35. BIOSYNTHESIS OF PURINE NUCLEOTIDES 311 



in this reaction by a nucleophilic attack of the nitrogen atom of glutamine 

 upon the carbon atom bearing the pyrophosphate substituent. 5-Phospho- 

 ribosyl pyrophosphate has been demonstrated to be of the a configuration. 

 5-Phosphoribosylamine is presumed to be a /3-ribofuranosyl derivative since 

 during the further reactions of purine synthesis the ^-nucleotides are 

 formed without any other apparent opportunity for change in configuration 

 on the part of the purine nucleotide intermediates. It has been suggested 

 that the several operations of the reaction, i.e., the release of pyrophos- 

 phate, the transfer of the amide group, and the introduction of the elements 

 of water are catalyzed by one enzyme without the formation of a discrete 

 covalent intermediate such as jV-(5-phosphoribosyl)glutamine. 



The second step in purine biosynthesis 41 • 43 involves the reaction of 

 5-phosphoribosylamine, ATP, and glycine to yield glycinamide ribonucleo- 



5-Phosphoribosyhimine + glycine + ATP «=* 



(3) 

 glycinamide ribonucleotide + ADP + orthophosphate 



tide according to Eq. (3). This reaction is freely reversible. The amide bond 

 is not broken in the splitting of glycinamide ribonucleotide unless both 

 ADP and orthophosphate are present. Arsenate may replace orthophos- 

 phate but ADP is still an essential component of the system. In addition 

 the oxygen of orthophosphate labeled with O 18 is transferred to the car- 

 boxyl carbon of glycine during its formation from glycinamide ribonucleo- 

 tide. These observations have led to the postulation that the synthesis and 

 breakdown of glycinamide ribonucleotide occurs when all three substrates 

 (either 5-phosphoribosylamine, glycine, and ATP or glycinamide ribo- 

 nucleotide, ADP, and orthophosphate depending upon the direction of re- 

 action) are present and can interact simultaneously. The enzyme system 

 responsible for this synthesis has been called glycinamide ribonucleotide 

 kinosynthase. 



The formylation of glycinamide ribonucleotide to formylglycinamide ri- 



iV 5 ,A ri0 -Anhydroformyl-FH 4 + glycinamide ribonucleotide + H 2 — » 



(4) 

 formylglycinamide ribonucleotide + FH 4 + H + 



bonucleotide 26 • 44 takes place according to Eq. (4). This reaction is very 

 easily measured by determining the amount of tetrahydrofolic acid (FH 4 ) 

 formed. Under the acid conditions of the Bratton and Marshall procedure 

 tetrahydrofolic acid breaks down to p-aminobenzoyl glutamate, which may 

 be diazotized and coupled with AM-naphthylethylenediamine to form a 



« S. C. Hartman and J. M. Buchanan, J. Biol. Chem. 233, 456 (1958). 

 " D. A. Goldthwait, R. A. Peabody, and G. R. Greenberg, J. Biol. Chem. 221, 569 

 (1956). 



