326 GEORGE W. CROSBIE 



formed in extracts of Streptococcus faecalis and that carbamyl phosphate is 

 a substrate of ornithine carbamyl transferase. Using an extract of rat liver 

 and a purified E. coli enzyme Reichard and Hanshoff 21 have confirmed the 

 role of carbamyl phosphate (compound X) in aspartate carbamyl trans- 

 ferase action and have established the nonidentity of the aspartate and 

 ornithine carbamyl transferases. The coliform aspartate carbamyl trans- 

 ferase has been considerably purified and good stoichiometry for the reac- 

 tion: carbamyl phosphate + aspartate — > carbamylaspartate + inorganic 

 phosphate, has been obtained. Evidence for reversibility was obtained only 

 by the use of inorganic phosphate-P 32 . The mechanism of the reaction ap- 

 parently does not involve a carbamyl-enzyme complex as indicated by nega- 

 tive isotope exchange experiments of the type (a) carbamyl phosphate + 

 inorganic phosphate-P 32 -f- enzyme and (b) aspartate-C 14 -f- carbamyl- 

 aspartate + enzyme. The mechanism probably involves a nucleophilic at- 

 tack of the N-atom of aspartic acid on the carbonyl group of carbamyl phos- 

 phate subsequent to the binding of the substrates on the enzyme. 



Lieberman and Kornberg 22 have described a ureidosuccinase enzyme ob- 

 tained from cells of Zymobacterium oroticum grown anaerobically on orotic 

 acid as the sole energy source. The enzyme effects the hydrolysis of car- 

 bamylaspartic acid to NH 3 , CO2 , and aspartic acid. A requirement for a 

 sulfhydryl compound and Mn ++ but not for adenosine-5'-diphosphate 

 (ADP) has been reported (cf. the ATP-yielding cleavage of citrulline 23 ). 

 The highly exergonic character of the reaction together with its effective 

 irreversibility led to the postulation of the synthesis of carbamylaspartic 

 acid from aspartic acid via arginosuccinate, a compound previously impli- 

 cated in pyrimidine biosynthesis on the basis of the observed utilization of 

 citrulline-ureido-C 14 for polynucleotide pyrimidine synthesis in Neurospora 24 

 and pigeon liver homogenates 25 and for orotic acid synthesis in rat liver 

 slices. 26 These observations on citrulline utilization have been integrated, 

 however, with the pathway described in Fig. 1 by the observation by Smith 

 and Reichard 27 of carbamyl phosphate formation from citrulline by a rat 

 liver mitochondrial extract in the presence of ATP, Mg ++ , and acetyl- 

 glutamate. The carbamyl group of citrulline may also be transferred to 

 aspartic acid to yield carbamylaspartate through the coupling of the or- 

 nithine and aspartate carbamyl transferases. The equilibrium of the coupled 

 system is in favor of carbamylaspartate formation. 27 ' 1 



21 P. Reichard and G. Hanshoff, Acta Chem. Srand. 10, 548 (1956). 



22 I. Lieberman and A. Kornberg, J. Biol. Chem. 212, 909 (1955). 



23 V. A. Knivett, Biochem. J. 56, 602 (1954). 



24 M. R. Heinrich, V. C. Dewey, and G. W. Kidder, J. Am. Chem. Soc. 76, 3102 (1954). 



25 M. P. Schulman and S. J. Badger, Federation Proc. 13, 292 (1954). 



26 L. H. Smith, Jr., and D. Stetten, J. Am. Chem. Soc. 76, 3864 (1954). 



27 L. H. Smith and P. Reichard, Acta Chem. Scand. 10, 1024 (1956). 

 273 P. Reichard, Ada Chem. Scand. 11, 523 (1957). 



