Ribonucleotide transformylase, glycinamide

Glycinamide ribonucleotide transformylase (GAR Tfase) is a folate-dependent enzyme essential to the de novo purine biosynthetic pathway. It utilizes the cofactor 10-formyl tetrahydrofohc acid (10-formyl-THF) to transfer a formyl group to the primary amine of its substrate a-glycinamide ribonucleotide. Potent, and potentially selective, inhibitors of GARTfase and de novo purine biosynthesis have been shown to be promising as antitumor drugs. 

Directly coupled HPLC-NMR spectroscopy has been used in a number of other studies of chemical impurities. An impurity in a bulk drug sample of the glycinamide ribonucleotide transformylase inhibitor AG2034, shown below ... 

Aimi,J., Qiu, H., Williams, J., Zalkin, H., and Dixon, J. E. (1990). De novo purine nucleotide biosynthesis cloning of human and avian cDNAs encoding the trifunctional glycinam-ide ribonucleotide synthetase-aminoimidazole ribonucleotide synthetase-glycinamide ribonucleotide transformylase by functional complementation in E. colt. Nucleic Acids Res., 18, 6665-6672. 

Fig. 9.8. a) Structural overlay of glycinamide ribonucleotide transformylase (PurN) [155] and the N-terminal domain of tRNA(fMet)-formyl-transferase (FMT) [156] from E. coli. Despite the low sequence identity (33 %), the two structures are almost perfectly superimposable. b) Structure-based multiple sequence alignment of PurN... 

J.B. Thoden, S. Firestine, A. Nixon, S. J. Benkovic, and H.M. Holden. 2000. Molecular structure of Escherichia coli PurT-encoded glycinamide ribonucleotide transformylase Biochemistry 39 8791-8802. (PubMed)... 

The transfer of 1-carbon units at this oxidation level originally was thought to involve two derivatives of H4-folate, 10-formyl-H4-folate and 5,10-methenyl-H4-folate which acted as cofactors for the two transformylases in de novo purine biosynthesis [67-69]. However, recent work has shown that the glycinamide ribonucleotide transformylase (GAR TFase) from E. coli as well as from avian liver utilize 10-formyl-H4-folate as the actual cofactor [70,71]. The preference for 10-for-myl-H 4-folate was masked by the presence of the opposite, unreactive diastereomer (R at C-6 in H4-folate) which is an excellent competitive inhibitor of the enzyme. The apparent reactivity of the 5,10-methenyl-H4-folate in the same assay arose because of a contaminating cyclohydrolase activity capable of selectively hydrolyzing it to the correct diastereomer of 10-formyl-H4-folate. 

Phosphoribosyl-1-pyrophosphate synthetase 2 aminophosphoribosyltransferase 3 phospho-ribosylglycinamide synthetase 4 glycinamide ribonucleotide transformylase 5 iV-formylglycin-amidine ribonucleotide amidoligase 6 5-aminoimidazole ribonucleotide synthetase 7 5-amino-imidazole ribonucleotide carboxylase 8 5-aminoimidazole-4-iV-succinocarboxamide ribonucleotide synthetase 9 adenylosuccinate lyase 10 5-aminoimidazole-4-carboxamide ribonucleotide transformylase 11 inosinicase... 

The 5-amino-4-imidazole carboxamide ribonucleotide transformylase and the glycinamide ribonucleotide transformylase reactions have been studied in chicken liver preparations by Hartman and Buchanan. 

Figure 44.1 Folate-mediated one carbon metabolism network. Enzymes and transport proteins are enclosed in rectangular boxes. AHCY S-adenosyDiomocys-teine hydrolase AICART 5-aminoimidazole carboxamide ribonucleotide transferase BHMT betaine homocysteine methyltransferase CBS cystathionine beta-synthase DHFR dihydrofolate reductase FR folate receptor FTCD formimidoyltransferase cyclodeaminase GART glycinamide ribonucleotide transformylase MATs (MATI/MATIII) adenosylmethionine transferase enzyme I/III MS methionine synthase MSR methionine synthase reductase MT methyltransferase MTHFD methylenetetrahydrofolate dehydrogenase MTHFR 5,10-methylenete-trahydrofolate reductase MTHFS 5,10-methylenetetrahydrofolate synthase. RFC reduced folate AdoMet 5-adenosylmethionine AdoHcy S-adenosylhomocysteine Hey homocysteine SHMT serine hydroxymethyltransferase TS thymidylate synthase.

The formylation of GAR to produce FGAR is catalyzed by glycinamide ribonucleotide transformylase. The immediate formyl donors are derivatives of tetrahydrofolic acid (FIR) (96, 107, 108). With purified enzyme preparations, iV, Ar -anhydroformyltetrahydrofolic acid alone donates its formyl group to GAR (/09). Because of their rapid interconversion by the enzyme cyclohydrolase (110), both JV -formyltetrahydrofolic acid and W, Ar -arjhydroformyltetrahydrofolio acid were reactive in crude enzyme... 

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