IV NUCLEOTIDE SYNTHESIS 99 



{b) Homogenate and enzyme experiments 



A labile compound formed from CO2,, NH3, and ATP is an intermediate in the synthesis 

 of USA (Fig. 45). This labile compound may be carbamyl phosphate (Reichard and 

 Hanshoff, 1955; Lowenstein and Cohen, 1955; Jones et al., 1955; Marshall el al., 1955), 

 or it may be an unknown complex form of carbamyl phosphate, "Compound X" (Grisolia 

 et al., 1955). Acetyl glutamate stimulates, but is not essential, for the formation of carbamyl 

 phosphate or "Compound X". The synthesis of carbamyl phosphate has been demonstrated 

 so far only in liver mitochondria, kidney and in Streptococci, but the subsequent carbamyl- 

 ation of aspartic acid is catalyzed by enzymes present in most rat organs, and in many 

 organisms. "Compound X" can also be formed in liver tissue or in microorganisms from 

 the ureido group of citrulline by the reversal of the steps involved in the formation of 

 citrulline from ornithine, CO2, NH.,, and ATP (Reichard and Smith, 1 955) . This explains the 

 observations that carbamyl labelled citrulline is incorporated into carbon atom 2 oi JVeurospora 

 nucleic acid cytosine and uracil (Heinrichf^a/., 1954), the nucleic acid pyrimidines of pigeon 

 liver (Schulman and Badger, 1954), and orotic acid of rat liver (Smith and Stetten, 1954). 



Liver homogenates convert ureidosuccinic acid to dihydroorotic acid and both these 

 compounds to orotic acid (Cooper et al., 1955). Enzymes catalyzing the interconversion 

 of USA, DHO, and orotic acid have been purified from the orotic acid fermenting 

 bacterium, ^ymobacterium oroticum (Lieberman and Kornberg, 1955) and from E. coli 

 B cells (Yates and Pardee, 1956). The enzyme, dihydroorotic dehydrogenase is lacking, 

 however, from an E. coli mutant which cannot produce orotic acid but which can utilize 

 it as a growth factor. Another mutant, which uses uracil and cytosine as growth factors, 

 excretes USA, DHO, and orotic acid. 



{c) Orotic acid utilization 



Orotic acid is readily utilized for the synthesis of acid soluble nucleotide and 

 nucleic acid pyrimidines by normal and tumor tissues of the rat in vivo, by bacteria 

 (Hurlbert and Potter, 1952; Spicer et al., 1952; Reichard and Lagerkvist, 1953) 

 yeast (Edmonds et al., 1952) rat spleen, rat liver, and tumor tissue slices (Lagerkvist 

 et al., 1955; Reichard, 1952; Weed and Wilson, 1951). Soluble preparations 

 obtained from pigeon liver, E. coli cells, rat liver, intestine, and the Flexner-Jobling 

 rat tumor incorporate labelled orotic acid into uridine nucleotides. In the case of 

 rat liver and tumor, the largest amount of label is found in UTP (Heidelberger 

 and Harbers, 1956). The conversion of labelled orotic to UDP, UTP, and UDPG 

 by a pigeon liver enzyme system is greatly diminished by the addition of unlabelled 

 U MP while the radioactivity of U MP is increased. Although some radioactive uracil 

 and uridine are also formed, the labelling of these substances can be attributed 

 to the action of hydrolytic enzymes. Further evidence against the role of uridine 

 as an intermediate in UMP synthesis in this system is the fact that non-labelled 

 uridine does not alter the conversion of orotic-^'^C to UMP (Hurlbert and Reichard, 

 1954). Two enzymes mediate the conversion of orotic acid to UMP (Lieberman 

 et al., 1955a), an enzyme catalyzing the condensation of orotic with PRPP and an 

 enzyme which is concerned with the decarboxylation of orotidine-5'-phosphate. 



yeast — CO2 



Orotic -L PRPP > orotidine-5'-phosphate > UMP 



liver 



5. Utilization of uracil, cytosine and thymine 

 (a) Introduction 



Exogenous uracil may be utilized for the synthesis of nucleic acid pyrimidines in 

 bacteria (Moore and Boylen, 1955; Rose and Schweigert, 1953 ; Wright and Miller, 



Literature p. 124 



