3--hydroxyisovalerate, and ultimately rt-ketoisovalerate, the keto analog of v a l i n e . Studies on Escherichia c o l l by Umbarger et a l . (1957) (1958) resulted i n three observations which provided strong evidence that «-acetolactate was Indeed the e a r l i e s t 5- carbon precursor of valine i n t h i s organism. One, oC-acetolactate was accumulated by a valine-requiring mutant of E. c o l l when supplied with l i m i t e d amounts of v a l i n e . Two, the end product valine exerted feedback control over the acetolactate forming system which was present i n t h i s organism. Three, cC-acetolaotate labeled i n the a-methyl p o s i t i o n was Incorporated into valine and into i t s precursor of-ketolsovalerate. The further conversion of ot-acetolactate to 06- ketoisovalerate by extracts of bakers* yeast (Strassman et a l . , 1958), and to tf,£-dihydroxyisovalerate by extracts of E. c o l l (Umbarger and Brown, 1958), and of Neurospora crassa (Wagner et a l . , 1958) confirmed i t s r o l e as a valine precursor. - 3 -From the subsequent work of Umbarger et a l . ( i 9 6 0 ) and Strassman et a l . ( i 9 6 0 ) the biosynthetic pathway of L-valine was established. I t involved an i n i t i a l conden-sation of two moles of pyruvate to form ot-acetolactate, followed by the conversion of acetolactate to ot,p-dihydroxy-isovalerate, and then a reduction of the l a t t e r to ot-ketoisovalerate, the keto analog of v a l i n e . Figure I shows the intermediates and enzymes i n the biosynthesis of isoleucine and v a l i n e . I I . Interrelationships Between L-Vallne and L-Isoleuclne Biosynthesis (a) Enzymes shared by the two pathways As I l l u s t r a t e d i n Figure 1, the biosynthetic pathways leading to valine and isoleucine share the same set of enzymes, the only difference being that the isoleucine pathway has an extra enzyme, threonine dehydratase, which regulates isoleucine biosynthesis. Since acetohydroxy a c i d synthetase i s shared by both biosynthetic routes and, at the same time, i s subject to i n h i b i t i o n by v a l i n e , i t i s apparent that valine may a f f e c t Isoleucine biosynthesis. (b) Feedback i n h i b i t i o n of acetohydroxy a c i d synthetase by valine i n E. c o l i K-12 - 4 -leucine pantoic a c i d 2 pyruvate ft-aceto-l a c t a t e I a,/?-dihydroxy-^"isovalerate tt-keto isovalerate" •> valine ACETO-HYDROXY ACID SYNTHETASE REDUCTO-ISOMERASE DEHYDRASE TRANSAM-INASE B Ct-keto-butyrate { flt-aceto--hydroxy-butyrate pyruvate i 0f» /?-dihydroxy-"-*"/£-methyl- ~ valerate tf-keto-^-methyl valerate | i s o -leucine THREONINE DEHYDRATASE threonine FIG. 1. Intermediates and enzymes i n the biosynthesis of isoleuclne and valine - 5 -I t had been reported by Bonner (1946) that the growth of a wild-type s t r a i n of E. c o l l K-12 on minimal medium was i n h i b i t e d by L-valine and that t h i s i n h i b i t i o n was overcome by L-isoleucine. Work by Umbarger and Adelberg (1953)» l a t e r confirmed by Temple et a l . (1965), revealed that valine exerts an i n h i b i t o r y e f f e c t on the growth of E. c o l l K-12 by i n h i b i t i n g the a c t i v i t y of acetohydroxy a c i d synthetase, and thus preventing the formation of acetohydroxybutyrate and isoleucine, acetohydroxy a c i d synthetase i s shared by both the valine and isoleucine biosynthetic pathways (Figure 1). In K-12, the extreme s e n s i t i v i t y of t h i s enzyme to end-product i n h i b i t i o n by valin e causes starvation f o r isoleucine when t h i s organism i s grown on glucose-salts medium supplemented with L-valine only. (c) Relation between threonine dehydratase a c t i v i t y and i n h i b i t i o n of E. c o l i K-12 by valine A second and perhaps a much more s i g n i f i c a n t cause of v a l i n e i n h i b i t i o n of the growth of E. c o l i K-12 i s the low l e v e l i n t h i s s t r a i n of threonine dehydratase, the f i r s t enzyme i n the isoleucine pathway (Desai, unpublished observations). I t was found by Ramakrishnan and Adelberg (1965a) that E. c o l l K-12 mutants having derepressed —2 threonine dehydratase were r e s i s t a n t to a high l e v e l (10 M) of v a l i n e , while mutants derepressed i n any one of the other enzymes i n the pathway except acetohydroxy a c i d synthetase and threonine dehydratase were a l l valine s e n s i t i v e . Mutants of E. c o l l K-12 derepressed i n acetohydroxy a c i d synthetase only were r e s i s t a n t to low -4 l e v e l s (10 M) of v a l i n e . The s e n s i t i v i t y of threonine dehydratase to feedback i n h i b i t i o n by isoleucine and of acetohydroxy a c i d synthetase to feedback i n h i b i t i o n by valine was not affected i n such mutants. These observations thus suggest that the extreme s e n s i t i v i t y of E. c o l l K-12 to valine r e s u l t s both from the e f f i c i e n c y with which valine represses acetohydroxy a c i d synthetase and from the low l e v e l of threonine dehydra-tase i n t h i s s t r a i n . I I I . E f f e c t of Streptomycin on the Isoleuclne-Vallne Biosynthetic Pathway In Streptomycin-mutants of E. c o l i . I t was independently discovered by Tirunarayanan et a l . (1962) and by Bragg and Polglase (1962) that streptomycin-dependent E. c o l l excreted r e l a t i v e l y large amounts of L-valine during growth on glucose-salts medium. The streptomycin-resistant ( i n d i f f e r e n t ) E. c o l l on the other hand produced l a c t i c a c i d from glucose when grown i n medium containing a n t i b i o t i c , but resembled the streptomycin-sensitive E. c o l i when grown i n the absence - 7 -of a n t i b i o t i c i n that they both f a i l e d to produce l a c t i c a c i d . Further observations on the formation of L-valine by streptomycin-dependent E. c o l i (Bragg and Polglase 1964a) has shown that l a c t i c a c i d production was a property of a n t i b i o t i c depletion or oxygen deprivation, whereas val i n e production (accounting f o r over 10% of the glucose carbon) was a property of streptomycin-supplemented c e l l s grown under aerobic conditions. These r e s u l t s suggested that valine and l a c t i c acid are alternate secondary products of glucose metabolism i n streptomycin-dependent E. c o l l the primary products being C0 2 and acetate. The formation of valine i n the catabolism of glucose thus appeared to be a unique property of streptomycin-dependent E. c o l l . Later studies by Bragg and Polglase (1965) and Coukell and Polglase (1965) on enzyme a c t i v i t i e s of several s t r a i n s of E. c o l i has revealed that the valine excretion correlated with an elevated acetohydroxy a c i d synthetase a c t i v i t y and that one action of the a n t i b i o t i c was to derepress the regulatory enzyme acetohydroxy a c i d synthetase i n the valine biosynthetic pathway of streptomycin-dependent mutants. In l i g h t of these findings together with e a r l i e r observations that streptomycin i s required f o r P-galactosidase induction i n dependent E. c o l i (Peretz and Polglase 1956), an action of the a n t i b i o t i c at the l e v e l of expression of - 8 -regulatory genes was suggested. I t was of i n t e r e s t therefore to study what e f f e c t streptomycin had on the a c t i v i t y of the remaining enzymes i n the valine biosynthetic pathway, some of which are controlled by the same operon. IV. Isoleuolne-Vallne Operon Ramakrishnan and Adelberg (1964) (1965b) observed that the enzymes of isoleuclne and valine biosynthesis ( M i l v w ) enzymes were controlled by a c l u s t e r of f i v e s t r u c t u r a l genes comprising three d i s t i n c t operons i n Escherichia c o l i K-12. I t was further observed that operator A controlled the formation of three of the f i v e enzymes: threonine dehydratase, dehydrase, and transaminase B. Operator B con t r o l l e d the formation of acetohydroxy a c i d synthetase, and a t h i r d operator locus (operon II) regulated the reductoisomerase formation, as shown i n Figure 2. The primary i n t e r e s t of t h i s study has been with the l a t t e r enzyme, reductoisomerase. V. Reductoisomerase Reductoisomerase catalyzes the NADPH requiring reduction reaction Involving a two-step conversion of cc-acetolactate to ct,ji-dihydroxyisovalerate. The mechanism of t h i s reaction was established by the work of Strassman -9-OPERON I I I I I GENE opr B i i l v B i l v C opr A i i l v A i i l v D i i l v SEQUENCE: — 1 ; 1 ; 1 1 7 1 ; 1 — ' / t • i / " i v i > \ \ , \ ' / r \ / \ t t <* V f f / \ ENZYME TP _ AHAS^ RI^ DH ^ TRB ^ SEQUENCE: ** *" *" *" **" F i g . 2 . Sequence of genes co n t r o l l i n g the formation of enzymes i n the isoleucine-valine biosynthetic pathway. TD, threonine dehydratase; AHAS, acetohydroxy acid synthetase; R l , reducto-isomerase; DH, dehydrase; TRB, transaminase B. - 10 -et a l . ( i 9 6 0 ) , Umbarger et a l . ( i 9 6 0 ) , and Radhakrishnan and S n e l l ( i 9 6 0 ) . An unusual pinacol-pinacolone rearrange-ment i s followed by reduction, both reactions being catalyzed by a single enzyme. C H 0 - 8 - C O H - G O O H * 3 CH 3 Ot-acetolactate «-ke to-^-hydroxy-isovalerate NADPH + H + NADP+ ^ : ^ y CH,-C0H-CH©H-C00H tf,p-dihydroxyisovalerate In order to study t h i s reaction i n streptomycin mutants of E. c o l i , i t was necessary to synthesize and to characterize the substrate rt-acetolactate. This was done both by chemical method of Krampitz and by an enzymatic method which u t i l i z e d the high enzymatic a c t i v i t y of streptomycin-dependent E. c o l i extracts. VI. Transaminase B Formation of valine from rt-ketoisovalerate i s catalyzed by transaminase B. According to Ramakrishnan and Adelberg (1965b), both transaminase B and threonine dehydratase are c ontrolled by the same operon. In streptomycin-dependent C H o - C O H - C - C O O H J C H 0 - l l -El. c o l i K-12, threonine dehydratase i s derepressed i n proportion to a n t i b i o t i c concentration (Desai and Polglase 1966). Transaminase B should also be derepressed i n streptomycin-dependent E. c o l l K-12 i f the a n t i b i o t i c acts by i n t e r f e r i n g with the formation or function of the repressor of operator A (Figure 2). This enzyme of the i s o l e u c i n e - v a l i n e pathway has therefore been investigated i n streptomycin-sensitive, - r e s i s t a n t and -dependent mutants of Escherichia c o l i K-12. - 12 -B. MATERIALS and METHODS I. Synthesis of cUAcetolactate The synthesis of ot-acetolaetate was achieved i n two ways: (a) Chemical method In t h i s synthesis, oxidation of the l a b i l e hydrogen of methyl-substituted acetoacetic ester was accomplished with lead tetraacetate, with subsequent hydrolysis of the acetoxy ester to give Ot-acetolactate. The o v e r a l l synthesis involved three major steps: ( i ) The methyl-substituted acetoacetic ester was prepared i n the manner given by Gilman and B l a t t ( 1 9 4 J ) . CHo-C-CHo-fi-OCH,, + CH 0I + CoH.0Na »- CH-5-C-CH-C-0CoH 2 i A5 3 2"5 ethyl acetoacetate t h y l oC-methylaceto-acetate + Nal + CgH-OH Procedure: In a 500-ml. round-bottomed f l a s k f i t t e d with a - 13 -mechanical s t i r r e r and r e f l u x condenser 7.7 g. (0 . 3 3 atoms) of m e t a l l i c sodium was added i n small pieces to 170 ml. of absolute ethanol under anhydrous conditions. A f t e r a l l the sodium had dissolved, 44 ml. (0 . 3 3 moles) of ethyl acetoacetate was added and the solution heated to gentle b o i l i n g . The s t i r r e r was started and 23 ml. (0 .36 moles) of methyl iodide was added over a period of about one hour. The r e f l u x i n g and s t i r r i n g was continued u n t i l a sample of the solution was neutral to moist litmus paper (about 5 hours). When the reaction was complete, the mixture was cooled and the solution decanted from the sodium iodide. The s a l t was washed 4 times with 10 ml. absolute alcohol and the washings were added to the main so l u t i o n . The alcohol was separated from the substituted acetoacetic ester by steam d i s t i l l a t i o n . The crude residue a f t e r removal of the alcohol was used for the oxidation step that followed. ( i i ) The oxidation of the methyl-substituted acetoacetic ester was performed according to the method of Krampitz ( 1948) . 0~C—CH 0 0 Pb(0Ac) 4 0 j 0 3 2 CH~-C-CH-C-0CoH= CHQ-C-C-C-0C..H. + Pb(0Ac)_ 3 CH 3 2 5 3 CH 3 2 5 2 ethyl flC-methyl- ethyl rf-methyl-acetoacetate ot-acetoxy-acetoacetate - 14 -Procedure: With vigorous mechanical s t i r r i n g under anhydrous conditions 52g. Ph(OAc)^ was added i n small portions to a mixture of 5&g. thiophene-free anhydrous benzene and l? g . (0.12 moles) methyl-substituted ethylacetoacetate. During the addition of the Pb(OAc)^ the temperature was not allowed to r i s e above 3 5 ° G , a f t e r which the mixture was heated to 40°C f o r 5 hours with vigorous s t i r r i n g . The mixture was allowed to stand overnight at room temperature, f i l t e r e d , and the p r e c i p i t a t e washed 5 times with 20 ml. portions of benzene. The washings were combined with the o r i g i n a l s o l u t i o n . To remove any ac e t i c a c i d present i n the benzene, i t was washed with 20 ml. portions of water u n t i l the water was neutral to bromothymal blue. The benzene layer was dried over MgSO^, f i l t e r e d , and the benzene d i s t i l l e d o f f under reduced pressure. The remaining ester, was d i s t i l l e d at a pressure of about 16 mm. A f r a c t i o n was obtained at 100-105GC which contained the methyl-acetoxy-substituted ester. Characterization of the methyl-acetoxy-substltuted ester was performed on t h i s f r a c t i o n . ( i i i ) Free ct-acetolactate was l i b e r a t e d by hydrolysis of the methyl-acetoxyacetoacetate i n the following manner: - 15 -p 0—(J—CH 0 I 0 3 2Na0H 0 OH 0 CH-C-C-C-OC H > CHo-C-p-C-ONa CH 3 * J CH^ ethyl oC-methyl- sodium ^-acetolactate a-acetoxy-acetoacetate 0 + CHyS-ONa + CgH^OH Procedure s To 10 ml. of 0.1M NaOH, c h i l l e d i n an i c e bath, was added 0.1 ml. of the methyl-acetoxy ester. The mixture was shaken constantly u n t i l a l l the ester had been hydrolyzed (about 30 min.). The r e s u l t i n g solution contained a mixture of oC-acetolactate, acetate and ethanol. (b) Enzymatic method. In t h i s method, tf-acetolactate was prepared enzymatically from sodium pyruvate using the acetolactate-forming system of E. c o l i DK-12 (streptomycin-dependent). CH3-(J-C00Na T F P sodium pyruvate CH3-CH-TPP 0 \ 0 OH CHjC-COONa Acetohyd?oxy ' C^-C-6-COONa sodium pyruvate a c i d synthetase C H ^ Na c<-acetolactate - 16 -Procedure: A 25-ml. reaction system, containing 2.5 moles of potassium phosphate, pH8; 4 umoles thymine pyrophosphate (TPP); 25 umoles MgCl 2; 250mumoles f l a v i n adenine dinucleo-t i d e (PAD); and 12.5 mmoles sodium pyruvate was prepared i n a 50-ml. p l a s t i c t e s t tube f i t t e d with a magnetic s t i r r e r . The reaction was started by introducing i n t o the system a d i a l y z l n g bag containing 20 ml. of fre s h l y prepared sonic extract of E. c o l l DK-12 (Ig. c e l l s per 15 ml. bu f f e r ) , followed by incubation at 30°C with s t i r r i n g . Samples were taken every h a l f hour f o r about 4 hours, and the concentrations of acetoin and tf-acetolactate were determined c o l o r i m e t r i c a l l y by the method of Westerfeld (1945). I I . Colorlmetrlc Determination of Acetoin and Pt-Acetolactate (a) Acetoin Acetoin (acetylmethylcarbinol) was determined c o l o r i m e t r i c a l l y by the method of Westfeld (1945). Procedure: To a 5 ml. sample of acetoin s o l u t i o n (concentration not higher than 0.2 umoles/5 ml.) 1 ml. of 0.5$ aqueous sol u t i o n of creatine was added and mixed. Then 1 ml. of fre s h l y prepared a l k a l i n e naphthol solution (5$ CK-naphthol - 1? -In 2.5N NaOH) was added. The color was developed i n the dark f o r one hour and the o p t i c a l densities were read at 5^0 mu i n a Beckman model B spectrophotometer. A "blank was prepared s i m i l a r l y with the exception that 5 ml. of water was substituted f o r the acetoin s o l u t i o n . A standard curve of acetoin with a concentration range of 0 to 0.2 umoles/5 ml. was plotted against o p t i c a l d e n s i t i e s . Acetylmethylcarbinol purchased from the "Ald r i c h Chemical Co." was used as the standard. (b) Ot>Acetolactate To determine the «-acetolactate concentration, i t was f i r s t converted to acetoin by decarboxylation. 0 OH „+ OH 0 CH^-C-jj-COONa >- CH^-CH-fi-Cl^ Na a-acetolactate acetoin Procedure: To a 5-ml. sample of a-acetolactate, one drop of cone. H-SO. was added and the solution incubated at 60°C f o r 15 min. The acetoin i n the r e s u l t i n g solution was determined with the procedure given i n (a). - 18 -I I I . Characterization of flt-Aoetolactate (a) Preparation of tf-acetolactate 2,4-dinitrophenylhydrazone Procedure: To 0 .5 ml. of a 0.01 M a l c o h o l i c s o l u t i o n of 2 , 4-dinitrophenylhydrazine i n 0.2N HC1,0.3 ml. (about 5.4 jimoles) of c(-acetolactate (chemically or enzymatically prepared) was added. The solu t i o n was shaken and the reaction allowed to proceed at 25°C f o r 30 min. I t was then centrifuged and the cl e a r supernatant was examined by paper chromatography, followed by elut i o n , and spectro-photometric determinations. (b) I d e n t i f i c a t i o n of hydrazones by paper chromatography Derivatives of 2 ,4-dinitrophenylhydrazone were spotted on Whatman No. 1 paper and the chromatographs (ascending i n a l l experiments) were developed i n n-butanol-water-ethanol (5:4:1) f o r 15 hours. The values of the hydrazones of b i o l o g i c a l cC-acetolactate were calculated and compared with that of the chemical product. (c) U l t r a v i o l e t and i n f r a r e d spectroscopy Each 2 ,4-dlnitrophenylhydrazone spot to be analyzed - 19 -was cut from the chromatograms and the yellow-colored hydrazone was extracted with a suitable solvent. For u l t r a v i o l e t analyses, the hydrazones were extracted with 0.2M sodium bicarbonate and the spectra were observed i n a Cary 15 spectrophotometer over a wavelength range of 250 to 400 millimicrons. For i n f r a r e d analyses, the hydrazones were extracted with absolute ethanol and the spectra were observed i n a Perkin-Elmer 137 spectrophotometer over a wavelength range of 2.5 to 14.5 microns. IV. Cultures and C e l l Extracts (a) Five s t r a i n s of E. c o l l used In t h i s work are summarized below. (I) E. c o l l A S t r a i n DA was a streptomycin-dependent s t r a i n which has been deposited with the American Type Culture C o l l e c t i o n (ATCC 15745), and which was o r i g i n a l l y obtained from T.F. Paine, J r . by W.J. Polglase. Strains SA and HA are streptomycin-sensitive and - r e s i s t a n t revertants, respectively, i s o l a t e d by back-mutation from streptomycin-dependent DA. (II) E. c o l l B S t r a i n SB was obtained as a wild type E. c o l i B from - 20 -the American Type Culture C o l l e c t i o n . S t r a i n DB and RB are streptomycin-dependent and -r e s i s t a n t mutants i s o l a t e d i n t h i s laboratory from se n s i t i v e E. c o l l SB. ( i i i ) E. c o l i Br St r a i n SBr was obtained as a streptomycin-sensitive s t r a i n from the American Type Culture C o l l e c t i o n (ATCC 12407). This s t r a i n i s a r a d i a t i o n r e s i s t a n t mutant of E. c o l i SB. Strains DBr and RBr are streptomycin-dependent and streptomycin-resistant mutants, respectively, i s o l a t e d from streptomycin-sensitive s t r a i n SBr (Coukell and Polglase 1965). (iv) E. c o l i E St r a i n SE was obtained as a streptomycin-sensitive s t r a i n from Dr. J . Stock of the Department of Bacteriology of t h i s University. Strains DE and RE are streptomycin-dependent and -r e s i s t a n t mutants i s o l a t e d from SE. (v) E. c o l i K-12 The wild-type (streptomycin-sensitive) s t r a i n was designated as SK-12. Streptomycin-resistant and -dependent mutants were i s o l a t e d i n t h i s laboratory (Polglase, 1965). - 21 -(b) Preparation of media The basal medium containing the following s a l t s : K2HPO^(0.?#), KHgPO^O^), sodium c i t r a t e (0 .05#), MgS02f(0.02^), (NH^gSO^O.l^), was adjusted to f i n a l pH of 7.0. Glucose (10$) was autoclaved separately and added to the s t e r i l i z e d basal medium to give a f i n a l concentration of 0.4$. Streptomycin-sensitive cultures were grown i n a n t i b i o t i c - f r e e medium while dependent mutants were grown i n medium containing 1000 units per m i l l i l i t e r of dihydro--3 streptomycin (1.71 x 10 M). Streptomycin-resistant cultures were grown i n both a n t i b i o t i c - f r e e medium (H** c e l l s ) and i n medium which had been supplemented with 1000 units per m i l l i l i t e r of dihydrostreptomycin ( B + c e l l s ) . (c) Growth of cultures To 1 l i t e r of medium i n a 2 - l i t e r Erlenmeyer f l a s k , 100 ml. of an 18-hr. stationary culture was added. Cultures were grown with vigorous aeration f o r 4 to 5 hrs. at 37°C and were harvested just before the end of the log phase. (d) Preparation of c e l l extracts C e l l s were harvested by centrifugation at 6000 X g i n a S e r v a l l centrifuge and suspended i n 0.1M potassium phosphate buffer (pH 7.0). The c e l l suspensions were c e n t r i -- 22 -fuged again at 6000 X g and resuspended i n 0.1M potassium phosphate buffer i n a. r a t i o of l g . packed c e l l s to 15 ml. of "buffer s o l u t i o n . The suspensions were then subjected to 3 min. of sonic disruption i n a Bronwill 20-kc sonic o s c i l l a t o r . The r e s u l t i n g crude extracts, which contained 2 .5 to 5.0 mg. per m i l l i l i t e r of protein, were used d i r e c t l y f o r enzyme assays. V. Colorlmetrio Determination of Proteins Proteins were determined by the method of Lowry et a l . ( 1 9 5 D . The following reagents were used: Reagent A: 2% NagCO^ i n 0.1 N NaOH. Reagent B: 0 .5$ CuS0^.5H20 i n 1% Na t a r t r a t e . Reagent C: Prepared just before protein determinations by mixing 50 ml. reagent A and 1 ml. reagent B. Reagent D: Diluted F o l i n reagent (1 volume of phenol reagent + 2 volumes of d i s t i l l e d water). Procedure: To determine the protein concentrations, a 1-ml. protein sample (concentration 0.25 to 0 .5 mg. per ml.) was mixed with 5 ml. of reagent C and the solu t i o n allowed to stand at room temperature f o r 10 min. Color was developed with 0.5 ml. of reagent D f o r 30 min. and the - 23 -o p t i c a l densities were read at 500 mu i n a Beckman model B spectrophotometer. VI. Enzyme Assays (a) Reductoisomerase This was measured spectrophotometrically by recording the disappearance of NADPH at room temperature, according to the method of Umbarger et a l . ( i 9 6 0 ) . 0 OH NADPH + H + NADP+ OH 9H CHo-C-C-COOH — r — 1 ^ CHo-C-CH-COOH 3 C H Reductoisomerase 3 <jg^ fc-acetolactate j3-dihydroxyi sovalerate Procedure: Assays were conducted i n 1.5-ml. systems containing 300 umoles potassium phosphate, pH 7.5; 10 umoles magnesium chloride; 0.3 umoles nicotinamide adenine dinucleotide phosphate (NADPH); substrate, 2 umoles of either chemically or enzymatlcally prepared rtr-acetolactate; and 0.2 ml. of b a c t e r i a l c e l l extract. The oxidation of NADPH (as Indicated by the decrease of o p t i c a l density at 3^0 mu) was recorded i n a Cary 15 spectrophotometer. S p e c i f i c a c t i v i t i e s of enzyme preparations were expressed i n mumoles oC-acetolactate reduced per mg. protein per minute. - 24 -(b) Transaminase B This a c t i v i t y was determined by measuring the production of valin e from the valine-glutamate transamination reaction, according to the method of Bragg and Polglase (1964b). glutamate tt-ketoglutarate J J J J ^ CH-- CH-8-C00H ^ ^ y- CH - CH-CH-COOH CH^ Transaminase B 3 CH^ a-ketoisovalerate valine Procedure: The system contained the following: 0.25 ml. (50 umoles) L-glutamlc ac i d (dissolved i n 0.1M potassium phosphate buffer, pH 8 ) ; 0.1 ml. (20 umoles) ff-keto-i s o v a l e r a t e ; 0.1 ml. (1 0 . 8 u m o l e s ) pyridoxal phosphate; 0.1 ml. ( 5 umoles) magnesium chloride; and 0.5 ml. c e l l extract ( l g . c e l l s plus 15 ml. b u f f e r ) . The mixture was centrifuged and the amino ac i d produced was determined from the supernatant by paper chromatography. Quantitative amounts of samples from enzyme assays and also several standard solutions of valine were spotted on Whatman No. 1 paper. Chromatograms, (ascending i n a l l experiments) were developed i n a solvent system containing - 25 -n-butanollacetic acidtwater ( 4 : l s l ) for about 20 hours. They were dried and dipped i n Ninhydrin s o l u t i o n (0.6 g. triketohydrindene hydrate, 6 ml. pyridine, and 294 ml. acetone). Chromatograms were dried and placed i n an oven f o r 15 min. at 90°C. They were dipped i n copper n i t r a t e s o l u t i o n (1 ml. saturated aqueous copper n i t r a t e , 0.02 ml. cone, n i t r i c a c i d , and 99 ml. acetone). Spots were then cut i n t o small pieces and the color was extracted with 2 ml. methanol. Optical densities were read at 530 mu i n a Beckman model B spectrophotometer. - 26 -C. RESULTS I. Synthesis of oUAoetolactate (a) Chemical synthesis Chemical synthesis by the method of Krampitz (1948) yielded an ester (ethyl ol-methyl-ct-acetoxy-acetoacetate) o having a b o i l i n g point of 100-105 C at 16 mm. Hg. The ester was s l i g h t l y yellow i n color and insoluble i n water, but hydrolyzed slowly ( i hr.) at 0°C i n 0.1M NaOH. When hydrolyzed, t h i s ester l i b e r a t e d an a c i d (ot-acetolactate) which gave a p o s i t i v e acetoin test upon a c i d i f i c a t i o n with s u l f u r i c a c i d . The y i e l d of the ester as calculated from the oxidation step was 39.6#. From the hydrolysis mixture (0.1 ml. ester plus 10 ml. 0.1M sodium hydroxide), the concentration of ot-acetolactate was 18.1 micromoles per ml. (b) Enzymatic synthesis B i o l o g i c a l a-acetolactate was prepared from sodium pyruvate using c e l l extracts of E. c o l i DK-12. Reactions were usually stopped a f t e r 4 to 5 hours. Figure 3 shows the time curves of ot-acetolactate formation i n t h i s reaction system. In the " c o n t r o l " system where c e l l extracts were - 27 -added d i r e c t l y to the reaction mixture, l i n e a r production of ot-acetolactate was observed f o r about 3^ hours. In the "standard" system where c e l l extracts were placed i n a d i a l y z i n g bag, there was an i n i t i a l l a g f o r 2 hours a f t e r which the production of a-acetolactate was l i n e a r . The rate of production of O-aeetolactate i n the " c o n t r o l " system was about 3 times that of the "standard". However, f o r a l l enzyme assays and characterization purposes, the a-acetolactate from the "standard" system was used since i t was not contaminated with c e l l extracts. I I . Colorlmetrlc Determination of Acetoin and a-Acetolactate Acetoin and a-acetolactate concentrations were determined by the method of Westfeld (1945) described under "Materials and Methods". This colorlmetrlc determina-t i o n gave a l i n e a r standard curve of acetoin i n the range 20 to 250 millimicromoles per 5 ml., as shown In Figure 4 . I I I . S t a b i l i t y of O-Acetolaotate Since rt-acetolactate was reported to be quite unstable and could undergo self-decarboxylation quite r e a d i l y (Umbarger 1958), the s t a b i l i t y of chemical a-acetolactate i n the hydrolysis mixture (pH 7-2) was determined at three d i f f e r e n t temperatures: room temperature (25°C), 5°C, and -10°C (frozen). -28-Time (hours) Fig. 3. Enzymic synthesis of fl£-acetolactate. "Control" represents system with cell extract introduced directly into the reaction mixture. "Standard" represents system with cell extract placed in a dialyzing bag. Cell extract was obtained from Escherichia coli K-12. -29-Concentration (milliraicromoles per 5 ml.) Fig. 4. Standard curve of acetoin (acetylmethylcarbinol). Each sample contained 5 ml.^acetoin solution, 1 ml. 0.5 % aqueous creatine, and 1 ml. of 5.0 % rt-naphthol in 2.5 N NaOH. Color was developed in the dark for 1 hour and the optical density was read at 5 AO mu in a Beckman model B spectrophotometer. - 30 -Figure 5 shows the % a-acetolactate present i n the hydrolysis mixture over a period of 20 days. As seen i n curve A, v i r t u a l l y a l l the O-acetolaetate had been converted to acetoin i n 10 days at room temperature. At -10°C, the a-acetolactate was r e l a t i v e l y stable i n t h i s mixture. IV. Characterization of rt-Acetolactate The procedure f o r the i d e n t i f i c a t i o n of 2,4-dinitrophenylhydrazone of a-acetolactate i s given on p. 18. The solvent system containing n-butanol-water-ethanol with a r a t i o of 5*4:1 was found to give best r e s u l t s . Spots could be separated without streaking i f the amount of the hydrazone solutions spotted on the paper d i d not exceed 0.05 ml. The R F values and the flmax of the hydrazone spots are given i n Tables I and I I . The in f r a r e d spectra are shown In Figures 7(a), 7(b) and 7(e). V. Enzyme Assays (a) Reductoisomerase Table I I I shows the s p e c i f i c a c t i v i t i e s of reductoisomerase from 5 s t r a i n s of E. c o l l . The rates of enzyme a c t i v i t i e s f o r most cases were of zero-order f o r the -31-100 Time (hours) Fig. 5. Stability of chemical ot-acetolactate at varies temperature: A, at room temperature (25°C); B, at 5°C; C, at -10°C. - 32 -TABLE I R Values of 2,4-Dinitrophenylhydrazone F Derivatives Substance Spot 1 Spot 2 Spot 3 Enzymatic a-acetolactate 0.46 0.57 Chemical a-acetolactate 0.47 0.59 0.91 Acetoin - - 0.93 Solvent system: n-butanol-ethanol-water (5:1*4) TABLE II Absorption Maxima and Minima of 2,4-Dinitrophenylhydrazone Derivatives Maxima (mu) Minima (mu) Substance Spot Spot Spot 1 2 3 Spot 1 Spot 2 Spot 3 Enzymatic tt-acetolactate 358 372 - 291 292 - • Chemical Ot-acetolactate 358 373 353 294 302 291 Acetoin - - 358 - - 295 Each hydrazone spot was cut from the chromatographs and the color was extracted with ,2M Na bicarbonate. Absorption spectra were run i n a Cary 15 spectrophotometer i n the u l t r a v i o l e t region. 4000 3000 2000 1500 FREQUENCY (CM**1) 1000 900 800 3 4 5 6 7 8 9 10 11 12 WAVELENGTH (MICRONS) F i g . 7(a). Infrared spectra of Ot-acetolactate 2,4-dinitrophenylhydrazone (Spot 1 of Table I ) . The heavy l i n e represents the derivative of the chemical fc-acetolactate and the l i g h t e r l i n e represents the derivative of the enzymic a-acetolactate. WAVELENGTH (MICRONS) Fig. 7(b). Infrared spectra of ft-acetolactate 2,4-dinitrophehylhydrazone (Spot 2 of Table I). The heavy line represents the derivative of the chemical ft-acetolactate and the lighter line represents the derivative of the enzymic OC-acetolactate. F i g . 7(c). Infrared spectra of acetylmethylcarbin6l(acetoin) 2,4.-dinitrophenylhydrazone. The heavy l i n e represents the derivative of commercial acetoin, and the l i g h t e r l i n e represents spot 3 from the chromatogram (see Table I ) . - 36 -i n i t i a l 10 to 15 minutes. A l l s p e c i f i c a c t i v i t i e s were determined from t h i s i n i t i a l period of zero-order k i n e t i c s . (b) Transaminase B Table IV shows the s p e c i f i c a c t i v i t i e s of trans-aminase B i n E. c o l i K-12. Three separate enzyme assays were performed with fresh extracts prepared from c e l l s grown on d i f f e r e n t days, and the r a t i o of a c t i v i t i e s i n dependent and sensitive extracts was determined to be 3 . 3 , 1.5 and 3 .6 . VI. Colorlmetrlc Determination of Proteins Proteins were determined by the method of Lowry (1951) described under "Materials and Methods". With c r y s t a l l i n e bovine serum albumin as the standard, t h i s method gave a l i n e a r standard curve over the range, 0 to 3 mg. per ml., as shown i n Figure 6. For t h i s reason, samples were usually d i l u t e d with water to give concentra-tions of approximately 1.5 to 3 mg. per ml. before proteins were determined. - 37 -TABLE III Reductoisomerase A c t i v i t y * Using E i t h e r Chemical or Enzymatic rt-Acetolactate as Substrate 0L- Acetolactate E. c o l l S t r a i n Chemical Enzymatic SB 6.7 5.6 DB 7.0 3.9 SBr 9.2 5.1 DBr 6.8 4.6 SA 16.5 DA 18.2 SE 19.2 DE 25.3 R"E 25.3 BPE 25.4 SK-12 13.3 6.1 DK-12 8.5 5.1 * A c t i v i t i e s are expressed as millimicromoles of substrates converted per milligram of protein per minute at room temperature (25°C). - 38 -TABLE IV Transaminase B A c t i v i t i e s i n Extracts of Streptomycin-Resistant, -Sensitive and -Dependent E. c o l l K-12. Resistant Sensitive Dependent D/S* mumoles of valine formed per mg. protein per min. Assay No. 1 21.2 6.4 20.8 3.3 Assay No. 2 20.? 11.4 1?.0 1.5 Assay No. 3 23.6 6.6 24.0 3.6 * D/S i s the r a t i o of a c t i v i t i e s i n dependent (D) and s e n s i t i v e (S) extracts. -39-0 .1 .2 .3 .A .5 .6 .7 Concentration (mg. protein per ml.) Fi g . 6. Standard curve of protein. C r y s t a l l i n e bovine albumin was used as the standard. Optical density was read i n a Beckman model B spectrophotometer at a wavelength of 500 m i l l i -microns . - 40 -D. DISCUSSION I. Relationship Between Streptomycin-Dependency and the Elevation of Aoetohydroxy Acid Synthetase, The biosynthesis of valine and isoleuclne i s c o n t r o l l e d by a c l u s t e r of f i v e s t r u c t u r a l genes comprising 3 d i s t i n c t operons (Ramakrishnan and Adelberg 1965b). These s t r u c t u r a l genes synthesize f i v e enzymes i n the i s o l e u c l n e - v a l i n e pathway. Threonine dehydratase and acetohydroxy a c i d synthetase are the f i r s t enzymes i n the synthesis of isoleuclne and valine respectively, while reductoisomerase, transaminase B as well as acetohydroxy a c i d synthetase are shared by both pathways, as shown i n Figure 1. A high degree of complexity can be r e a l i z e d from the f a c t that a branch point exists at the tf-keto-isovalerate intermediate leading to the formation of leucine and pantoic a c i d . I t i s not surprising therefore that t h i s complex biosynthetic pathway has provided quite an unique system i n studying enzyme regulation and biosynthetic control mechanisms. An i n t e r e s t i n the isoleucine-valine pathway was r e s u l t e d from the independent discovery by Tirunarayanan et al . ( 1962) and by Bragg and Polglase (1962) that strepto-- 41 -mycin-dependent mutants excreted r e l a t i v e l y large amounts of L-valine (accounting f o r 10% of the glucose carbon) during growth on glucose-salts medium. This has lead to the suggestion of a f a i l u r e of control mechanisms i n dependent s t r a i n s and that the r o l e of the a n t i b i o t i c appeared to regulate an anomalous pathway of glucose catabolism, leading to the secretion of v a l i n e . A d e t a i l e d study of acetohydroxy a c i d synthetase (the f i r s t enzyme i n the valine pathway) by Coukell and Polglase (1965) revealed that there was an elevation (2 to 6 times) of t h i s enzyme i n the dependent mutants which could account f o r the increase of valine formation. The sensitive and r e s i s t a n t mutants showed no elevation i n acetohydroxy a c i d synthetase when grown eithe r with or without added streptomycin. I t was apparent therefore that a close r e l a t i o n s h i p e x i s t s between streptomycin-dependency and the elevation of acetohydroxy a c i d synthetase a c t i v i t y i n these mutants. In order to v e r i f y the l a t t e r point and to e s t a b l i s h the p o s i t i o n (or positions) In the isoleucine-valine pathway aff e c t e d by streptomycin, a study was undertaken to determine the a c t i v i t y of reductoisomerase i n the streptomycin-mutants of Escherichia c o l l . - 42 -I I . Synthesis and Characterization of 06-Acetolaotate To obtain a r e l i a b l e r e s u l t from an enzymic reaction, i t i s e s s e n t i a l to ensure that the substrates are authentic. One way to achieve t h i s i s to synthesize a substrate chemically and then compare i t s properties with that of the enzyme product. I f the chemically-synthesized product i s i d e n t i c a l with that synthesized enzymatically the authenticity of the material i s confirmed. In t h i s study, ct-acetolactate was synthesized chemically by the method of Krampitz (1948) and the product was characterized and compared with the b i o l o g i c a l OC-acetolactate as t h e i r hydrazone d e r i v a t i v e s . (a) General properties of keto-acid 2,4-dini trophenylhydrazones Hydrazones of keto-acids are colored providing the advantage that these compounds are s e l f - i n d i c a t i n g on paper chromatograms. However, complications may a r i s e from the f a c t that a keto-acid w i l l often give r i s e to two or more hydrazone d e r i v a t i v e s . Studies by Altman ejb a l . (195D» C a v a l l i n i et al.(1954), Mortimer et al.(1954), Cruickshank ( 1 9 5 4 ) , and Towers et a l . ( 1 9 5 4 ) revealed that a common phenomenon among keto-acids encountered frequently i n the b i o l o g i c a l f i e l d i s t h e i r a b i l i t y to form isomeric hydrazones. - 43 -Detailed studies by Isherwood (1955) on several keto-acids have provided confirmatory data f o r the s t r u c t u r a l assignment of the hydrazones as geometrical isomers. For example, pyruvic a c i d gave r i s e to two hydrazone spots i n the chromatograms, having R values F of G.6 and 0.4. The absorption maxima of the two hydra-zones In 0.2N sodium bicarbonate were very s i m i l a r (flmax 380mu .and 3?0mu). The difference lOmu was very s i m i l a r to that observed f o r isomeric hydrazones of other keto-acids such as GL-ketoglutaric and oxaloacetic a c i d s . From these observations together with the findings from the i n f r a r e d spectra, Isherwood proposed that these two hydrazones of pyruvic a c i d represented a p a i r of c i s - t r a n s geometrical Isomers around the C=N double bond as shown i n Figure 8(a). The c i s configuration i s s t a b i l i z e d by the formation of a hydrogen bond between the carboxyl and the imlno groups, while the trans configuration has a free carboxyl d e r i v a t i v e . The i n f r a r e d spectra of the isomers were found to be very s i m i l a r . Emphasis i s focussed here on the carboxyl function of the molecule. In the trans-isomer of pyruvic a c i d hydrazone, i t was observed that a strong absorption band occurred at 1722 cm"1, while the cis-isomer had a subsidiary band at 1672 cm . This suggested that some form - 44 -of chelation had taken place i n a substantial proportion of the absorbing molecules. This phenomenon was also reported by P l e t t (1951) who observed that whereas normal saturated a l i p h a t i c acids absorbed strongly i n the region 1725-1700 cm'1, i n a s i g n i f i c a n t number of cases where chelat i o n could occur between carboxyl linkage and an amino or imino hydrogen, absorptions occurred below 1680 cm"1. The higher R value of the cis-isomer on paper F chromatograms i s a r e f l e c t i o n of the s t e r i c interference of neighboring phenyl groups on the a b i l i t y of the ionized carboxyl to a t t r a c t water molecules and become Incorporated i n the water-cellulose complex. Such s t e r i c factors are not present i n the trans-isomer. The higher flmax of the cis-isomer i n the u l t r a -v i o l e t as compared with the trans-isomer (hyperchromic e f f e c t ) was probably due to the s t a b i l i z a t i o n of an excited state as a r e s u l t of e l e c t r o s t a t i c a t t r a c t i o n , as shown i n Figure 8(c). (b) Hydrazones of Ot-acetolactate Results obtained from the study of OC-acetolactate hydrazones also indicated the formation of isomeric deriva-t i v e s . As seen i n Table I, two hydrazones were separated - A5 -CH 3 I \ HO—C' N N0 2 A 1 H A HO, / \ / - c w CHj N0 2 N0 2 ! I K HO -N02 || 0 c i s - trans-F i g . 8(a). Cis-trans geometrical isomers of pyruvic acid 2, ^ .-dinitrophenylhydrazone. ° H ° H 3 OH C H 3 N0 2 \ I \ I H }=\ H 3 C - ? C - C ^ H 3 C - C - C N - ^ - N 0 2 1 N N0 2 p n _ _ / \ / HO-C 2 H 0 _ c ' if 0.. -N02 I c i s - trans-F i g . 8(b). Cis-trans geometrical isomers of flt-acetolactate 2,4,-dinitrophenylhydrazone. F i g . 8(c). Cis-isomer of pyruvic acid 2,4,-dinitrophenylhydrazone, showing s t a b i l i z e d excited state by e l e c t r o s t a t i c a t t r a c t i o n . - 46 -from the enzymic (X-acetolactate having R values of 0.46 and O.57. The absorption maxima of these two hydrazones were very s i m i l a r (flmax 358 and 372 mu). The difference 14 mu i s comparable with values obtained by Isherwood (1955) on pyruvic, Ot-ketoglutaric and oxaloacetic acids. Figure 7(a) and 7(b) shows the i n f r a r e d spectra of the two hydra-zones. The fa s t e r moving hydrazone has a weak absorption — 1 band at 1670 cm while the slower hydrazone has a strong absorption band at 1700 cm'1. Similar r e s u l t s were obtained with the chemical Ot-acetolactate except i n t h i s case, a t h i r d hydrazone was i s o l a t e d . This compound was i d e n t i f i e d with the hydrazone of acetoin and thus represented the decarboxylation product of Ot-acetolactate hydrazone. As expected, the i n f r a r e d spectrum of t h i s compound d i d not display the carboxyl peak, c h a r a c t e r i s t i c of the other two keto-acids hydrazones. From the observations on ot-acetolactic acid hydrazone and a comparison with the observations by Isherwood on other keto-acid hydrazones, i t i s apparent that the two hydrazones from each Ot-acetolactate (chemical or enzymic) represented a p a i r of geometrical isomers. Their probable structures are shown i n Figure 8(b). Thus Spot 2 i n Table I represents the cis-isomer, which i s s t a b i l i z e d by a hydrogen-bond between the carboxyl and the imlno groups. The disappearance - 47 -of the carboxyl absorption band at 1700 cm probably indicates that chelation was complete i n the hydrazone molecule. Spot 1 represents the trans-isomer having a lower R value and flmax. P The i d e n t i t y of the hydrazones of enzymatically prepared (X-acetolactate with hydrazones of the synthetic product was established, as shown i n Table I, by comparison of R values f o r both isomeric derivatives, and by the F i d e n t i c a l absorption maxima and minima as shown i n Table I I . In addition, i n f r a r e d spectra of the hydrazones from these two keto-acids were p r a c t i c a l l y superimposable, as shown i n Figure 7(a) and 7(b). I t can be concluded therefore that the products synthesized chemically by the method of Krampitz and enzymatically with c e l l extracts of E. c o l l K-12 represented the same compound, Ot-acetolactate. I I I . Reductoisomerase A c t i v i t i e s i n Streptomycin-Mutants of E. c o l i Reductoisomerase a c t i v i t i e s i n c e l l extracts from streptomycin mutants of E. c o l l were determined e i t h e r with enzymatically synthesized Ot-acetolactate or with the chemically synthesized product as substrate. Table I I I shows the s p e c i f i c a c t i v i t i e s from extracts of f i v e s t r a i n s of E. c o l l . The main i n t e r e s t here i s - 48 -concerned with a comparison of a c t i v i t i e s between sensitive and dependent mutants of each s t r a i n . Each p a i r of dependent and sensitive mutants was grown and studied a t the same time under the same conditions i n order to f a c i l i t a t e d i r e c t comparison of the enzymatic a c t i v i t i e s i n these mutants. As seen i n Table I I I , there i s no appreciable difference i n the a c t i v i t i e s of reductoisomerase between the dependent and the sensi t i v e (wild type) mutants i n any of the f i v e s t r a i n s . Reductoisomerase i s thus not elevated i n the streptomycin-dependent mutants, unlike acetohydroxy a c i d synthetase which precedes reductoisomerase i n the biosynthetic sequence leading to the formation of v a l i n e . IV, Transaminase B A c t i v i t i e s i n Streptomycin-Resistant, -Sensitive, and -Dependent E. c o l l K-12 The a c t i v i t y of transaminase B i n streptomycin-dependent and streptomycin r e s i s t a n t E. c o l l K-12 was found to be 2 to 3 times higher than i n the sensitive mutants. Since threonine dehydratase was observed to be derepressed also (about 9-fold) i n the dependent K-12 as compared to both the sensitive and r e s i s t a n t mutants of t h i s s t r a i n (Desai and Polglase 1966), the r e s u l t obtained f o r transaminase B thus indicates that these two enzymes - 49 -are coordinately derepressed (although not to the same degree) i n streptomycin-dependent K-12. V. Interpretation of the Results This study establishes that reductoisomerase i s not derepressed. E a r l i e r observations (Coukell and Polglase, 1965) showed an elevation i n acetohydroxy a c i d synthetase i n streptomycin-dependent E. c o l l . Thus mutation to streptomycin-dependence a f f e c t s only the regulatory ( i . e . r a t e - l i m i t i n g ) enzyme of valine biosynthesis. Derepression of acetohydroxy acid synthetase i n the streptomycin mutants of E. c o l i B, A, Br and E can explain the observed over production of valin e , provided that the other enzymes of the pathway are not r a t e - l i m i t i n g ( i . e . are present i n excess). The s e l e c t i v e advantage to a streptomycin-dependent mutant of valin e excretion might be that t h i s compound i s a neutral end-product of pyruvate metabolism from glucose oxidation. Bragg and Polglase (1964a) had observed that i n the an t i b i o t i c - d e p l e t e d dependent c e l l s , excess pyruvic a c i d (or l a c t i c acid) accumulated during glucose oxidation while r e l a t i v e l y l i t t l e valine was found. When streptomycin was added to the growth medium of the depleted c e l l s , however, the s i t u a t i o n was reversed so that a large amount of valine - 50 -was excreted" with the concomitant decrease i n pyruvic a c i d . These observations indicate that the streptomycin-dependent c e l l s are not capable of regulating the oxidation of glucose to pyruvate. As a r e s u l t , the l a t t e r would accumulate when there i s an abundant supply of glucose. Streptomycin, by derepressing acetohydroxy a c i d synthetase i n the dependent mutants, permits the d i s s i m i l a t i o n of excess pyruvate v i a valine formation. A question concerning the nature of derepression might be r a i s e d with regard to the magnitude of elevation of acetohydroxy ac i d synthetase observed i n these dependent mutants. A M t r u e w genetic derepression, according to the "Jacob-Monod" operon concept can be pictured as an i n a c t i v a t l o n of the regulatory gene or i t s product by a corepressor so that no repressor (or a non-functional repressor) i s produced. As a r e s u l t the operator can function maximally i n d i r e c t i n g messenger synthesis, leading to an increased production of protein (enzyme i n t h i s case). Genetic derepression of acetohydroxy a c i d synthetase resulted i n a 20 f o l d elevation of t h i s enzyme, which was observed by Ramakrishnan and Adelberg ( 1 9 6 5 a ) . In the case of acetohydroxy ac i d synthetase, however, an elevation of only 2 to 6 f o l d was observed by Coukell and Polglase ( 1 9 6 5 ) . These investigators postulated that - 51 - i. the enzyme was induced by pyruvate to provide an alternate pathway of catabolism. From these observations, i t may be suggested that the r o l e of streptomycin as an enzyme derepressor r e s t s on i t s a b i l i t y to a f f e c t the streptomycin-dependent E. c o l l i n a manner such that the metabolite, pyruvate, i s able to act as an inducer of acetohydroxy a c i d synthetase. Escherichia c o l l K-12 requires a spe c i a l discussion because i t has properties d i f f e r e n t from other s t r a i n s . Streptomycin-dependent K-12 was found to be valine r e s i s t a n t (Polglase, 1965)» whereas the growth of the streptomycin-s e n s i t i v e (wild-type) s t r a i n was i n h i b i t e d by valine and t h i s i n h i b i t i o n could be reversed by isoleucine (Bonner, 1 9 4 6 ) . Studies on enzyme a c t i v i t i e s by Desal and Polglase (1966) indicated that there was a 9 f o l d or higher de-repression of threonine dehydratase a c t i v i t y i n the dependent mutants of K-12 as compared to the sensitive parent. However, when the s p e c i f i c a c t i v i t i e s of threonine dehydratase a c t i v i t i e s of several s t r a i n s of E. c o l i were compared, i t was found that the a c t i v i t y from dependent K-12 was s i m i l a r to that of dependent-mutants from other s t r a i n s . I t became evident therefore that t h i s high r a t i o of threonine dehydratase a c t i v i t y was due to the unusually low l e v e l of threonine dehydratase i n the wild-type s t r a i n rather than to a high - 52 -l e v e l of t h i s enzyme i n the dependent mutant. Since mutants of K-12 having derepressed threonine dehydratase alone exhibited a high l e v e l of valine resistance (Ramakrishnan and Adelberg, 1965b), i t became probable that threonine dehydratase i s the key enzyme i n determining the s e n s i t i v i t y to v a l i n e . Only by derepressing threonine dehydratase can s u f f i c i e n t isoleuclne be made to overcome the i n h i b i t i o n of t h i s s t r a i n by v a l i n e . The extreme s e n s i t i v i t y to valine of sen s i t i v e K-12 can thus be explained i n terms of the low l e v e l of threonine dehydratase i n t h i s s t r a i n . The discovery by Ramakrishnan and Adelberg (1965b) that the three s t r u c t u r a l genes c o n t r o l l i n g threonine dehydratase, dehydrase and transaminase B are i n the same operon provided a c r i t i c a l c r i t e r i o n i n determining the r o l e of streptomycin at the l e v e l of expression of the regulatory genes as follows: a mutant having derepressed threonine dehydratase should show coordinate derepression of the other two enzymes i n t h i s operon, the dehydrase (see Figure 2) and transaminase B. The r e s u l t from t h i s study indicates that threonine dehydratase and transaminase B are coordinately derepressed i n E. c o l l K-12. However, since the degree of derepression - 53 -of transaminase B (about 2 to 3 fold) was much l e s s than that of threonine dehydratase (about 9 f o l d ) , i t i s evident therefore that these two enzymes were not derepressed to the same degree. Contrary to what would be expected of a derepression from a non-functional gene product (based on the operon model of Jacob and Monod, 1961), the r e s u l t obtained from t h i s study shows a derepression where two enzymes (threonine dehydratase and transaminase B) i n the same operon were not "coordinately" derepressed to the same degree. - 54 -E. BIBLIOGRAPHY Altman, S.M., Crook, E.M., and Datta, S.P., Biochem. J . , 4£ I X i i i ( 195D. Bonner, 0., J . B i o l . Chem., 166 545 (1946). Bragg, P.D., and Polglase, W.J., J . Bact., 84 370 (1962). Bragg, P.D., and Polglase, W.J., J . Bact., 88 1006 (1964a). Bragg, P.D., and Polglase, W.J., J . Bact., 88 1399 (1964b). Bragg, P.D., and Polglase, W.J., J . Bact., 8£ 1158 (1965). C a v a l l i n i , D., and P r o n t a l i , N., Biochem. Biophys. Acta, H 439 (195*0. Coukell, M.B., and Polglase, W.J., Can. J . Microbiol., 11 905 (1965). Cruickshank, D.H., and Isherwood, F.A., Nature, 173 121 (1954). 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Biochemistry and Molecular Biology
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Enzyme activities of the isoleucine-valine biosynthetic pathway in streptomycin mutants of Escherichia coli
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