35. BIOSYNTHESIS OF PURINE NUCLEOTIDES 317 



contains an enzymic component, inosinicase, which is capable of cyclizing 

 the formamido compound to inosinic acid [Eq. (12)]. 59 The transformylase 

 requires potassium ions for activity while inosinicase does not. 



5-Formamido-4-imidazolecarboxamide ribonucleotide «=* inosinie acid + H>(J (12) 



5-Amino-4-imidazolecarboxamide ribonucleotide transformylase 59 may 

 be determined by measuring the disappearance of nonacetylatable diazo- 

 tizable amine catalyzed by the enzyme in the presence of iV 1 °-f ormyltetra- 

 hydrofolic acid. Since tetrahydrofolic acid, which is produced in the reac- 

 tion, is decomposed to an acetylatable arylamine during the course of the 

 Bratton and Marshall procedure an acetylation step must be introduced to 

 prevent its reaction in the test. 



Although 5-formamido-4-imidazolecarboxamide ribonucleotide does not 

 accumulate in the enzymic reactions it may be synthesized chemically by 

 formylating the free amine in the presence of a mixture of concentrated 

 formic acid and acetic anhydride. The formamido compound has the same 

 spectral characteristics as the free amine with an absorption maximum at 

 267 nui- Since this material is a substituted amine it does not react in the 

 Bratton and Marshall procedure but may readily be converted quantita- 

 tively to the free amine by a brief hydrolysis in 0.1 N HC1 at 100°. 



The formamido compound is readily converted enzymically to inosinic 

 acid. The enzyme is specific for the phosphoribosyl compound and does not 

 catalyze the cyclization of the corresponding ribonucleoside or the free base. 

 It has been possible to demonstrate the reversibility of Eq. (12) even though 

 the equilibrium of the reaction greatly favors the formation of inosinic acid. 

 At equilibrium the ratio of inosinic acid to 5-formamido-4-imidazolecar- 

 boxamide ribonucleotide is 16,000 to l. 60 



At the present time it has not been possible to separate the carboxamide 

 ribonucleotide transformylase from inosinicase. During purification of the 

 enzyme from chicken liver the ratio of the two activities remains constant. 

 In certain mutants of Neurospora crassa loss of one activity will be accom- 

 panied by loss of the other. There are a few instances, however, where 

 inosinicase activity may be retained while the transformylase is lost. It 

 thus is difficult to decide whether both activities reside in one protein or 

 not. In any event carboxamide ribonucleotide transformylase and ino- 

 sinicase are closely associated with each other structurally, functionally, 

 and genetically. 



In contrast to the formylation of glycinamide ribonucleotide, the for- 

 mylation of 5-amino-4-imidazolecarboxamide ribonucleotide requires spe- 

 cifically the Af 10 -formyltetrahydrofolic acid as the formyl donor. 46 Prepara- 

 tions of the carboxamide ribonucleotide transformylase which contain 

 cyclohydrolase may utilize either the A^-formyl or the ./V 5 ,./V 10 -anhydro- 



