Biosynthesis and Utilization of Acetyl Phosphate, Formyl Phosphate

10 Biosynthesis and Utilization of Acetyl Phosphate, Formyl Phosphate, and Carbamyl Phosphate and their Relations to the Urea Cycle Downloaded by TUFTS UNIV on November 19, 2016 | http://pubs.acs.org Publication Date: January 1, 1964 | doi: 10.1021/ba-1964-0044.ch010

SANTIAGO GRISOLIA AND LUISA RAIJMAN Department of Biochemistry, School of Medicine, University of Kansas, Kansas City, Kan.

Animal and bacterial enzymes that utilize or synthesize carbamyl phosphate have activity with acetyl phosphate. Acyl phosphatase hydrolyzes both substrates, and maybe involved in the specific dynamic action of proteins. Ornithine and aspartic transcarbamylases also synthesize acetylornithine and acetyl aspartate. Finally, bacterial carbamate kinase and animal carbamyl phosphate synthetase utilize acetyl phosphate as well as carbamyl phosphate in the synthesis of adenosine triphosphate. The synthesis of acetyl phosphate and of formyl phosphate by carbamyl phosphate synthetases is described. The mechanism of carbon dioxide activation by animal carbamyl phosphate synthetase is reviewed on the basis of the findings concerning acetate and formate activation.

y h i s a r t i c l e d i s c u s s e s the p r e s e n t status of the m e c h a n i s m of c a r b a m y l phosphate ( c a r b a m y l - P ) f o r m a t i o n and i l l u s t r a t e s that the reagents a c e t y l phosphate ( a c e t y l - P ) and c a r b a m y l - P c a n r e p l a c e e a c h other w i t h a n u m b e r of w e l l defined a n d / o r highly p u r i f i e d e n z y m e s . W h i l e quite apparent to us now, i t has taken many y e a r s to r e a l i z e that a c e t y l - P and c a r b a m y l - P c o u l d be s u b s t r a t e s f o r the s a m e e n z y m e s . A c e t y l - P was c o n s i d e r e d , f o r some y e a r s , an i m p o r t a n t i n t e r mediate f o r a c e t y l a t i o n . H o w e v e r , no evidence f o r e i t h e r i t s s y n t h e s i s o r i t s u t i l i z a t i o n , other than by phosphatase a c t i o n (26) w i t h a n i m a l

128

Stekol; Amino Acids and Serum Proteins Advances in Chemistry; American Chemical Society: Washington, DC, 1964.

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GRISOU A AND RAIJMAN

Ρ h os pate Biosynthesis

129

p r e p a r a t i o n s , c o u l d be d e m o n s t r a t e d u n t i l r e c e n t l y . T h e d i s c o v e r y of c o e n z y m e A and a c e t y l c o e n z y m e A changed the status of t h i s reagent f r o m a p o s s i b l e i n t e r m e d i a t e to m o r e o r l e s s a c u r i o s i t y , as f a r as a n i m a l s y s t e m s w e r e c o n c e r n e d . T h e advances made w i t h a c e t y l C o A and the fact that c o e n z y m e A i s not i n v o l v e d i n c i t r u l l i n e s y n t h e s i s (12) p e r haps justify the d e l a y i n t e s t i n g the p o s s i b l e s u b s t i t u t i o n of a c e t y l - P for c a r b a m y l - P as a s u b s t r a t e . T h i s p r e s e n t a t i o n a l s o i n d i c a t e s that a l l c a r b a m y l - P and a c e t y l - P r e a c t i o n s thus f a r studied a r e r e l a t e d v i a the u r e a c y c l e . T h e newer findings and t h e i r r e l a t i o n to the u r e a c y c l e a r e outlined i n F i g u r e 1.

Downloaded by TUFTS UNIV on November 19, 2016 | http://pubs.acs.org Publication Date: January 1, 1964 | doi: 10.1021/ba-1964-0044.ch010

ACYL-PHOSPHATASE-

• ICITRULLINË1

g-ACETYL-ORNITHINE*

• A R GI N O S U C C I N A T E — » JURE A|

t»ADP

CONFORMATION

^ Cc /A R B A M Y L - P

•

A G / M ^ M Ç

4

-

f AG SYNTHETASE

1

A C E T A T E + ATP FORMATE + ATP

;θ3+ΝΗ *+ΐτρ» 4

AGV • A [ H C O 3 + A T P * AG — • "ACTIVE COgl B C H C 0 5 + ATP C [AG + A T P

•

C0 -P]

•

AG-P]

D [AG-P+C0 -P+NH3-* 2

D'CC0 -P+NH3 2

+

Mn**,Mg*

FORMYL-P

x

2

AG « A C E T Y L G L U T A M A T E

ΑΤΡ-»

Figure 1. Interrelations of carbamyl-P, acetyl-P, formyl-P metabolism with the urea cycle

Studies with Acyl

+

r*ADP

and

Phosphatase

The l a r g e c o n c e n t r a t i o n s of a c y l phosphatase p r e s e n t i n m o s t t i s sues l e d to the b e l i e f that i t prevented the d e m o n s t r a t i o n of a c e t y l - P b i o s y n t h e s i s and a c c u m u l a t i o n i n a n i m a l t i s s u e s . W e p u r i f i e d e x t e n s i v e l y , s o m e y e a r s ago, what we f i r s t thought to be a s p e c i f i c c a r b a m y l - P phosphatase. T h i s e n z y m e i n t e r e s t e d us as a t o o l f o r b e t t e r u n d e r s t a n d i n g the m e c h a n i s m of c i t r u l l i n e s y n t h e s i s ; however, on t e s t i n g for s p e c i f i c i t y , we found that i t attacked a c e t y l - P j u s t as r e a d i l y as carbamyl-P.


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ADVANCES IN CHEMISTRY SERIES

T h e b r a i n e n z y m e has been p u r i f i e d o v e r 1000-fold and shown to be homogeneous by u l t r a c e n t r i f u g a t i o n and e l e c t r o p h o r e s i s c r i t e r i a (36); the a c t i v i t y r a t i o f o r a c e t y l - P o v e r c a r b a m y l - P r e m a i n s unchanged w i t h p u r i f i c a t i o n . T h i s e n z y m e i s one of the s m a l l e s t on r e c o r d ; the m o l e c u l a r weight f r o m p h y s i c a l data i s 13,200 and f r o m a m i n o a c i d a n a l y s i s i s 12,600; the a m i n o a c i d c o m p o s i t i o n of the e n z y m e i s given i n T a b l e I. The t e r m i n a l a m i n o a c i d i s a s p a r t i c a c i d (25). C y s t i n e has not been detected. Table I. Amino Acid Composition of Brain Acyl Phosphatase


Amino Acid Lysine Histidine Ammonia Arginine Methionine sulfoxides Cysteic acid Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine 1/2 cystine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Tryptophan

μ moles/0.7 8 Mg. Protein

Calculated No. of Residues per Mole Protein

0.64 0.10 0.68 0.20 0.05 0.04 0.54 0.38 0.42 0.82 0.44 0.51 0.43 0d 0.31 trace 0.27 0.32 0.09 0.12

13 2 14 4 lb ic 11 7 8 16 9 10 9 0 6 lb 5 6 2 2

a

—

a

Assuming cysteine = methionine = 1 mole/mole enzyme, k Methionine sulfoxides calculated as methionine. Cysteic acid = cysteine. d As little as 0.01 μπιοΐβ would have been detected. Small amount of degradation product of tryptophan found. At least 1 residue tryptophan assumed to be present. c

e

T h e great s t a b i l i t y of a c y l phosphatase, together w i t h the l a c k of d i s u l f i d e b r i d g e s , i s u n u s u a l i n e n z y m e c h e m i s t r y . T h e high s p e c i f i c rotation, — = 89.3, i s of i n t e r e s t a l s o , suggesting that the e n z y m e does not have an a p p r e c i a b l e n u m b e r of r e s i d u e s i n the a - h e l i x c o n f i g u r a t i o n . T h i s i s c o n s i s t e n t w i t h the r e l a t i v e l y l a r g e n u m b e r of p r o l i n e r e s i d u e s w h i c h , i f evenly d i s t r i b u t e d , w o u l d p r e v e n t a - h e l i x f o r m a t i o n (7). T h e a c y l phosphatase m a y p l a y a s i g n i f i c a n t r o l e i n p h y s i o l o g i c a l phenomena s u c h as h i b e r n a t i o n and s p e c i f i c d y n a m i c a c t i o n (4).


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Phospate Biosynthesis

Heat s t a b i l i t y and other c h a r a c t e r i s t i c s i n d i c a t e that t h i s e n z y m e m a y be i d e n t i c a l w i t h the a c e t y l phosphatase of L i p p m a n (26), w h i c h a l s o c a t a l y z e s the h y d r o l y s i s of 1,3-diphosphoglycerate (21). In spite of the i n d i c a t i o n that the s a m e enzyme u t i l i z e d c a r b a m y l - P and a c e t y l - P , we d i d not r e a l i z e the p o s s i b i l i t y , u n t i l s o m e 4 y e a r s l a t e r , that a c e t y l - P c o u l d be s y n t h e s i z e d o r used f o r synthetic p u r p o s e s in animal tissues. Studies with Ornithine

Transcarbamylase


A s i n d i c a t e d i n R e a c t i o n s 1 and 2 , both c a r b a m y l - P and a c e t y l - P can r e a c t w i t h o r n i t h i n e to f o r m e i t h e r c i t r u l l i n e o r ô - a c e t y l o r n i t h i n e w i t h o r n i t h i n e t r a n s c a r b a m y l a s e (15). C a r b a m y l - P + ornithine — c i t r u l l i n e + P i

(1)

A c e t y l - P + ornithine — δ - a c e t y l o r n i t h i n e + P i

(2)

W h i l e the a - a m i n o group of o r n i t h i n e c o u l d a l s o r e a c t w i t h a c e t y l P , t h i s p o s s i b i l i t y w a s e l i m i n a t e d i n a n u m b e r of w a y s . C h r o m a t o g raphy i n the automatic r e c o r d i n g a m i n o a c i d a n a l y z e r by the p r o c e d u r e of S p a c k m a n , S t e i n , and M o o r e (41) y i e l d s e x c e l l e n t s e p a r a t i o n of Ôacety l o r n i thine and a?-acetylornithine. Only t r a c e s of a - a c e t y l o r n i t h i n e have been detected w i t h o r n i t h i n e t r a n s c a r b a m y l a s e and a c e t y l - P ; the p r o d u c t i s a l w a y s better than 95% ô - a c e t y l o r n i t h i n e . T h e s e p a r a t i o n of a- and ô - a c e t y l o r n i t h i n e by paper c h r o m a t o g r a p h y i s not p r a c t i c a l l y f e a s i b l e , although both a c e t y l d e r i v a t i v e s a r e v e r y r e a d i l y s e p a r a t e d f r o m o r n i t h i n e (15). δ - A c e t y l o r n i t h i n e i s a n a t u r a l product f i r s t i s o lated f r o m a S i b e r i a n plant 26 y e a r s ago (28) and now known to be p r e s ent i n many plants (39). T h e product of the s t o i c h i o m e t r i c r e a c t i o n of a c e t y l - P w i t h o r n i thine, c a t a l y z e d by o r n i t h i n e t r a n s c a r b a m y l a s e , has been shown u n e q u i v o c a l l y to be ô - a c e t y l o r n i t h i n e ; the t r a n s c a r b a m y l a s e s f r o m r a t l i v e r , f r o g l i v e r , and b a c t e r i a , however, even though y i e l d i n g the s a m e product, appear to differ i n t h e i r r a t i o s of a c t i v i t y w i t h c a r b a m y l - P and a c e t y l - P (Table Π ) . W h i l e i t i s p o s s i b l e that the s y n t h e s i s of ô - a c e t y l o r n i t h i n e i s c a t a l y z e d by other e n z y m e s (16), the different r a t i o s m a y be due to s p e c i e s d i f f e r e n c e s ; we know now that the r a t i o s of a c t i v i t y w i t h c a r b a m y l - P and a c e t y l - P of a l l o r n i t h i n e t r a n s c a r b a m y l a s e s thus f a r tested r e m a i n constant w i t h p u r i f i c a t i o n . F u r t h e r , the r a t i o of c i t r u l l i n e to a c e t y l o r n i t h i n e f o r m a t i o n does not change w i t h a number of t r e a t m e n t s , s u c h as heat i n a c t i v a t i o n of p r e p a r a t i o n s c o n t a i n i n g o r n i -

Table II. Relative Rates of Utilization of Acetyl-P and Carbamyl-P with Animal and Bacterial Ornithine Transcarbamylases

Carbamyl-P Acetyl-P

Rat Liver

Frog Liver

34 4.1

245 7.4

Bacteria 900 5.4


132


thine t r a n s c a r b a m y l a s e (15). A c e t y l - P s e e m s to be u s e d m u c h m o r e effectively b y a n i m a l p r e p a r a t i o n s than by b a c t e r i a l p r e p a r a t i o n s of both o r n i t h i n e t r a n s c a r b a m y l a s e and a s p a r t i c t r a n s c a r b a m y l a s e (see below). L y s i n e r e a c t s , although v e r y s l o w l y , w i t h e i t h e r a c e t y l - P o r c a r b a m y l - P and o r n i t h i n e t r a n s c a r b a m y l a s e .


Studies with Aspartic

Transcarbamylase

Aspartate + c a r b a m y l - P — c a r b a m y l aspartate + P i

(3)

Aspartate + a c e t y l - P — Ν - a c e t y l aspartate + P i

(4)

R e a c t i o n s 3 and 4 i n d i c a t e that w i t h a s p a r t i c a c i d , a s p a r t i c t r a n s c a r b a m y l a s e , and c a r b a m y l - P o r a c e t y l - P , e i t h e r c a r b a m y l a s p a r t a t e o r a c e t y l a s p a r t a t e c a n be f o r m e d . C a r b a m y l a s p a r t a t e i s the f i r s t i n t e r m e d i a t e i n the f o r m a t i o n of p y r i m i d i n e s , and a c e t y l a s p a r t a t e , of u n known function, i s the a m i n o a c i d d e r i v a t i v e p r e s e n t i n the l a r g e s t c o n c e n t r a t i o n i n b r a i n of m o s t s p e c i e s (43). P r e p a r a t i o n s of a s p a r t a t e t r a n s c a r b a m y l a s e f r o m dog i n t e s t i n a l m u c o s a , r a t l i v e r , E . c o l i B , and E . c o l i 185-482 c a n u t i l i z e a c e t y l - P , although at m u c h s l o w e r r a t e s than c a r b a m y l - P . T h e r a t i o of c a r b a m y l - P to a c e t y l - P t r a n s f e r i s of the o r d e r of 20 w i t h m a m m a l i a n e n z y m e s , and 400 w i t h b a c t e r i a l p r e p a r a t i o n s (as i n d i c a t e d above, the r a t i o s of c a r b a m y l - P to a c e t y l - P t r a n s f e r r i n g a c t i v i t y a r e a l s o s m a l l e r w i t h m a m m a l i a n than w i t h b a c t e r i a l o r n i t h i n e t r a n s c a r b a m y l a s e ) . T h e a c t i v i t y of the m a m m a l i a n e n z y m e s i s v e r y l o w , even w i t h c a r b a m y l - P , and i t has not y e t been a s c e r t a i n e d beyond doubt that R e a c t i o n s 3 and 4 a r e c a t a l y z e d b y a n i m a l a s p a r t i c t r a n s c a r b a m y l a s e . H o w e v e r , the s p e c i f i c a c t i v i t y f o r a c e t y l - P and c a r b a m y l - P u t i l i z a t i o n r e m a i n s constant w i t h p u r i f i c a t i o n of the t r a n s c a r b a m y l a s e f r o m E . c o l i 185-482, suggesting that both a c t i v i t i e s a r e c a t a l y z e d by the s a m e e n z y m e (11). W h e t h e r a c e t y l - P functions i n the a c e t y l a t i o n of l y s i n e r e s i d u e s of p r o t e i n s — e . g . , c y t o c h r o m e C — o r of polypeptides [suggested by M e i s t e r and H o s p e l h o r n (31)], o r i n other a c e t y l a t i o n r e a c t i o n s r e m a i n s to be i n v e s t i g a t e d . A c e t y l h y d r o x y o r n i t h i n e f o r m s a l a r g e p e r c e n t a g e of f e r r i c h r o m e (9). E x t r a c t s of s o n i c a l l y d i s r u p t e d U s t i l a g o sphaerogena w e r e found to contain o r n i t h i n e t r a n s c a r b a m y l a s e (1 m g . of p r o t e i n s y n t h e s i z e d 40 μ π ι ο ΐ β β of c i t r u l l i n e i n 15 m i n u t e s at 3 7 ° C ) . T h e p o s s i b i l i t y that o r n i t h i n e t r a n s c a r b a m y l a s e m a y be r e q u i r e d to a c e t y l a t e N - h y d r o x y o r n i t h i n e i s a n a t t r a c t i v e one. Since the fungus c a n a l s o s y n t h e s i z e the glutaconic d e r i v a t i v e of δ - h y d r o x y o r n i t h i n e , i t i s of i n t e r e s t to find out i f the p r e s e n c e of t r a n s c a r b a m y l a s e i n t h i s o r g a n i s m m a y be r e s p o n s i b l e f o r the s y n t h e s i s of one o r the other d e r i v a t i v e of h y droxyornithine. Studies with Carbamate

Kinase

C a r b a m a t e k i n a s e f r o m s t r a i n D , group D s t r e p t o c o c c i u t i l i z e s a c e t y l - P f o r A T P s y n t h e s i s (16, 17) at a p p r o x i m a t e l y the s a m e r a t e as 1 0


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133

c a r b a m y l - P , a c c o r d i n g to R e a c t i o n s 5 and 6. Acetate + A T P


Carbamate + A T P

(5)

acetyl-P + A D P

M gΛ 2 1

M g

(6)

carbamyl-P + A D P

T o test the p o s s i b i l i t y that the c a r b a m a t e k i n a s e might be i d e n t i c a l w i t h acetokinase i n other m i c r o o r g a n i s m s , t h r e e deficient E . c o l i mutants, R 1 8 5 - 8 2 3 , R p r o t o t r o p h , and K ^ - w t , known to have l o w c a r b a m a t e k i n ase a c t i v i t y , w e r e tested f o r a c e t y l - P u t i l i z a t i o n (17). Synthesis of A T P f r o m a c e t y l - P o c c u r r e d m u c h f a s t e r than f r o m c a r b a m y l - P ; the r a t i o s of a c t i v i t y f o r a c e t y l - P - c a r b a m y l - P ranged f r o m 8 to 20; on the other hand, the e n z y m e f r o m s t r e p t o c o c c u s O u t i l i z e s c a r b a m y l - P t w i c e as fast as a c e t y l - P ; t h i s a c t i v i t y r a t i o . r e m a i n e d unchanged throughout a 4 0 - f o l d p u r i f i c a t i o n of the e n z y m e (17). It a p p e a r s then that although a n u m b e r of e n z y m e s that u t i l i z e o r s y n t h e s i z e c a r b a m y l - P can a l s o u t i l i z e o r s y n t h e s i z e a c e t y l - P , i t c a n not be a s s u m e d that a l l a c e t y l - P e n z y m e s a r e a l s o able to u s e c a r b a m y l - P (11). m

Studies with Carbamyl-P

Synthetase

F o r many y e a r s we have been i n t e r e s t e d i n the m e c h a n i s m of c i t r u l l i n e s y n t h e s i s f r o m o r n i t h i n e . T h e m e c h a n i s m of the r e a c t i o n w a s c l a r i f i e d c o n s i d e r a b l y by J o n e s , Spector, and L i p m a n n , when they s y n t h e s i z e d c a r b a m y l - P c h e m i c a l l y (24) and suggested that the m a t e r i a l w h i c h we had f i r s t i s o l a t e d f r o m a n i m a l p r e p a r a t i o n s (19) w a s c a r b a myl-P. Acetyl Glutamate-Induced Activation and Inactivation of Carbamyl-P\ Synthetase T h e i n v e s t i g a t i o n of the m e c h a n i s m of a c t i o n of c a r b a m y l - P s y n thetase has been a l w a y s a s s o c i a t e d w i t h the study of the r o l e of a c e t y l glutamate o r r e l a t e d c o f a c t o r s i n the r e a c t i o n : HCO3- + N H

+ 4

+ 2 ATP

a c e t y l glutamate ,

c

a

r

b

a

m

y

l

.

p

+

2

+ Pi

ADP (7)

In extension of p r e v i o u s o b s e r v a t i o n s f r o m t h i s l a b o r a t o r y (20) M e t z e n b e r g , M a r s h a l l , and Cohen (32) d e m o n s t r a t e d that the f r o g l i v e r c a r b a m y l - P synthetase can be a c t i v a t e d by p r e i n c u b a t i o n w i t h a c e t y l g l u tamate. T h e a c t i v a t i o n phenomenon has been e x t e n s i v e l y studied i n o u r laboratory. T h e a c t i v a t i o n i s not i m m e d i a t e ; under the conditions of T a b l e Ι Π , n e a r l y m a x i m a l effect w a s obtained i n 2 m i n u t e s . S a m p l e s c o n t a i n i n g 0.8 m g . of c a r b a m y l - P synthetase (35), 50


134

ADVANCES IN CHEMISTRY SERIES Table III. Effect of Length of Preincubation on Activation of Frog Liver Carbamyl-P Synthetase by Acetyl Glutamate. (37)

Acetyl Glutamate Present during Preincubation Preincubation Time, Minutes

Citrulline Synthesis, μ moles


2 5 10

0.021 0.021 0.021

0.142 0.149 0.153

μ m o l e s of T r i s - C I " , p H 7.4, and 2 μ m o l e s of a c e t y l glutamate w e r e p r e i n c u b a t e d at 3 8 ° f o r the i n d i c a t e d length of t i m e . The tubes w e r e then c o o l e d . Incubations f o r the a s s a y of c a r b a m y l - P synthetase w e r e conducted i n a v o l u m e of 2 m l . The f i n a l c o n c e n t r a t i o n of reagents w a s : A T P , 0.004M; K H C O , 0.05M; N H C 1 , 0.025M; M g S 0 , 0.01M; acetyl glutamate, 0 . 0 0 5 M ; o r n i t h i n e t r a n s c a r b a m y l a s e , 25 units (4); o r n i t h i n e , 0 . 0 0 5 M ; T r i s - C l " , p H 7.4, 0 . 0 5 M ; and the e n z y m e . W h e n any of the above reagents w a s p r e s e n t i n p r e i n c u b a t i o n m i x t u r e s , f u r t h e r a d d i tions w e r e adjusted to t e m p e r a t u r e and to y i e l d the i n d i c a t e d f i n a l c o n c e n t r a t i o n s d u r i n g the a s s a y . Incubation was f o r 1 minute at 2 5 ° . C i t r u l l i n e w a s m e a s u r e d as p r e v i o u s l y d e s c r i b e d (4). T h i s r e l a t i v e l y r a p i d type of a c t i v a t i o n e x p l a i n s the a u t o c a t a l y t i c c u r v e s obtained when i n i t i a l r a t e s of c i t r u l l i n e s y n t h e s i s a r e c o m p a r e d w i t h those obtained after s e v e r a l m i n u t e s ' i n c u b a t i o n . T a b l e I V d e m o n s t r a t e s that by i n c r e a s i n g the a c e t y l glutamate c o n c e n t r a t i o n and the length of i n c u b a t i o n f o r the o v e r - a l l s y n t h e s i s , the a c t i v a t i o n w a s o b s c u r e d . T h i s i s why the a c t i v a t i o n effect r e m a i n e d undetected u n t i l e x t r e m e l y s h o r t incubation t i m e s and low t e m p e r a t u r e s w e r e used. B r i n g i n g a c o m p l i c a t e d r e a c t i o n m i x t u r e to i n c u b a t i o n t e m p e r a t u r e , w a i t i n g f o r e q u i l i b r a t i o n , and c o m p l e t i n g w i t h a l a s t component, as i s often done, m a y o b s c u r e effects s u c h as the ones d e s c r i b e d h e r e . s

4

4

Table IV. Initial Rate of Synthesis of Preincubated and Unmodified Frog Liver Carbamyl-P Synthetase at Different Concentrations of Acetyl Glutamate (37)

μ moles of Acetyl Glutamate Added Incubation Time, Seconds

2 Exptl.

10

Control

a

Exptl.

40 Control a

Exptl.

Control a

μ moles Citrulline Synthesized 12 30 60 120 240

0.13 0.34 0.77 1.38 2.46

0.01 0.02 0.16 0.39 1.22

0.17 0.41 0.94 1.60 2.69

0.02 0.07 0.34 0.94 2.14

0.17 0.51 1.03

0.03 0.22 0.72

Control tubes contained no acetyl glutamate during preincubation period; indicated amount of this cofactor added prior to final incubation.


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Ρ ho spa te Biosynthesis

In the e x p e r i m e n t s of T a b l e I V 3.2 m g . of p r o t e i n f r o m f r o g l i v e r p r e p a r a t i o n s (35), 100 μ ι η ο ΐ β β of T r i s - C l " at p H 7.4, and the i n d i c a t e d amount of a c e t y l glutamate, i n a v o l u m e of 1 m l . , w e r e p r e i n c u b a t e d at 3 8 ° f o r 5 m i n u t e s . The tubes w e r e c o o l e d to 2 5 ° and c o m p l e t e d , at def i n i t e i n t e r v a l s , w i t h the components and c o n c e n t r a t i o n of reagents i n d i cated i n T a b l e Ι Π , and then incubated at 2 5 ° f o r the i n d i c a t e d length of time. T h e a c t i v a t i o n of the e x t r e m e l y unstable r a t l i v e r e n z y m e i s l e s s m a r k e d than that of the f r o g l i v e r e n z y m e and i s thus m o r e d i f f i c u l t to d e m o n s t r a t e . H o w e v e r , as i l l u s t r a t e d i n T a b l e V , w i t h the a i d of C 0 1 4


2

Table V. Effect of Preincubation of Rat Liver Carbamyl-P Synthetase with Acetyl Glutamate, A T P , Mg and HC0 ~. (37) +2

3

Reagents Present during Preincubation

Total Counts per Minute

None Acetyl Glutamate Acetyl Glutamate + ATP + Mg

143 549 1890

a

+2

Figures in parenthesis correspond extended to 6 seconds.

Calculated C 0 Fixation, Μμπιοΐββ 2

6.4 24.4 84

(12.3)a (37.5) (129)

experiments where incubation time was

and v e r y s h o r t i n c u b a t i o n t i m e , i t w a s p o s s i b l e to d e m o n s t r a t e that the r a t l i v e r e n z y m e w a s a l s o a c t i v a t e d by a c e t y l glutamate. In the e x p e r i m e n t s of T a b l e V 4.5 m g . of p r o t e i n f r o m an acetone powder of r a t l i v e r m i t o c h o n d r i a (4) c o n t a i n i n g 100 μ π ι ο ΐ β β of T r i s - C l " at p H 7.4, w e r e p r e i n c u b a t e d at 2 5 ° , f o r 10 m i n u t e s , w i t h the f o l l o w i n g i n a v o l u m e of 1 m l . : a c e t y l glutamate, 5 μ m o l e s ; A T P , 4 μ m o l e s ; M g S 0 , 10 μ ι η ο ΐ β β ; and H C O â , 100 μ π ι ο ΐ β β (containing 50 μ c u r i e s ) . T h e tubes w e r e c o m p l e t e d at definite i n t e r v a l s w i t h the components and c o n c e n t r a t i o n of r e a g e n t s i n d i c a t e d i n T a b l e Ι Π , except that H C O g w a s used. The c o m p l e t e d tubes w e r e incubated at 2 5 ° f o r 3 s e c o n d s . R a d i o a c t i v i t y w a s d e t e r m i n e d i n a l i q u o t s f r o m the d e p r o t e i n i z e d supernatant fluids. 14

4

14

Effect of Acetyl

Glutamate

Concentration

The degree of a c t i v a t i o n i s a function of the c o n c e n t r a t i o n of a c e t y l glutamate, as shown i n F i g u r e 2. The m a x i m a l effect o c c u r s at 2 to 5 χ 10 " M a c e t y l glutamate; the c o n c e n t r a t i o n of e n z y m e i n these e x p e r i m e n t s w a s a p p r o x i m a t e l y 4 x 1 0 " M , 1 x 1 0 " M , and 2.2 x 1 0 " M f o r c u r v e s I , Π , and Ι Π , r e s p e c t i v e l y . H a l f - m a x i m a l a c t i v a t i o n o c c u r s at a p p r o x i m a t e l y 5 χ 10 "* M a c e t y l glutamate, a v a l u e v e r y c l o s e to the K m f o r the o v e r - a l l r e a c t i o n , and f o r the a c e t y l g l u t a m a t e - i n d u c e d i n a c t i v a t i o n . T h e s i g n i f i c a n c e of t h i s finding w a s pointed out by C a r a v a c a and G r i s o l i a (4). S i m i l a r findings w i t h other e n z y m e s (1, 14) i n d i c a t e s

6

5


5


136


nl

I I0"

7

MOLARITY

Figure

I IO"

6

I IO"

5

OF

1

I0"

4

1 IO"

1

1 3

IO'

2

ACETYL6LUTAM ATE

2. Dependence of activation phenomenon concentration of acetyl glutamate

on

1.45, 3.45, and 7.1 mg. of frog liver enzyme (bottom, middle, and top curves, respectively) in 0.1M TrisCl", pH 7.4, preincubated with acetyl glutamate at indicated molarities, at 38° for 10 minutes. Tubes cooled to 25°, completed at definite intervals, and assayed under conditions of Table III. Incubation at 25°, 1 minute the usefulness of the technique of substrate and cof actor-induced inactivation. We have shown that there is good agreement between dissociation constants obtained by kinetic studies, and by measurements of the sub strate-induced enzyme inactivation (4); the latter may yield true equi librium constants, as is the case for Κτ determinations, free from k i netic variables that may be present in estimations of Km (6). However, the constants determined by inducing inactivations cannot be accepted without properly evaluating other factors (14). Influence of pH on Activation. The pH dependence of the activation phenomenon is illustrated in Figure 3. The fact that the activation be gan to appear at pH 5.6, became maximal at pH near 7.4, and remained maximal at pH as high as 10.0, indicated that a certain ionic species of acetyl glutamate might be involved in the activation of the enzyme. Since the amino group of glutamate becomes more acidic on acetylation than on carbamylation, and since both acetyl and carbamyl glutamate



70.

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Ρ hot pa te Biosynthesis

i

Biosynthesis and Utilization of Acetyl Phosphate, Formyl Phosphate

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