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Enzyme activities of the isoleucine-valine biosynthetic pathway in streptomycin mutants of Escherichia… Lau, D.C.C. 1966

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ENZYME ACTIVITIES OF THE ISOLEUCINE-VALINE BIOSYNTHETIC PATHWAY IN STREPTOMYCIN MUTANTS OF ESCHERICHIA COLI by D.C.C. LAU B. Sc., U n i v e r s i t y o f 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 t h e s i s as conforming t o the r e q u i r e d standard f o r the degree o f MASTER OF SCIENCE.  The U n i v e r s i t y o f B r i t i s h April, 1966.  Columbia  In presenting  this thesis i n p a r t i a l fulfilment of  the requirements for an advanced degree at the University of  ' •  B r i t i s h Columbia, I agree that the Library shall make i t f r e e l y 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  his representatives.  by.  I t i s understood that; copying or publi-  cation of this thesis for f i n a n c i a l gain shall: not be allowed without my written permission.  ;- *;  Department of The University of B r i t i s h Columbia,: Vancouver 8, Canada. j " Date  $  ,  :  ,%-v,x- .-/, ;  •. '  - 1 ABSTRACT The compound oU-acetolactate has been prepared by the chemical method of Krampltz procedure.  (19^8) and by an enzymatic  The products o b t a i n e d by each method were  c h a r a c t e r i z e d c h r o m a t o g r a p h i c a l l y and s p e c t r o p h o t o m e t r i c a l l y by c o n v e r s i o n t o t h e i r 2 , 4 — d i n i t r o p h e n y l h y d r a z o n e as w e l l as by c o n v e r s i o n t o a c e t o i n .  derivatives  The I d e n t i t y o f the  hydrazone o f e n z y m a t i c a l l y prepared OC-acetolactate w i t h hydrazones  o f the chemical product was e s t a b l i s h e d by  comparison  o f Ep v a l u e s , the i d e n t i c a l a b s o r p t i o n maxima,  and by the i n f r a r e d s p e c t r a o f these compounds. The a - a c e t o l a c t a t e so o b t a i n e d was used as s u b s t r a t e i n a comparison  o f the a c t i v i t y i n streptomycin mutants o f  the reductoisomerase  enzyme which c a t a l y z e s the rearrangement  and r e d u c t i o n o f a - a c e t o l a c t a t e to acid.  T h i s r e a c t i o n i s the second Streptomycin-dependent  at,^-dihydroxyisovaleric step i n v a l i n e b i o s y n t h e s i s .  mutants o f E s c h e r i c h i a  coll  p r e v i o u s l y has been shown t o be d e r e p r e s s e d i n acetohydroxy a c i d s y n t h e t a s e , the enzyme which i n i t i a t e s v a l i n e b i o s y n t h e s i s . I n a d d i t i o n , i t had been r e p o r t e d p r e v i o u s l y 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 b i o s y n t h e s i s o f i s o l e u c i n e , i s derepressed.  also  I n c o n t r a s t , reductoisomerase, which i s  common t o both the v a l i n e and i s o l e u c i n e pathway, has been found i n t h i s work to be normal ( i . e . , not derepressed) i n  - i istreptomycin mutants. An a d d i t i o n a l enzyme o f the common pathway, transaminase  B, was  found to be about 2 to 3 times h i g h e r  i n streptomycin-dependent resistant strains.  mutants than i n s e n s i t i v e or  The s t r u c t u r a l genes f o r both  aminase B and t h r e o n i n e dehydratase  trans-  of E. c o l l K-12 have  been shown by g e n e t i c s t u d i e s (Ramakrishnan and A d e l b e r g , 1965b)  t o be c o o r d i n a t e l y r e g u l a t e d ( i . e . , on the same  operon).  The o b s e r v a t i o n s made i n t h i s study w i t h s t r e p t o m y c i n -  dependent E . c o l i K-12 workers.  support the o b s e r v a t i o n s of these  That i s , transaminase  B of  streptomycin-dependent  E . c o l i K-12 i s derepressed c o o r d i n a t e l y w i t h t h r e o n i n e dehydratase.  However, the degree of d e r e p r e s s i o n of t r a n s -  aminase B (about 2 t o 3 f o l d ) was t h r e o n i n e dehydratase Polglase, I 9 6 6 ) .  much l e s s than t h a t of  (about 9 f o l d , a c c o r d i n g t o D e s a i  I t may  and  be concluded from these s t u d i e s t h a t  the type of d e r e p r e s s i o n of c e r t a i n enzymes which has been observed i n streptomycin-dependent  E . c o l l has  contrasting  f e a t u r e s t o the type o f d e r e p r e s s i o n which would be  expected  on the b a s i s of the Jacob and Monod model ( I 9 6 I ) from a nonf u n c t i o n a l r e g u l a t o r y gene o r product t h e r e o f .  iii  -  ACKNOWLEDGMENT  The author wishes t o express h i s s i n c e r e thanks and deep a p p r e c i a t i o n t o Dr. W.J. P o l g l a s e f o r h i s guidance and encouragement d u r i n g the course o f t h i s  research.  - iv-  TABLE OP CONTENTS PAGE A.  INTRODUCTION I.  II.  The M e t a b o l i c Role o f Ct-Acetolactate  1  (a)  Formation o f a c e t o i n  1  (b)  Formation o f L - v a l i n e  1  I n t e r r e l a t i o n s h i p s Between L - v a l i n e and L-Isoleucine  Biosynthesis  (a)  Enzymes shared by the two pathways  (b)  Feedback  3 3  i n h i b i t i o n o f acetohydroxy  a c i d synthetase by v a l i n e i n E. c o l l K-12 (c)  3  R e l a t i o n between t h r e o n i n e dehydratase a c t i v i t y and i n h i b i t i o n o f E . c o l l K-12 by v a l i n e  III.  5  E f f e c t o f Streptomycin on the I s o l e u c i n e V a l i n e B i o s y n t h e t i c Pathway i n Streptomycin-Mutants o f E . c o l l  6  IV.  I s o l e u c i n e - V a l i n e Operon  8  V.  Reductoisomerase  8  VI.  Transaminase B  10  -  V  -  PAGE B.  MATERIALS AND I.  II.  METHODS  S y n t h e s i s of OC-Acetolaotate  12  (a)  Chemical method  12  (b)  Enzymatic method  15  C o l o r l m e t r l c Determination  of A c e t o i n 16  and a - A c e t o l a c t a t e  III.  (a)  Acetoin  16  (b)  Oc-Acetolactate  17  C h a r a c t e r i z a t i o n of a - A c e t o l a c t a t e (a)  18  P r e p a r a t i o n of a c e t o l a c t a t e 2,4-dinitrophenylhydrazone  (b)  ........  I d e n t i f i c a t i o n o f hydrazones by paper chromatography  (c) IV.  18  18  U l t r a v i o l e t and i n f r a r e d spectroscopy.  18  C u l t u r e s and C e l l E x t r a c t s  19  (a)  Summary o f E . c o l l  19  (b)  P r e p a r a t i o n o f media  (c)  Growth o f c u l t u r e s  (d)  P r e p a r a t i o n of c e l l e x t r a c t s  s t r a i n s used  21 21  • ........  21  of P r o t e i n s ....  22  V.  C o l o r l m e t r l c Determination  VI.  Enzyme Assays  23  (a)  Reductoisomerase  23  (b)  Transaminase B  - vi -  PAGE C.  RESULTS Ii  II.  Synthesis of a-Acetolactate  26  (a)  Chemical s y n t h e s i s  26  (b)  Enzymatic s y n t h e s i s  26  C o l o r l m e t r l c Determination  of Acetoin  and a - A c e t o l a c t a t e III.  S t a b i l i t y of a-Acetolactate  IV.  Characterization of a-Acetolactate  V.  Enzyme Assays  30  (a)  Reductoisomerase  30  (b)  Transaminase B  36  VI.  D.  27  C o l o r i m e t r i c Determination  27 ........  of Proteins  30  36  DISCUSSION I.  R e l a t i o n s h i p Between Streptomycin-Dependency and  the E l e v a t i o n o f Acetohydroxy A c i d  Synthetase II.  40  S y n t h e s i s and C h a r a c t e r i z a t i o n o f a-Acetolactate  42  (a) G e n e r a l p r o p e r t i e s o f k e t o - a c i d 2,4-dinitrophenylhydrazones  42  (b) Hydrazones o f a - A c e t o l a c t a t e  44  - vii-  PAGE I I I . Reductoisomerase A c t i v i t i e s i n Streptomycin-Mutants o f E . p o l l IV.  Transaminase  B Activities i n  Streptomycin-Resistant,  V.  E.  -Sensitive,  and -Dependent E . c o l l K-12  48  I n t e r p r e t a t i o n o f the R e s u l t s  49  BIBLIOGRAPHY  5k  - viii  -  LIST OP FIGURES FIGURE 1.  PAGE Intermediates and enzymes i n the b i o s y n t h e s i s o f i s o l e u c i n e and v a l i n e  2.  4  Sequence of genes c o n t r o l l i n g the f o r m a t i o n of enzymes i n the i s o l e u c i n e - v a l i n e b l o s y n t h e t i c pathway  9  3.  Enzymie s y n t h e s i s of a - a c e t o l a c t a t e  4.  Standard  5.  S t a b i l i t y of chemical a - a c e t o l a c t a t e  31  6.  Standard  39  7(a).  Infrared spectra of a-acetolactate  curve o f a c e t o i n ( a c e t y l m e t h y l c a r b i n o l )  curve o f p r o t e i n  2,4-dlnitrophenylhydrazone 7(b).  (Spot 1 )  (Spot 2 )  34  35  C i s - t r a n s g e o m e t r i c a l isomers o f  ............  45  ^acetolactic  a c i d 2,4-dinitrophenylhydrazone 8(c).  33  C l s - t r a n s g e o m e t r i c a l isomers of p y r u v i c a c i d 2,4-dinitrophenylhydrazone  8(b).  29  Infrared spectra of acetoin 2 , 4 - d i n i t r ophenylhydrazone  8(a).  28  Infrared spectra of a-acetolactate 2,4-dinltrophenylhydrazone  7(c).  ....  45  C i s - i s o m e r of p y r u v i c a c i d 2 , 4 - d i n i t r o p h e n y l hydrazone, showing s t a b i l i z e d e x c i t e d s t a t e 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  - ix -  LIST OP TABLES TABLE I.  PAGE R  F  V a l u e s of 2 , 4 - D i n i t r o p h e n y l h y d r a z o n e Derivatives  II.  32  A b s o r p t i o n Maxima and Minima of 2,4-Dinitrophenylhydrazone D e r i v a t i v e s . . . .  III.  Reductoisomerase A c t i v i t i e s Using E i t h e r Chemical  or Enzymatic  GK-Acetolactate  as S u b s t r a t e IV.  32  Transaminase B A c t i v i t i e s i n E x t r a c t s  37 of  S t r e p t o m y c i n - R e s i s t a n t , -Dependent and - S e n s i t i v e E. c o l l K-12  38  -  A.  I.  The M e t a b o l i c (a) The  has  1 -  INTRODUCTION  Role o f  tt-Acetolaotate  Formation o f a c e t o i n formation  of acetoin  (acetylmethylcarbinol)  been observed i n a v a r i e t y o f b i o l o g i c a l  systems:  i n y e a s t , by Neuberg and Simon ( 1 9 2 5 ) , i n p l a n t s , by Tomiyasu ( 1 9 3 7 ) * and i n A e r o b a c t e r aerogenes by Silverman and  Werkman ( 1 9 4 1 ) .  Studies  on p y r u v i c a c i d metabolism  o f A. aerogenes l e d Watt and Krampitz ( 1 9 4 7 ) t o suggest oC-acetolactate acetoin.  as an i n t e r m e d i a t e  i n the formation o f  I n 1 9 4 8 , Krampitz f i r s t p o s t u l a t e d a condensation  r e a c t i o n i n v o l v i n g acetaldehyde and p y r u v i c a c i d t o form ot-acetolactate which upon d e c a r b o x y l a t i o n By  the e x c e l l e n t work o f J u n i  J u n i and Heym ( 1 9 5 6 )  (1952),  i t was e s t a b l i s h e d t h a t oC-acetolactate an i n t e r m e d i a t e  i n a c e t o i n formation  yielded acetoin.  played  the r o l e o f  both i n a c e t o i n -  p r o d u c i n g b a c t e r i a (A. aerogenes) and i n pigeon b r e a s t muscle. (b)  Formation o f L - v a l i n e  On the b a s i s o f i s o t o p i c s t u d i e s by Strassman e t a l .  - 2 -  (1953) a c e t o l a c t a t e was then suggested as an Intermediate i n the b i o s y n t h e s i s o f L - v a l i n e i n T o r u l o p s l s u t l l l s . These authors proposed a scheme i n v o l v i n g a keto condensation of pyruvate with acetaldehyde t o y i e l d  '  t X - a c e t o l a c t a t e , f o l l o w e d by an I n t r a m o l e c u l a r m i g r a t i o n of the oc-methyl group t o the /B-carbon  t o y i e l d #-keto-/3-  - h y d r o x y i s o v a l e r a t e , and u l t i m a t e l y  rt-ketoisovalerate,  the keto a n a l o g of v a l i n e . S t u d i e s on E s c h e r i c h i a c o l l by Umbarger e t a l .  (1957) (1958) r e s u l t e d i n t h r e e o b s e r v a t i o n s which p r o v i d e d s t r o n g evidence t h a t «-acetolactate was  Indeed  5- carbon p r e c u r s o r of v a l i n e i n t h i s organism. oC-acetolactate was  accumulated  the e a r l i e s t One,  by a v a l i n e - r e q u i r i n g mutant  of E . c o l l when s u p p l i e d w i t h l i m i t e d amounts o f v a l i n e . Two,  the end product v a l i n e e x e r t e d feedback  the a c e t o l a c t a t e forming system which was organism.  control  over  present i n t h i s  Three, cC-acetolaotate l a b e l e d i n the a-methyl  p o s i t i o n was  I n c o r p o r a t e d i n t o v a l i n e and i n t o i t s p r e c u r s o r  of-ketolsovalerate. The  f u r t h e r c o n v e r s i o n of ot-acetolactate t o  06- k e t o i s o v a l e r a t e by e x t r a c t s o f bakers* y e a s t et  a l . , 1958), and t o  of E . c o l l  tf,£-dihydroxyisovalerate  (Umbarger and Brown, 1958), and of  c r a s s a (Wagner e t a l . , 1958) precursor.  (Strassman by  extracts  Neurospora  confirmed i t s r o l e as a v a l i n e  - 3 -  From the subsequent work o f Umbarger e t a l . ( i 9 6 0 ) and  Strassman e t a l . ( i 9 6 0 ) the b i o s y n t h e t i c pathway of  L - v a l i n e was e s t a b l i s h e d .  I t i n v o l v e d an i n i t i a l conden-  s a t i o n o f two moles o f pyruvate to form o t - a c e t o l a c t a t e , f o l l o w e d by the c o n v e r s i o n  o f a c e t o l a c t a t e t o ot,p-dihydroxy-  i s o v a l e r a t e , and then a r e d u c t i o n of the l a t t e r t o o t - k e t o i s o v a l e r a t e , the k e t o a n a l o g o f v a l i n e . shows the i n t e r m e d i a t e s  Figure I  and enzymes i n the b i o s y n t h e s i s o f  i s o l e u c i n e and v a l i n e . II.  I n t e r r e l a t i o n s h i p s Between L - V a l l n e 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 F i g u r e  1, the b i o s y n t h e t i c  pathways l e a d i n g t o v a l i n e and i s o l e u c i n e share the same s e t o f enzymes, the o n l y d i f f e r e n c e being pathway has an e x t r a enzyme, threonine regulates isoleucine biosynthesis.  t h a t the i s o l e u c i n e  dehydratase, which  Since acetohydroxy a c i d  synthetase i s shared by both b i o s y n t h e t i c r o u t e s and, a t the same time, i s s u b j e c t t o i n h i b i t i o n by v a l i n e , i t i s apparent t h a t v a l i n e may a f f e c t I s o l e u c i n e (b)  biosynthesis.  Feedback i n h i b i t i o n of acetohydroxy a c i d synthetase by v a l i n e i n E . c o l i K-12  - 4-  leucine  ft-acetolactate  2 pyruvate  ACETOHYDROXY ACID SYNTHETASE  a,/?-dihydroxy^"isovalerate  I  REDUCTOISOMERASE  {  tt-keto isovalerate"  DEHYDRASE  flt-acetoi 0f» /?-dihydroxy"-*"/£-methyl~ -hydroxyv a l e r a t e butyrate pyruvate  Ct-ketobutyrate  tf-keto^-methyl valerate  THREONINE DEHYDRATASE  threonine  FIG. 1.  pantoic acid  Intermediates and enzymes i n the b i o s y n t h e s i s o f i s o l e u c l n e and v a l i n e  •> v a l i n e  TRANSAMINASE B  | isoleucine  - 5 -  I t had been r e p o r t e d by Bonner (1946) t h a t the growth of a w i l d - t y p e s t r a i n o f E . c o l l K-12 medium was was  on minimal  i n h i b i t e d by L - v a l i n e and t h a t t h i s  overcome by L - i s o l e u c i n e .  inhibition  Work by Umbarger and  A d e l b e r g (1953)» l a t e r confirmed by Temple e t a l . (1965), r e v e a l e d t h a t v a l i n e e x e r t s an i n h i b i t o r y e f f e c t on the growth o f 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 o f  acetohydroxy a c i d s y n t h e t a s e , and thus p r e v e n t i n g the f o r m a t i o n o f acetohydroxybutyrate and  isoleucine,  acetohydroxy a c i d synthetase i s shared by both the v a l i n e and i s o l e u c i n e b i o s y n t h e t i c pathways ( F i g u r e 1 ) . K-12,  In  the extreme s e n s i t i v i t y o f t h i s enzyme t o end-product  i n h i b i t i o n by v a l i n e causes s t a r v a t i o n f o r i s o l e u c i n e when t h i s organism i s grown on g l u c o s e - s a l t s medium  supplemented  with L-valine only. (c)  R e l a t i o n between t h r e o n i n e dehydratase a c t i v i t y and i n h i b i t i o n o f E . c o l i K-12 by v a l i n e  A second and perhaps a much more s i g n i f i c a n t of v a l i n e i n h i b i t i o n o f the growth of E . c o l i K-12  cause i s the  low l e v e l i n t h i s s t r a i n o f t h r e o n i n e dehydratase, the  first  enzyme i n the i s o l e u c i n e pathway (Desai, u n p u b l i s h e d observations).  I t was  found by Ramakrishnan and A d e l b e r g  (1965a) t h a t E . c o l l K-12  mutants having d e r e p r e s s e d —2  t h r e o n i n e dehydratase were r e s i s t a n t to a h i g h l e v e l  (10  M)  of v a l i n e , w h i l e mutants d e r e p r e s s e d i n any one o f the o t h e r enzymes i n the pathway except acetohydroxy a c i d s y n t h e t a s e and t h r e o n i n e dehydratase were a l l v a l i n e sensitive.  Mutants  o f 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 levels  (10  M) o f v a l i n e .  The s e n s i t i v i t y o f t h r e o n i n e  dehydratase to feedback i n h i b i t i o n by i s o l e u c i n e and o f acetohydroxy a c i d synthetase t o feedback i n h i b i t i o n by v a l i n e was not a f f e c t e d i n such  mutants.  These o b s e r v a t i o n s thus suggest t h a t the extreme s e n s i t i v i t y o f E. c o l l K-12 the  t o v a l i n e r e s u l t s both from  e f f i c i e n c y w i t h which v a l i n e r e p r e s s e s acetohydroxy  a c i d synthetase and from the low l e v e l o f t h r e o n i n e dehydratase i n t h i s III.  E f f e c t o f Streptomycin on the I s o l e u c l n e - V a l l n e B i o s y n t h e t i c Pathway I n Streptomycin-mutants of E . c o l i . I t was  et  strain.  i n d e p e n d e n t l y d i s c o v e r e d by T i r u n a r a y a n a n  a l . (1962) and by Bragg and P o l g l a s e (1962) t h a t  streptomycin-dependent E . c o l l e x c r e t e d r e l a t i v e l y  large  amounts o f L - v a l i n e d u r i n g growth on g l u c o s e - s a l t s medium. The s t r e p t o m y c i n - r e s i s t a n t ( i n d i f f e r e n t ) E . c o l l on the o t h e r hand produced l a c t i c a c i d from g l u c o s e when grown i n medium c o n t a i n i n g a n t i b i o t i c , but resembled  the  s t r e p t o m y c i n - s e n s i t i v e E . c o l i when grown i n the absence  -  7  -  o f a n t i b i o t i c i n t h a t they both f a i l e d t o produce acid.  lactic  F u r t h e r o b s e r v a t i o n s on the f o r m a t i o n o f L - v a l i n e  by streptomycin-dependent E . c o l i  (Bragg and P o l g l a s e  1964a) has shown t h a t l a c t i c a c i d p r o d u c t i o n was a p r o p e r t y o f a n t i b i o t i c d e p l e t i o n o r oxygen d e p r i v a t i o n , whereas v a l i n e p r o d u c t i o n ( a c c o u n t i n g f o r over 10% o f the g l u c o s e carbon) was a p r o p e r t y o f streptomycin-supplemented grown under a e r o b i c c o n d i t i o n s .  cells  These r e s u l t s suggested  t h a t v a l i n e and l a c t i c a c i d a r e a l t e r n a t e secondary products o f g l u c o s e metabolism i n streptomycin-dependent E . c o l l the primary p r o d u c t s b e i n g C 0  2  and a c e t a t e .  The f o r m a t i o n  o f v a l i n e i n the c a t a b o l i s m o f g l u c o s e thus appeared t o be a unique p r o p e r t y o f streptomycin-dependent E . c o l l . L a t e r s t u d i e s by Bragg and P o l g l a s e (1965) and C o u k e l l and P o l g l a s e (1965) on enzyme a c t i v i t i e s o f s e v e r a l s t r a i n s o f E . c o l i has r e v e a l e d t h a t the v a l i n e e x c r e t i o n  correlated  w i t h an e l e v a t e d acetohydroxy a c i d synthetase a c t i v i t y and t h a t one a c t i o n of the a n t i b i o t i c was t o d e r e p r e s s the r e g u l a t o r y enzyme acetohydroxy a c i d synthetase i n the v a l i n e b i o s y n t h e t i c pathway o f streptomycin-dependent  mutants.  I n l i g h t o f these f i n d i n g s t o g e t h e r w i t h e a r l i e r observations that streptomycin i s required f o r P-galactosidase i n d u c t i o n i n dependent E . c o l i  ( P e r e t z and P o l g l a s e 1956),  an a c t i o n o f the a n t i b i o t i c a t the l e v e l o f e x p r e s s i o n o f  - 8 -  r e g u l a t o r y genes was  suggested.  I t was  of i n t e r e s t  t h e r e f o r e t o study what e f f e c t s t r e p t o m y c i n had on the a c t i v i t y of the remaining enzymes i n the v a l i n e b i o s y n t h e t i c pathway, some o f which a r e c o n t r o l l e d by the same operon. IV.  I s o l e u o l n e - V a l l n e Operon Ramakrishnan and A d e l b e r g ( 1 9 6 4 ) ( 1 9 6 5 b ) observed  t h a t the enzymes of i s o l e u c l n e and v a l i n e b i o s y n t h e s i s ( i l v ) enzymes were c o n t r o l l e d by a c l u s t e r of f i v e M  w  s t r u c t u r a l genes comprising t h r e e d i s t i n c t operons i n E s c h e r i c h i a c o l i K-12.  I t was  f u r t h e r observed  that  o p e r a t o r A c o n t r o l l e d the f o r m a t i o n o f t h r e e o f the f i v e enzymes: B.  t h r e o n i n e dehydratase, dehydrase, and  transaminase  Operator B c o n t r o l l e d the f o r m a t i o n of acetohydroxy  a c i d synthetase, and a t h i r d o p e r a t o r l o c u s (operon I I ) r e g u l a t e d the reductoisomerase f o r m a t i o n , as shown i n Figure 2.  The primary i n t e r e s t o f t h i s study has been w i t h  the l a t t e r enzyme, reductoisomerase. V.  Reductoisomerase Reductoisomerase  c a t a l y z e s the NADPH r e q u i r i n g  r e d u c t i o n r e a c t i o n I n v o l v i n g a two-step  c o n v e r s i o n of  cc-acetolactate to ct,ji-dihydroxyisovalerate. of t h i s r e a c t i o n was  The mechanism  e s t a b l i s h e d by the work of  Strassman  -9-  OPERON  II  GENE SEQUENCE:  opr B i i l v B — ; 1  \  III  ilvC ;  1  , \  / \  /  TP _ **  t "  • i  i  '  >  /  r t  f  V  \ AHAS^ *"  1  /  \  <*  ENZYME SEQUENCE:  1  1  ' i v  \  /  opr A i i l v A i i l v D i i l v 7 ; —  1  RI^ *"  DH ^ *"  t  f  TRB ^ **"  F i g . 2 . Sequence of genes c o n t r o l l i n g the formation of enzymes i n the i s o l e u c i n e - v a l i n e b i o s y n t h e t i c pathway. TD, threonine dehydratase; AHAS, acetohydroxy a c i d synthetase; R l , reductoisomerase; DH, dehydrase; TRB, transaminase B.  - 10 -  e t a l . ( i 9 6 0 ) , Umbarger e t a l . ( i 9 6 0 ) , and Radhakrishnan and S n e l l  (i960).  An unusual p i n a c o l - p i n a c o l o n e r e a r r a n g e -  ment i s f o l l o w e d by r e d u c t i o n , both r e a c t i o n s b e i n g c a t a l y z e d by a s i n g l e enzyme.  CH0-8-COH-GOOH CH  * CHo-COH-C-COOH J  3  3  Ot-acetolactate  NADPH + H ^ :  +  C H  0  «-ke to-^-hydroxyisovalerate  NADP ^  +  y  CH,-C0H-CH©H-C00H  tf,p-dihydroxyisovalerate In  o r d e r t o study t h i s r e a c t i o n i n s t r e p t o m y c i n  mutants o f E . c o l i ,  i t was n e c e s s a r y t o s y n t h e s i z e and t o  c h a r a c t e r i z e the s u b s t r a t e  rt-acetolactate.  T h i s was done  both by chemical method o f Krampitz and by an enzymatic method which u t i l i z e d the h i g h enzymatic a c t i v i t y o f streptomycin-dependent E . c o l i VI.  Transaminase  extracts.  B  Formation o f v a l i n e from c a t a l y z e d by transaminase B. Adelberg are  rt-ketoisovalerate  is  A c c o r d i n g t o Ramakrishnan and  (1965b), both transaminase B and t h r e o n i n e dehydratase  c o n t r o l l e d by the same operon.  I n streptomycin-dependent  - l l -  El. c o l i K-12,  threonine  dehydratase i s derepressed  p r o p o r t i o n to a n t i b i o t i c c o n c e n t r a t i o n 1966).  Transaminase B should a l s o be derepressed  streptomycin-dependent E. c o l l K-12 by i n t e r f e r i n g with  the f o r m a t i o n  i s o l e u c i n e - v a l i n e pathway has streptomycin-sensitive,  of E s c h e r i c h i a c o l i  K-12.  Polglase in  i f the a n t i b i o t i c or f u n c t i o n o f  r e p r e s s o r o f operator A ( F i g u r e 2 ) .  in  (Desai and  in  acts  the  T h i s enzyme of  the  t h e r e f o r e been i n v e s t i g a t e d  - r e s i s t a n t and  -dependent mutants  -  B.  I.  MATERIALS and METHODS  Synthesis of cUAcetolactate The  two  12 -  s y n t h e s i s o f ot-acetolaetate was achieved i n  ways: (a)  Chemical method  In t h i s s y n t h e s i s , o x i d a t i o n o f the l a b i l e hydrogen of m e t h y l - s u b s t i t u t e d  a c e t o a c e t i c e s t e r was accomplished  w i t h l e a d t e t r a a c e t a t e , with subsequent h y d r o l y s i s o f the acetoxy e s t e r t o g i v e Ot-acetolactate.  The o v e r a l l  synthesis  i n v o l v e d three major s t e p s : (i)  The m e t h y l - s u b s t i t u t e d  a c e t o a c e t i c e s t e r was  i n the manner g i v e n by Gilman and B l a t t  CHo-C-CHo-fi-OCH,, + 2 5 i A  ethyl  CH I 3 0  +  C H.0Na o  2"5  prepared  (194J).  »- CH-5-C-CH-C-0C H o  acetoacetate  t h y l oC-methylacetoacetate +  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) o f m e t a l l i c sodium was added i n small p i e c e s absolute  ethanol  under anhydrous c o n d i t i o n s .  t o 170 ml. o f After a l l  the sodium had d i s s o l v e d , 44 ml. ( 0 . 3 3 moles) o f e t h y l a c e t o a c e t a t e was added and the s o l u t i o n heated t o g e n t l e boiling.  The s t i r r e r was s t a r t e d and 23 ml. ( 0 . 3 6 moles)  o f methyl i o d i d e was added over a p e r i o d o f 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  o f the s o l u t i o n was n e u t r a l to moist l i t m u s paper (about 5 hours).  When the r e a c t i o n was complete, the mixture was  c o o l e d and t h e s o l u t i o n decanted from the sodium i o d i d e . The  s a l t was washed 4 times w i t h 10 ml. a b s o l u t e  and  the washings were added t o the main s o l u t i o n .  a l c o h o l was separated from the s u b s t i t u t e d e s t e r by steam d i s t i l l a t i o n .  alcohol  acetoacetic  The crude r e s i d u e  after  removal o f t h e a l c o h o l was used f o r t h e o x i d a t i o n that  followed.  (ii)  The o x i d a t i o n o f the m e t h y l - s u b s t i t u t e d e s t e r was performed a c c o r d i n g  step  acetoacetic  t o the method o f  (1948).  Krampitz  0 0 Pb(0Ac) 2 CH~-C-CH-C-0C H= CH o  3  The  2  5  3  ethyl flC-methylacetoacetate  0~C—CH 0 j 0 CH -C-C-C-0C..H. + CH 3  4  Q  3  2  5  3  ethyl rf-methylot-acetoxy-acetoacetate  Pb(0Ac)_ 2  - 14 -  Procedure: With v i g o r o u s mechanical  s t i r r i n g under anhydrous  c o n d i t i o n s 52g. Ph(OAc)^ was added i n s m a l l p o r t i o n s t o a mixture o f 5&g. t h i o p h e n e - f r e e anhydrous benzene and (0.12 moles) m e t h y l - s u b s t i t u t e d e t h y l a c e t o a c e t a t e .  l?g.  D u r i n g the a d d i t i o n o f the Pb(OAc)^ the temperature was n o t a l l o w e d t o r i s e above 3 5 ° G , a f t e r which the mixture was  heated t o 40°C f o r 5 hours w i t h v i g o r o u s s t i r r i n g .  mixture was a l l o w e d t o stand o v e r n i g h t a t room  The  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 w i t h 20 ml. p o r t i o n s o f benzene. original solution.  The washings were combined w i t h the To remove any a c e t i c a c i d p r e s e n t i n  the benzene, i t was washed w i t h 20 ml. p o r t i o n s o f water until  the water was n e u t r a l t o bromothymal b l u e .  The  benzene l a y e r was d r i e d 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 p r e s s u r e . ester, was d i s t i l l e d a t a p r e s s u r e o f about was  The remaining  16 mm.  A fraction  o b t a i n e d a t 100-105 C which c o n t a i n e d the methyl-acetoxyG  substituted ester.  C h a r a c t e r i z a t i o n o f the methyl-acetoxy-  s u b s t l t u t e d e s t e r was performed (iii)  on t h i s  fraction.  Free ct-acetolactate was l i b e r a t e d by h y d r o l y s i s of  the methyl-acetoxyacetoacetate i n the f o l l o w i n g  manner:  -  15 -  p 0—(J—CH 0 I 0  2Na0H  3  CH-C-C-C-OC H CH  *  3  0 OH 0  > CHo-C-p-C-ONa J  e t h y l oC-methyla-acetoxy-acetoacetate  CH^  sodium ^ - a c e t o l a c t a t e 0  + CHyS-ONa  +  CgH^OH  Procedure s To 10 ml. o f 0.1M NaOH, c h i l l e d i n an i c e bath, was added 0.1 ml. o f the methyl-acetoxy  ester.  The  mixture was shaken c o n s t a n t l y u n t i l a l l the e s t e r had been h y d r o l y z e d (about 30 min.).  The r e s u l t i n g  solution  c o n t a i n e d a mixture o f oC-acetolactate, a c e t a t e and e t h a n o l . (b) In  Enzymatic method. t h i s method,  tf-acetolactate  was prepared  e n z y m a t i c a l l y from sodium pyruvate u s i n g the a c e t o l a c t a t e forming system o f E . c o l i DK-12 (streptomycin-dependent).  CH -(J-C00Na  T  F  P  CH -CH-TPP  3  3  sodium pyruvate 0 CHjC-COONa sodium pyruvate  A  \ cetohyd?oxy ' a c i d synthetase  0 OH C^-C-6-COONa ^ C  H  Na c<-acetolactate  - 16 -  Procedure: A 25-ml. r e a c t i o n system, c o n t a i n i n g 2.5 moles o f potassium phosphate, pH8; (TPP);  25 umoles M g C l ; 2  t i d e (PAD);  4 umoles thymine pyrophosphate  250mumoles f l a v i n adenine d i n u c l e o -  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 The  stirrer.  r e a c t i o n was s t a r t e d by i n t r o d u c i n g i n t o the system a  d i a l y z l n g bag c o n t a i n i n g 20 ml. o f f r e s h l y prepared s o n i c e x t r a c t o f E . c o l l DK-12 ( I g . c e l l s p e r 15 ml. b u f f e r ) , f o l l o w e d by i n c u b a t i o n a t 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  o f a c e t o i n and  tf-acetolactate  c o l o r i m e t r i c a l l y by the method o f W e s t e r f e l d II.  were determined (1945).  C o l o r l m e t r l c D e t e r m i n a t i o n o f A c e t o i n and Pt-Acetolactate (a)  Acetoin  Acetoin  ( a c e t y l m e t h y l c a r b i n o l ) was determined  c o l o r i m e t r i c a l l y by the method o f Westfeld  (1945).  Procedure: To a 5 ml. sample o f a c e t o i n s o l u t i o n ( c o n c e n t r a t i o n not h i g h e r than 0.2 umoles/5 ml.)  1 ml. o f 0.5$ aqueous  s o l u t i o n o f c r e a t i n e was added and mixed.  Then 1 ml. o f  f r e s h l y prepared a l k a l i n e naphthol s o l u t i o n (5$ CK-naphthol  -  I n 2.5N  1?  NaOH) was added.  -  The c o l o r was  developed i n the  dark f o r one hour and the o p t i c a l d e n s i t i e s were r e a d a t 5^0  mu i n a Beckman model B spectrophotometer.  was  prepared s i m i l a r l y w i t h the e x c e p t i o n t h a t 5 ml.  o f water was  A "blank  s u b s t i t u t e d f o r the a c e t o i n s o l u t i o n .  A  s t a n d a r d curve of a c e t o i n w i t h a c o n c e n t r a t i o n range o f 0 to 0.2  umoles/5 ml. was  plotted against optical densities.  A c e t y l m e t h y l c a r b i n o l purchased Co." was  Chemical  used as the s t a n d a r d .  (b)  Ot>Acetolactate  To determine was  from the " A l d r i c h  the «-acetolactate c o n c e n t r a t i o n , i t  f i r s t converted to a c e t o i n by d e c a r b o x y l a t i o n .  0 OH CH^-C-jj-COONa  „+  Na a - a c e t o l a c t a t e  OH 0 >- CH^-CH-fi-Cl^ acetoin  Procedure: To a 5-ml. cone. H-SO. f o r 15  min.  was  sample of a - a c e t o l a c t a t e , one drop of  added and the s o l u t i o n i n c u b a t e d a t 60°C  The a c e t o i n i n the r e s u l t i n g s o l u t i o n  determined w i t h the procedure  given i n ( a ) .  was  - 18 -  III.  C h a r a c t e r i z a t i o n of (a)  flt-Aoetolactate  P r e p a r a t i o n 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 - d i n i t r o p h e n y l h y d r a z i n e i n 0.2N 5.4  HC1,0.3 ml.  (about  jimoles) of c(-acetolactate ( c h e m i c a l l y o r e n z y m a t i c a l l y  prepared) was  added.  The  s o l u t i o n was  shaken and the  r e a c t i o n a l l o w e d t o proceed a t 25°C f o r 30 min. then c e n t r i f u g e d and the c l e a r supernatant was  I t was examined  by paper chromatography, f o l l o w e d by e l u t i o n , and s p e c t r o photometric d e t e r m i n a t i o n s . (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 s p o t t e d on Whatman No.  1 paper and the chromatographs  (ascending i n a l l experiments) water-ethanol hydrazones  were  were developed i n n-butanol-  ( 5 : 4 : 1 ) f o r 15 hours.  The  v a l u e s o f the  o f b i o l o g i c a l cC-acetolactate were c a l c u l a t e d  and compared w i t h t h a t of the chemical p r o d u c t . (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 - d l n i t r o p h e n y l h y d r a z o n e  spot to be a n a l y z e d  - 19 -  was  cut from the chromatograms and the y e l l o w - c o l o r e d  hydrazone was For  extracted with a s u i t a b l e solvent.  u l t r a v i o l e t a n a l y s e s , the hydrazones were  e x t r a c t e d with 0.2M  sodium b i c a r b o n a t e and the s p e c t r a  were observed i n a Cary 15 wavelength For  range o f 250  spectrophotometer over a  t o 400  millimicrons.  i n f r a r e d a n a l y s e s , the hydrazones were e x t r a c t e d  w i t h a b s o l u t e e t h a n o l and the s p e c t r a were observed i n a P e r k i n - E l m e r 137 of  2.5  IV.  t o 14.5  range  microns.  C u l t u r e s and C e l l (a)  (I)  spectrophotometer over a wavelength  Extracts  F i v e s t r a i n s o f E. c o l l used I n t h i s work a r e summarized below.  E. c o l l A S t r a i n DA was a streptomycin-dependent  strain  which  has been d e p o s i t e d with the American Type C u l t u r e C o l l e c t i o n (ATCC 15745), and which was P a i n e , J r . by W.J.  o r i g i n a l l y o b t a i n e d from  Polglase.  S t r a i n s SA and HA a r e s t r e p t o m y c i n - s e n s i t i v e - r e s i s t a n t r e v e r t a n t s , r e s p e c t i v e l y , i s o l a t e d by mutation from streptomycin-dependent (II)  E. c o l l  T.F.  and  back-  DA.  B  S t r a i n SB was  o b t a i n e d as a w i l d type E. c o l i B from  - 20 -  the American Type C u l t u r e C o l l e c t i o n . S t r a i n DB and RB a r e 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 l a b o r a t o r y  from  s e n s i t i v e E . c o l l SB. (iii)  E . c o l i Br S t r a i n SBr was o b t a i n e d as a  streptomycin-sensitive  s t r a i n from the American Type C u l t u r e C o l l e c t i o n (ATCC 12407). T h i s 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 o f E . c o l i SB. S t r a i n s DBr and RBr a r e streptomycin-dependent and s t r e p t o m y c i n - r e s i s t a n t mutants, r e s p e c t i v e l y , i s o l a t e d streptomycin-sensitive (iv)  from  s t r a i n SBr ( C o u k e l l and P o l g l a s e  1965).  E. c o l i E S t r a i n SE was o b t a i n e d as a  streptomycin-sensitive  s t r a i n from Dr. J . Stock o f the Department o f B a c t e r i o l o g y of t h i s U n i v e r s i t y . S t r a i n s DE and RE a r e 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 w i l d - t y p e ( s t r e p t o m y c i n - s e n s i t i v e ) d e s i g n a t e d as SK-12.  Streptomycin-resistant  s t r a i n was and -dependent  mutants were i s o l a t e d i n t h i s l a b o r a t o r y ( P o l g l a s e , 1965).  - 21 -  (b) The  P r e p a r a t i o n o f media b a s a l medium c o n t a i n i n g the f o l l o w i n g s a l t s :  K HPO^(0.?#), K H g P O ^ O ^ ) , sodium c i t r a t e 2  (0.05#),  MgS0 (0.02^), ( N H ^ g S O ^ O . l ^ ) , was a d j u s t e d t o f i n a l 2f  o f 7.0.  Glucose (10$) was a u t o c l a v e d  s e p a r a t e l y and added  t o the s t e r i l i z e d b a s a l medium t o g i v e a f i n a l o f 0.4$.  Streptomycin-sensitive  pH  concentration  c u l t u r e s were grown i n  a n t i b i o t i c - f r e e medium w h i l e dependent mutants were grown i n medium c o n t a i n i n g 1000 u n i t s p e r m i l l i l i t e r  of d i h y d r o -  -3 streptomycin  (1.71 x 10  M).  Streptomycin-resistant  c u l t u r e s were grown i n both a n t i b i o t i c - f r e e medium and  (H** c e l l s )  i n medium which had been supplemented w i t h 1000 u n i t s  per m i l l i l i t e r (c)  of dihydrostreptomycin  (B  +  cells).  Growth of c u l t u r e s  To 1 l i t e r o f medium i n a 2 - l i t e r Erlenmeyer f l a s k , 100 ml. o f an 18-hr. s t a t i o n a r y c u l t u r e was added.  Cultures  were grown w i t h v i g o r o u s a e r a t i o n f o r 4 t o 5 h r s . a t 37°C and were h a r v e s t e d (d)  j u s t before the end o f the l o g phase.  Preparation of c e l l  C e l l s were h a r v e s t e d in a Servall  extracts  by c e n t r i f u g a t i o n a t 6000 X g  c e n t r i f u g e and suspended i n 0.1M  phosphate b u f f e r (pH 7.0).  The c e l l  potassium  suspensions were c e n t r i -  - 22 -  fuged a g a i n a t 6000 X g and resuspended phosphate  ml.  The suspensions were then s u b j e c t e d  3 min. of s o n i c d i s r u p t i o n i n a B r o n w i l l 20-kc s o n i c  oscillator. 2.5  potassium  b u f f e r i n a. r a t i o o f l g . packed c e l l s t o 15  of "buffer s o l u t i o n . to  i n 0.1M  t o 5.0  The r e s u l t i n g crude e x t r a c t s , which c o n t a i n e d mg.  per m i l l i l i t e r o f p r o t e i n , were used  directly  f o r enzyme a s s a y s . V.  Colorlmetrio Determination of Proteins P r o t e i n s were determined by the method o f Lowry  et a l . (195D. The f o l l o w i n g reagents were used: Reagent A:  2% NagCO^ i n 0.1  N NaOH.  Reagent B:  0.5$  Reagent C:  Prepared j u s t b e f o r e p r o t e i n d e t e r m i n a t i o n s  CuS0^.5H 0 i n 1% Na 2  tartrate.  by mixing 50 ml. reagent A and 1 ml. reagent B. Reagent D:  D i l u t e d F o l i n reagent (1  volume o f phenol  reagent + 2 volumes o f d i s t i l l e d w a t e r ) . Procedure: 1-ml.  To determine the p r o t e i n c o n c e n t r a t i o n s , a p r o t e i n sample ( c o n c e n t r a t i o n 0.25 was to  t o 0.5  mg.  per  ml.)  mixed with 5 ml. of reagent C and the s o l u t i o n a l l o w e d s t a n d a t room temperature  developed w i t h 0.5  f o r 10 min.  C o l o r was  ml. o f reagent D f o r 30 min. and  the  - 23 -  o p t i c a l d e n s i t i e s were read a t 500 mu i n a Beckman model B  spectrophotometer.  VI.  Enzyme Assays (a)  Reductoisomerase  T h i s was measured s p e c t r o p h o t o m e t r i c a l l y by r e c o r d i n g the disappearance o f NADPH a t room temperature, a c c o r d i n g t o the method o f Umbarger e t a l . ( i 9 6 0 ) .  0 OH CHo-C-C-COOH 3 C H  NADPH + H NADP — r — 1 ^ Reductoisomerase +  +  OH 9H CHo-C-CH-COOH 3 <jg^  fc-acetolactate  j3-dihydroxyi s o v a l e r a t e  Procedure: Assays were conducted i n 1.5-ml. systems 300 umoles potassium phosphate, pH 7 . 5 ; chloride; phosphate  containing  10 umoles magnesium  0.3 umoles n i c o t i n a m i d e adenine d i n u c l e o t i d e (NADPH);  s u b s t r a t e , 2 umoles o f e i t h e r c h e m i c a l l y  o r e n z y m a t l c a l l y prepared bacterial c e l l extract.  rtr-acetolactate;  and 0.2 m l . o f  The o x i d a t i o n o f NADPH (as I n d i c a t e d  by the decrease o f o p t i c a l d e n s i t y a t 3^0 mu) was r e c o r d e d i n a Cary 15 spectrophotometer. S p e c i f i c a c t i v i t i e s o f enzyme p r e p a r a t i o n s were expressed i n mumoles oC-acetolactate reduced p e r mg. p r o t e i n p e r minute.  - 24 -  (b)  Transaminase  This a c t i v i t y  was  B determined by measuring the  p r o d u c t i o n o f v a l i n e from the v a l i n e - g l u t a m a t e t r a n s a m i n a t i o n r e a c t i o n , a c c o r d i n g to the method o f Bragg and P o l g l a s e (1964b). glutamate CH--  CH-8-C00H  tt-ketoglutarate  ^ ^ Transaminase B  CH^  JJJJ^  y- CH - CH-CH-COOH CH^ 3  a-ketoisovalerate  valine  Procedure: The system c o n t a i n e d the f o l l o w i n g :  0 . 2 5 ml.  ( 5 0 umoles) L - g l u t a m l c a c i d ( d i s s o l v e d i n 0.1M potassium phosphate b u f f e r , pH 8 ) ; isovalerate;  0.1 ml. ( 2 0 umoles)  0.1 ml. ( 1 0 . 8 u m o l e s ) p y r i d o x a l  0.1 ml. ( 5 umoles) magnesium c h l o r i d e ; e x t r a c t ( l g . c e l l s p l u s 15 ml. b u f f e r ) .  phosphate;  and 0.5 ml. The mixture  c e n t r i f u g e d and the amino a c i d produced was from the supernatant by paper  ff-keto-  cell was  determined  chromatography.  Q u a n t i t a t i v e amounts o f samples from enzyme a s s a y s and a l s o s e v e r a l s t a n d a r d s o l u t i o n s o f v a l i n e were s p o t t e d on Whatman No. 1 paper.  Chromatograms, ( a s c e n d i n g i n a l l  experiments) were developed i n a s o l v e n t system c o n t a i n i n g  - 25 -  n-butanollacetic  a c i d t w a t e r ( 4 : l s l ) f o r about 20  hours.  They were d r i e d and dipped i n N i n h y d r i n s o l u t i o n (0.6 t r i k e t o h y d r i n d e n e hydrate, 6 ml. p y r i d i n e , acetone). f o r 15  ml.  Chromatograms were d r i e d and p l a c e d i n an oven  min. a t 90°C.  solution  and 294  g.  (1  They were dipped i n copper  nitrate  ml. s a t u r a t e d 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. a c e t o n e ) .  Spots were  then c u t i n t o s m a l l p i e c e s and the c o l o r was  e x t r a c t e d with  2 ml. methanol. a Beckman model B  O p t i c a l d e n s i t i e s were r e a d a t 530 spectrophotometer.  mu i n  - 26  C. I.  Synthesis (a)  -  RESULTS  of o U A o e t o l a c t a t e  Chemical  synthesis  Chemical s y n t h e s i s by the method of Krampitz (1948) y i e l d e d an e s t e r ( e t h y l  ol-methyl-ct-acetoxy-acetoacetate) o  h a v i n g a b o i l i n g p o i n t of 100-105 C a t 16 mm. was  s l i g h t l y yellow  hydrolyzed hydrolyzed,  slowly  i n c o l o r and  ( i hr.) a t 0°C  Hg.  The  ester  i n s o l u b l e i n water, but i n 0.1M  NaOH.  t h i s e s t e r l i b e r a t e d an a c i d  When  (ot-acetolactate)  which gave a p o s i t i v e a c e t o i n t e s t upon a c i d i f i c a t i o n w i t h sulfuric acid.  The  y i e l d of the e s t e r as c a l c u l a t e d from  the o x i d a t i o n step was  39.6#.  From the h y d r o l y s i s mixture (0.1 10 ml. 0.1M  sodium h y d r o x i d e ) ,  ot-acetolactate was (b)  18.1  Enzymatic  ml. e s t e r p l u s  the c o n c e n t r a t i o n  micromoles per  ml.  synthesis  B i o l o g i c a l a-acetolactate  was  prepared from sodium  pyruvate u s i n g c e l l e x t r a c t s o f E. c o l i DK-12. were u s u a l l y stopped a f t e r 4 to 5 hours.  Reactions  F i g u r e 3 shows  the time curves of ot-acetolactate formation system.  of  i n this reaction  In the " c o n t r o l " system where c e l l e x t r a c t s were  - 27 -  added d i r e c t l y to the r e a c t i o n mixture, of  ot-acetolactate was  the "standard"  linear  production  f o r about 3^ hours.  observed  system where c e l l e x t r a c t s were p l a c e d  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 p r o d u c t i o n of a - a c e t o l a c t a t e was r a t e of p r o d u c t i o n of O - a e e t o l a c t a t e i n the  The  system was for  In  linear. "control"  about 3 times t h a t of the " s t a n d a r d " .  However,  a l l enzyme assays and c h a r a c t e r i z a t i o n purposes, the  a - a c e t o l a c t a t e from the "standard" i t was II.  system was  used s i n c e  not contaminated w i t h c e l l e x t r a c t s .  C o l o r l m e t r l c Determination  of A c e t o i n and a - A c e t o l a c t a t e  A c e t o i n and a - a c e t o l a c t a t e c o n c e n t r a t i o n s were determined by the method o f W e s t f e l d under " M a t e r i a l s and Methods".  (1945) d e s c r i b e d  T h i s c o l o r l m e t r l c determina-  t i o n gave a l i n e a r standard curve o f a c e t o i n i n the range 20 to  250  III.  m i l l i m i c r o m o l e s per 5 ml., as shown I n F i g u r e  4.  S t a b i l i t y of O - A c e t o l a o t a t e Since  rt-acetolactate  was  r e p o r t e d to be q u i t e u n s t a b l e  and c o u l d undergo s e l f - d e c a r b o x y l a t i o n q u i t e r e a d i l y (Umbarger 1958), the s t a b i l i t y of chemical i n the h y d r o l y s i s mixture (pH 7-2) d i f f e r e n t temperatures: and -10°C  (frozen).  was  a-acetolactate  determined a t three  room temperature (25°C),  5°C,  -28-  Time (hours) Fig. 3.  Enzymic synthesis of fl£-acetolactate.  "Control" represents system with c e l l extract introduced directly into the reaction mixture. "Standard" represents system with c e l l extract placed in a dialyzing bag. C e l l extract was obtained from Escherichia c o l i  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 i n the dark for 1 hour and the optical density was read at 5 AO mu in a Beckman model B spectrophotometer.  - 30 -  F i g u r e 5 shows the % a - a c e t o l a c t a t e p r e s e n t i n the h y d r o l y s i s mixture over a p e r i o d o f 20 days.  As seen i n  curve A, v i r t u a l l y a l l the O - a c e t o l a e t a t e had been converted  t o a c e t o i n i n 10 days a t room temperature.  A t -10°C, the a - a c e t o l a c t a t e was r e l a t i v e l y  stable i n this  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 o f 2,4dinitrophenylhydrazone The  o f a - a c e t o l a c t a t e i s g i v e n on p. 18.  s o l v e n t system c o n t a i n i n g n-butanol-water-ethanol  a r a t i o o f 5*4:1  was found t o g i v e b e s t r e s u l t s .  c o u l d be separated without  with  Spots  s t r e a k i n g i f the amount o f the  hydrazone s o l u t i o n s s p o t t e d on the paper d i d n o t exceed  0.05 ml. The R  F  v a l u e s and the flmax o f the hydrazone  a r e g i v e n i n Tables I and I I .  The i n f r a r e d  spots  spectra are  shown I n F i g u r e s 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 o f reductoisomerase  from 5 s t r a i n s o f E . c o l l .  The r a t e s o f  enzyme a c t i v i t i e s f o r most cases were o f z e r o - o r d e r 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  F  V a l u e s of 2 , 4 - D i n i t r o p h e n y l h y d r a z o n e Derivatives  Substance  Spot 1  Spot 2  Enzymatic a - a c e t o l a c t a t e  0.46  0.57  Chemical a - a c e t o l a c t a t e  0.47  0.59  0.91  -  -  0.93  Acetoin S o l v e n t system:  Spot 3  n-butanol-ethanol-water (5:1*4)  TABLE I I A b s o r p t i o n Maxima and Minima o f 2,4-Dinitrophenylhydrazone D e r i v a t i v e s  Maxima (mu) Substance Enzymatic  tt-acetolactate  Chemical Ot-acetolactate Acetoin  Spot Spot Spot 1  2  Minima  (mu)  Spot Spot Spot  3  1  2  358  372  -  291  292  358  373  353  294  302  -  -  358  -  -  3  - • 291 295  Each hydrazone spot was c u t from the chromatographs and the c o l o r was e x t r a c t e d w i t h ,2M Na b i c a r b o n a t e . A b s o r p t i o n s p e c t r a 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  3  2000  4  5  1500  6  FREQUENCY  7  8  (CM** ) 1  9  1000  10  900  11  800  12  WAVELENGTH (MICRONS)  F i g . 7 ( a ) . Infrared spectra o f Ot-acetolactate 2,4-dinitrophenylhydrazone (Spot 1 of Table I ) . The heavy l i n e represents the d e r i v a t i v e of the chemical fc-acetolactate and the l i g h t e r l i n e represents the d e r i v a t i v e o f 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 d e r i v a t i v e 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 -  initial  10 t o 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 p e r i o d o f z e r o - o r d e r kinetics. (b)  Transaminase  B  T a b l e IV shows the s p e c i f i c a c t i v i t i e s o f t r a n s aminase B i n E . c o l i K-12.  Three separate enzyme assays  were performed with f r e s h e x t r a c t s 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 o f a c t i v i t i e s i n dependent and s e n s i t i v e e x t r a c t s was determined  t o be  3 . 3 , 1.5 and 3 . 6 . VI.  Colorlmetrlc Determination of Proteins P r o t e i n s were determined by the method o f Lowry  (1951) d e s c r i b e d under " M a t e r i a l s 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 t o 3 mg. p e r ml., as shown i n F i g u r e 6.  F o r t h i s reason,  samples were u s u a l l y d i l u t e d w i t h water t o g i v e c o n c e n t r a t i o n s o f approximately 1.5 were determined.  t o 3 mg. per ml. b e f o r e p r o t e i n s  - 37 -  TABLE I I I  Reductoisomerase A c t i v i t y * U s i n g E i t h e r Chemical o r Enzymatic rt-Acetolactate as S u b s t r a t e 0L- A c e t o l a c t a t e E. c o l l S t r a i n  *  Chemical  SB DB  6.7 7.0  SBr DBr  9.2 6.8  SA DA  16.5 18.2  SE DE R"E  BPE  19.2 25.3 25.3 25.4  SK-12 DK-12  13.3 8.5  Enzymatic  5.6 3.9 5.1  4.6  6.1 5.1  A c t i v i t i e s a r e expressed as m i l l i m i c r o m o l e s o f substrates converted per m i l l i g r a m of p r o t e i n per minute a t room temperature (25°C).  -  38  -  TABLE IV  Transaminase B A c t i v i t i e s i n E x t r a c t s of Streptomycin-Resistant, - S e n s i t i v e and -Dependent E . c o l l K-12.  Resistant  Sensitive  Dependent  mumoles o f v a l i n e formed p e r p r o t e i n per min.  Assay No.  D/S*  mg.  1  21.2  6.4  20.8  3.3  Assay No. 2  20.?  11.4  1?.0  1.5  Assay No.  23.6  6.6  24.0  3.6  3  * D/S i s the r a t i o o f a c t i v i t i e s i n dependent and s e n s i t i v e (S) e x t r a c t s .  (D)  -39-  0  .1  .2  .3  .A  .5  .6  .7  Concentration (mg. p r o t e i n per ml.)  F i g . 6.  Standard curve of p r o t e i n .  C r y s t a l l i n e bovine albumin was used as the standard. O p t i c a l d e n s i t y was read i n a Beckman model B spectrophotometer at a wavelength o f 500 m i l l i microns .  - 40  D. I.  -  DISCUSSION  R e l a t i o n s h i p Between Streptomycin-Dependency and the E l e v a t i o n of Aoetohydroxy A c i d Synthetase, The  biosynthesis  of v a l i n e 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 A d e l b e r g 1965b). These s t r u c t u r a l genes s y n t h e s i z e 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.  and  Threonine dehydratase  acetohydroxy a c i d synthetase are synthesis  of i s o l e u c l n e and  the  f i r s t enzymes i n  valine respectively,  the  while  reductoisomerase, transaminase B as w e l l as acetohydroxy a c i d synthetase are F i g u r e 1. from the  shared by both pathways, as shown i n  A h i g h degree of complexity can be f a c t t h a t a branch p o i n t  i s o v a l e r a t e intermediate leading l e u c i n e and  pantoic a c i d .  e x i s t s a t the t o the  I t i s not  t h a t t h i s complex b i o s y n t h e t i c  An  tf-keto-  f o r m a t i o n of  surprising  pathway has  therefore  provided  an unique system i n s t u d y i n g enzyme r e g u l a t i o n biosynthetic  realized  quite  and  c o n t r o l mechanisms.  i n t e r e s t i n the i s o l e u c i n e - v a l i n e pathway  r e s u l t e d from the independent d i s c o v e r y e t a l . ( 1 9 6 2 ) and  by Bragg and  Polglase  by  was  Tirunarayanan  (1962) t h a t  strepto-  - 41  -  mycin-dependent mutants e x c r e t e d r e l a t i v e l y l a r g e amounts of L - v a l i n e  ( a c c o u n t i n g f o r 10%  d u r i n g growth on g l u c o s e - s a l t s t o the  of the g l u c o s e carbon) medium.  T h i s has  lead  s u g g e s t i o n of a f a i l u r e of c o n t r o l mechanisms i n  dependent s t r a i n s and appeared to r e g u l a t e catabolism, leading  t h a t the r o l e of the a n t i b i o t i c an anomalous pathway of glucose  to the  s e c r e t i o n of v a l i n e .  A  detailed  study of acetohydroxy a c i d synthetase (the f i r s t enzyme in  the v a l i n e pathway) by C o u k e l l  revealed  t h a t t h e r e was  and  Polglase (2  an e l e v a t i o n  (1965)  to 6 times) of t h i s  enzyme i n the dependent mutants which c o u l d account f o r increase  of v a l i n e f o r m a t i o n .  mutants showed no  The  s e n s i t i v e and  resistant  e l e v a t i o n i n acetohydroxy a c i d synthetase  when grown e i t h e r w i t h or without added s t r e p t o m y c i n . was  the  apparent t h e r e f o r e  that a close r e l a t i o n s h i p  between streptomycin-dependency and  the  elevation  It  exists of  acetohydroxy a c i d synthetase a c t i v i t y i n these mutants. In o r d e r to v e r i f y the l a t t e r p o i n t and the p o s i t i o n (or p o s i t i o n s ) a f f e c t e d by  In the i s o l e u c i n e - v a l i n e  s t r e p t o m y c i n , a study was  coll.  pathway  undertaken to determine  the a c t i v i t y of reductoisomerase i n the of E s c h e r i c h i a  to e s t a b l i s h  streptomycin-mutants  - 42  II.  Synthesis  and  -  C h a r a c t e r i z a t i o n of  06-Acetolaotate  To o b t a i n a r e l i a b l e r e s u l t from an enzymic r e a c t i o n , it One  i s e s s e n t i a l to ensure t h a t the s u b s t r a t e s a r e way  to a c h i e v e t h i s i s to s y n t h e s i z e a  c h e m i c a l l y and  authentic.  substrate  then compare i t s p r o p e r t i e s w i t h t h a t  the enzyme p r o d u c t .  I f the c h e m i c a l l y - s y n t h e s i z e d  i s i d e n t i c a l with that synthesized  enzymatically  of  product the  a u t h e n t i c i t y o f the m a t e r i a l i s c o n f i r m e d . In t h i s study, c t - a c e t o l a c t a t e was chemically was  by the method of Krampitz ( 1 9 4 8 ) and  c h a r a c t e r i z e d and  OC-acetolactate (a)  synthesized  compared w i t h the  the  product  biological  as t h e i r hydrazone d e r i v a t i v e s .  General p r o p e r t i e s of k e t o - a c i d 2 , 4 - d i n i trophenylhydrazones  Hydrazones of k e t o - a c i d s  are colored p r o v i d i n g  advantage t h a t these compounds are s e l f - i n d i c a t i n g paper chromatograms.  However, c o m p l i c a t i o n s  may  on  arise  from the f a c t t h a t a k e t o - a c i d w i l l o f t e n g i v e r i s e two  o r more hydrazone d e r i v a t i v e s .  the  to  S t u d i e s by Altman ejb a l .  (195D» C a v a l l i n i e t al.(1954), Mortimer e t al.(1954), Cruickshank ( 1 9 5 4 ) ,  and  Towers e t a l . ( 1 9 5 4 ) r e v e a l e d  a common phenomenon among k e t o - a c i d s  encountered  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 hydrazones.  to form  that  frequently isomeric  -  43  -  D e t a i l e d s t u d i e s by Isherwood (1955) on s e v e r a l k e t o - a c i d s have p r o v i d e d c o n f i r m a t o r y data f o r the s t r u c t u r a l assignment o f the hydrazones as isomers.  geometrical  For example, p y r u v i c a c i d gave r i s e to  hydrazone spots i n the chromatograms, h a v i n g R  two values  F o f G.6  and 0.4.  zones I n 0.2N  The a b s o r p t i o n maxima o f the two  sodium b i c a r b o n a t e were very  hydra-  similar  (flmax 380mu .and 3?0mu).  The d i f f e r e n c e lOmu was  very  s i m i l a r to t h a t observed  f o r i s o m e r i c hydrazones of o t h e r  k e t o - a c i d s such as GL-ketoglutaric and o x a l o a c e t i c a c i d s . From these o b s e r v a t i o n s t o g e t h e r w i t h the f i n d i n g s from the i n f r a r e d s p e c t r a , Isherwood proposed t h a t these two hydrazones of p y r u v i c a c i d r e p r e s e n t e d a p a i r of c i s - t r a n s g e o m e t r i c a l Isomers around the C=N double as shown i n F i g u r e 8 ( a ) .  The  bond  c i s configuration i s stabilized  by the formation o f a hydrogen bond between the c a r b o x y l and the imlno groups, w h i l e the t r a n s c o n f i g u r a t i o n has a free carboxyl d e r i v a t i v e . The i n f r a r e d s p e c t r a of the isomers were found be v e r y s i m i l a r .  to  Emphasis i s focussed here on the c a r b o x y l  f u n c t i o n of the molecule. a c i d hydrazone, i t was band o c c u r r e d a t 1722 s u b s i d i a r y band a t 1672  I n the trans-isomer of p y r u v i c  observed cm" , 1  cm  that a strong absorption  while the c i s - i s o m e r had .  T h i s suggested  a  t h a t some form  - 44 -  o f c h e l a t i o n had taken p l a c e i n a s u b s t a n t i a l p r o p o r t i o n o f the a b s o r b i n g molecules.  T h i s phenomenon was  r e p o r t e d by P l e t t  observed  (1951) who  s a t u r a t e d a l i p h a t i c a c i d s absorbed 1725-1700 cm' , 1  also  t h a t whereas normal  s t r o n g l y i n the r e g i o n  i n a s i g n i f i c a n t number o f cases where  c h e l a t i o n c o u l d occur between c a r b o x y l l i n k a g e and amino o r imino hydrogen, a b s o r p t i o n s o c c u r r e d below  an 1680  cm" . 1  The h i g h e r R  F  v a l u e o f the c i s - i s o m e r on paper  chromatograms i s a r e f l e c t i o n of the s t e r i c i n t e r f e r e n c e of n e i g h b o r i n g phenyl groups on the a b i l i t y o f the i o n i z e d c a r b o x y l t o a t t r a c t water molecules and become I n c o r p o r a t e d i n the w a t e r - c e l l u l o s e complex.  Such s t e r i c f a c t o r s a r e  not p r e s e n t i n the t r a n s - i s o m e r . The h i g h e r flmax o f the c i s - i s o m e r i n the u l t r a v i o l e t as compared w i t h the t r a n s - i s o m e r e f f e c t ) was  p r o b a b l y due  (hyperchromic  to the s t a b i l i z a t i o n o f an  excited  s t a t e as a r e s u l t o f 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 o f Ot-acetolactate  R e s u l t s o b t a i n e d from the study of OC-acetolactate hydrazones a l s o i n d i c a t e d the f o r m a t i o n of i s o m e r i c d e r i v a tives.  As seen i n Table I , two hydrazones were separated  - A5 -  CH  I  3  A\  1  NN HO, N0  !  I  HO—C'  2  K -N0  CHj  N0  H  / \  HO- c || 0  2  cis-  /  A  2  N0  2  w  trans-  F i g . 8(a). C i s - t r a n s geometrical isomers o f pyruvic a c i d 2, ^.-dinitrophenylhydrazone.  °  H  \  °  I  3  H  OH C H 3  \I H3C-C-C  H C- C-C^ 3  ?  HO-C 1  N N0  0..  22  H  -N0  2  p _n _'_ / 0  I c  cisF i g . 8(b). C i s - t r a n s geometrical isomers o f 2,4,-dinitrophenylhydrazone.  N0 H }=\ N-^-N0 2  2  if\ /  transflt-acetolactate  F i g . 8(c). Cis-isomer o f pyruvic a c i d 2,4,-dinitrophenylhydrazone, showing s t a b i l i z e d e x c i t e d 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 and O.57.  The a b s o r p t i o n maxima o f these two  were very s i m i l a r (flmax 358 14 mu  v a l u e s of  and 372  mu).  The  0.46  hydrazones difference  i s comparable w i t h v a l u e s o b t a i n e d by Isherwood (1955)  on p y r u v i c , O t - k e t o g l u t a r i c and o x a l o a c e t i c a c i d s . 7(a) and 7(b) zones.  The  Figure  shows the i n f r a r e d s p e c t r a of the two  hydra-  f a s t e r moving hydrazone has a weak a b s o r p t i o n  —1 band a t 1670  cm  w h i l e the slower hydrazone has a s t r o n g  a b s o r p t i o n band a t 1700  cm' . 1  S i m i l a r r e s u l t s were o b t a i n e d with the chemical Ot-acetolactate except i n t h i s case, a t h i r d hydrazone isolated.  T h i s compound was  was  i d e n t i f i e d w i t h the hydrazone  o f a c e t o i n and thus r e p r e s e n t e d the d e c a r b o x y l a t i o n product of Ot-acetolactate hydrazone. spectrum  As expected,  the i n f r a r e d  o f t h i s compound d i d not d i s p l a y the c a r b o x y l peak,  c h a r a c t e r i s t i c o f the o t h e r two k e t o - a c i d s  hydrazones.  From the o b s e r v a t i o n s on o t - a c e t o l a c t i c a c i d hydrazone and a comparison w i t h the o b s e r v a t i o n s by Isherwood on o t h e r k e t o - a c i d hydrazones,  i t i s apparent  t h a t the two  hydrazones  from each Ot-acetolactate (chemical or enzymic) r e p r e s e n t e d a p a i r o f g e o m e t r i c a l isomers. a r e shown i n F i g u r e 8 ( b ) .  T h e i r probable s t r u c t u r e s  Thus Spot 2 i n Table I r e p r e s e n t s  the c i s - i s o m e r , which i s s t a b i l i z e d by a hydrogen-bond between the c a r b o x y l and the imlno groups.  The  disappearance  - 47  -  of the c a r b o x y l a b s o r p t i o n band a t 1700  cm  probably  i n d i c a t e s t h a t c h e l a t i o n was  complete i n the hydrazone  molecule.  the trans-isomer  lower R  P  Spot 1 r e p r e s e n t s  having  a  value and flmax.  The  i d e n t i t y of the hydrazones of  enzymatically  prepared (X-acetolactate w i t h hydrazones o f the s y n t h e t i c product of R  was  e s t a b l i s h e d , as shown i n Table I , by  comparison  v a l u e s f o r both i s o m e r i c d e r i v a t i v e s , and by  the  F i d e n t i c a l a b s o r p t i o n maxima and minima as shown i n Table II.  In a d d i t i o n , i n f r a r e d s p e c t r a o f the hydrazones from  these two k e t o - a c i d s were p r a c t i c a l l y superimposable, as shown i n F i g u r e 7(a) and  7(b).  I t can be concluded  t h e r e f o r e t h a t the  products  s y n t h e s i z e d c h e m i c a l l y by the method of Krampitz  and  e n z y m a t i c a l l y w i t h c e l l e x t r a c t s of E . c o l l K-12  represented  the same compound, O t - a c e t o l a c t a t e . III.  Reductoisomerase A c t i v i t i e s i n Mutants of E . c o l i  Streptomycin-  Reductoisomerase a c t i v i t i e s i n c e l l e x t r a c t s from streptomycin  mutants o f E . c o l l were determined e i t h e r  w i t h e n z y m a t i c a l l y s y n t h e s i z e d Ot-acetolactate o r w i t h c h e m i c a l l y s y n t h e s i z e d product  as  the  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 e x t r a c t s o f 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 w i t h a comparison o f a c t i v i t i e s between s e n s i t i v e and dependent  mutants o f each s t r a i n .  Each p a i r o f dependent  and s e n s i t i v e mutants was grown and s t u d i e d a t the same time under the same c o n d i t i o n s i n o r d e r t o f a c i l i t a t e comparison  o f the enzymatic a c t i v i t i e s i n these  direct mutants.  As seen i n Table I I I , t h e r e i s no a p p r e c i a b l e d i f f e r e n c e i n the a c t i v i t i e s o f reductoisomerase between the dependent  and the s e n s i t i v e  any o f the f i v e s t r a i n s .  ( w i l d type) mutants i n  Reductoisomerase  i s thus n o t  e l e v a t e d i n the streptomycin-dependent mutants,  unlike  acetohydroxy a c i d synthetase which precedes reductoisomerase i n the b i o s y n t h e t i c sequence  l e a d i n g t o the f o r m a t i o n o f  valine. IV,  Transaminase B A c t i v i t i e s i n S t r e p t o m y c i n - R e s i s t a n t , - S e n s i t i v e , and -Dependent E . c o l l K-12 The a c t i v i t y o f transaminase B i n s t r e p t o m y c i n -  dependent  and s t r e p t o m y c i n r e s i s t a n t E . c o l l K-12 was  found t o be 2 t o 3 times h i g h e r than i n the s e n s i t i v e mutants. Since t h r e o n i n e dehydratase was observed t o be d e r e p r e s s e d a l s o (about 9 - f o l d ) i n the dependent  K-12 as  compared to both the s e n s i t i v e and r e s i s t a n t mutants o f this strain  (Desai and P o l g l a s e 1966), the r e s u l t o b t a i n e d  f o r transaminase B thus i n d i c a t e s t h a t these two enzymes  -  are  49  -  c o o r d i n a t e l y d e r e p r e s s e d (although not t o the same  degree) i n streptomycin-dependent V.  K-12.  I n t e r p r e t a t i o n of the R e s u l t s T h i s study e s t a b l i s h e s t h a t  not  derepressed.  1965)  reductoisomerase i s  E a r l i e r o b s e r v a t i o n s ( C o u k e l l and P o l g l a s e ,  showed an e l e v a t i o n i n acetohydroxy a c i d synthetase  i n streptomycin-dependent E. c o l l . streptomycin-dependence  Thus mutation t o  a f f e c t s o n l y the r e g u l a t o r y ( i . e .  r a t e - l i m i t i n g ) enzyme o f v a l i n e b i o s y n t h e s i s .  Derepression  of acetohydroxy a c i d synthetase i n the s t r e p t o m y c i n mutants of E . c o l i B, A, Br and E can e x p l a i n the observed over p r o d u c t i o n o f v a l i n e , p r o v i d e d t h a t the o t h e r enzymes o f the  pathway a r e not r a t e - l i m i t i n g  excess).  The s e l e c t i v e advantage  ( i . e . are present i n to a  streptomycin-dependent  mutant o f v a l i n e e x c r e t i o n might be t h a t t h i s compound i s a n e u t r a l end-product o f pyruvate metabolism from g l u c o s e oxidation. Bragg and P o l g l a s e ( 1 9 6 4 a ) had observed t h a t i n the a n t i b i o t i c - d e p l e t e d dependent c e l l s , excess p y r u v i c a c i d ( o r l a c t i c a c i d ) accumulated d u r i n g glucose o x i d a t i o n w h i l e r e l a t i v e l y l i t t l e v a l i n e was  found.  When s t r e p t o m y c i n was  added t o the growth medium o f the d e p l e t e d c e l l s , however, the  s i t u a t i o n was r e v e r s e d so t h a t a l a r g e amount o f v a l i n e  -  was  50  -  excreted" with the concomitant decrease  i n pyruvic a c i d .  These o b s e r v a t i o n s i n d i c a t e t h a t the streptomycin-dependent c e l l s are not capable to p y r u v a t e .  of r e g u l a t i n g the o x i d a t i o n of  As a r e s u l t , the l a t t e r would accumulate when  t h e r e i s an abundant supply of g l u c o s e . d e r e p r e s s i n g acetohydroxy a c i d synthetase mutants, permits valine  glucose  Streptomycin,  by  i n the dependent  the d i s s i m i l a t i o n of excess pyruvate  via  formation. A question concerning  the nature of d e r e p r e s s i o n  might be r a i s e d with r e g a r d to the magnitude of e l e v a t i o n o f acetohydroxy a c i d synthetase mutants.  A  M  true  w  observed  i n these dependent  g e n e t i c d e r e p r e s s i o n , a c c o r d i n g t o the  "Jacob-Monod" operon concept  can be p i c t u r e d as  an  i n a c t i v a t l o n of the r e g u l a t o r y gene or i t s product by a c o r e p r e s s o r so t h a t no r e p r e s s o r (or a n o n - f u n c t i o n a l r e p r e s s o r ) i s produced.  As a r e s u l t the o p e r a t o r  can  f u n c t i o n maximally i n d i r e c t i n g messenger s y n t h e s i s , l e a d i n g to an i n c r e a s e d p r o d u c t i o n of p r o t e i n (enzyme i n t h i s c a s e ) . G e n e t i c d e r e p r e s s i o n of acetohydroxy a c i d synthetase r e s u l t e d i n a 20 f o l d e l e v a t i o n of t h i s enzyme, which was by Ramakrishnan and A d e l b e r g  observed  (1965a).  I n the case of acetohydroxy a c i d synthetase, an e l e v a t i o n of only 2 t o 6 f o l d was and P o l g l a s e ( 1 9 6 5 ) .  observed  however,  by C o u k e l l  These i n v e s t i g a t o r s p o s t u l a t e d t h a t  -  51  -  the enzyme was induced by pyruvate  i.  t o p r o v i d e an a l t e r n a t e  pathway o f c a t a b o l i s m . From these o b s e r v a t i o n s , i t may be suggested  that  the r o l e o f streptomycin as an enzyme d e r e p r e s s o r r e s t s on i t s a b i l i t y t o a f f e c t the streptomycin-dependent  E. c o l l  i n a manner such t h a t the m e t a b o l i t e , pyruvate, i s a b l e t o a c t as an i n d u c e r o f acetohydroxy  acid  synthetase.  E s c h e r i c h i a c o l l K-12 r e q u i r e s a s p e c i a l d i s c u s s i o n because i t has p r o p e r t i e s d i f f e r e n t from o t h e r  strains.  Streptomycin-dependent K-12 was found t o be v a l i n e  resistant  ( P o l g l a s e , 1 9 6 5 ) » whereas the growth o f the s t r e p t o m y c i n s e n s i t i v e ( w i l d - t y p e ) s t r a i n was i n h i b i t e d by v a l i n e and t h i s i n h i b i t i o n c o u l d be r e v e r s e d by i s o l e u c i n e 1946). (1966)  (Bonner,  S t u d i e s on enzyme a c t i v i t i e s by Desal and P o l g l a s e i n d i c a t e d t h a t t h e r e was a 9 f o l d o r h i g h e r de-  r e p r e s s i o n o f threonine dehydratase  a c t i v i t y i n the dependent  mutants o f K-12 as compared t o the s e n s i t i v e p a r e n t .  However,  when the s p e c i f i c a c t i v i t i e s o f threonine dehydratase  activities  o f s e v e r a l s t r a i n s o f E . c o l i were compared, i t was  found  t h a t the a c t i v i t y from dependent K-12 was s i m i l a r t o t h a t o f dependent-mutants from o t h e r s t r a i n s .  I t became e v i d e n t  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 t o the u n u s u a l l y low l e v e l o f t h r e o n i n e dehydratase  i n the w i l d - t y p e s t r a i n r a t h e r than t o a h i g h  - 52 -  l e v e l of t h i s enzyme i n the dependent mutant. Since mutants o f K-12 dehydratase  h a v i n g derepressed  alone e x h i b i t e d a h i g h l e v e l o f v a l i n e r e s i s t a n c e 1965b), i t became probable t h a t  (Ramakrishnan and A d e l b e r g , t h r e o n i n e dehydratase s e n s i t i v i t y to v a l i n e . dehydratase  i s the key enzyme i n d e t e r m i n i n g  the  Only by d e r e p r e s s i n g threonine  can s u f f i c i e n t i s o l e u c l n e be made t o 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 . s e n s i t i v i t y to v a l i n e of s e n s i t i v e K-12 in  threonine  The  extreme  can thus be  terms of the low l e v e l o f threonine dehydratase  explained i n this  strain. The d i s c o v e r y by Ramakrishnan and A d e l b e r g t h a t the three s t r u c t u r a l genes c o n t r o l l i n g dehydratase,  dehydrase and transaminase  operon p r o v i d e d a c r i t i c a l  (1965b)  threonine  B a r e i n the same  c r i t e r i o n i n determining  the  r o l e of s t r e p t o m y c i n a t the l e v e l of e x p r e s s i o n of the r e g u l a t o r y genes as f o l l o w s : t h r e o n i n e dehydratase of  a mutant h a v i n g  derepressed  should show c o o r d i n a t e d e r e p r e s s i o n  the o t h e r two enzymes i n t h i s operon, the dehydrase  (see F i g u r e 2)  and transaminase  B.  The r e s u l t from t h i s study i n d i c a t e s t h a t t h r e o n i n e dehydratase  and transaminase  i n E . c o l l K-12.  B a r e c o o r d i n a t e l y derepressed  However, s i n c e the degree o f d e r e p r e s s i o n  - 53 -  of transaminase B (about 2 t o 3 f o l d ) was much l e s s than t h a t o f t h r e o n i n e dehydratase (about 9 f o l d ) , i t i s e v i d e n t t h e r e f o r e t h a t these two enzymes were n o t d e r e p r e s s e d t o the same degree.  C o n t r a r y t o what would  be expected o f a d e r e p r e s s i o n from a n o n - f u n c t i o n a l gene p r o d u c t (based on the operon model o f Jacob and Monod, 1961), the r e s u l t o b t a i n e d from t h i s study shows a d e r e p r e s s i o n where two enzymes ( t h r e o n i n e dehydratase and transaminase B) i n the same operon were not " c o o r d i n a t e l y " d e r e p r e s s e d t o the same degree.  - 54 -  E.  BIBLIOGRAPHY  Altman, S.M., Crook, E.M., and D a t t a , S.P., Biochem. J . ,  4£ I X i i i  (195D.  Bonner, 0., J . B i o l . Chem., 166 545 (1946). 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