Transformylase activity

The possibility that the iV °-formyl derivative (III.163) might serve as a substrate for two important enzymes of the de novo purine pathway that utilize reduced folates as their natural substrates, namely 5-aminoimidazole-4-car-boxamide ribonucleotide (AICAR) transformylase and glycinamide ribonucleotide (GAR) transformylase, was examined [61]. While the affinity of (III. 163) (AT, (app) = 29/xM) for AICAR transformylase appeared to be greater than that of the natural substrate 10-formyltetrahydrofolate (A (app) = 68 /xM), the reaction was slow, resulting in a 750-fold lower Frei/Am(app) ratio for the quinazoline. The affinity of (III. 163) (/frn(app) = 1.9 /xM) for GAR transformylase was likewise several times greater than that of the natural substrate, in this case 5,10-methenyltetra-hydrofolate (/if,n(app) = 8.9/xM). However, (III. 163) was also used rather efficiently in the reaction by GAR transformylase, resulting in a 4-fold higher V.J Ai, (app) for the quinazoline than for 5,10-methenyltetrahydrofolate. The authors concluded from these results that the 5,10-methenyl structure is not needed for GAR transformylase activity. 

A bifunctional enzyme, comprising the activities of AIR carboxylase and SAICAR synthetase, catalyzes reactions 6 and 7 of the purine pathway (AIR—> CAIR— SAICAR Fig. 15-16). A second bifunctional enzyme, IMP synthase, containing the activities of AICAR transformylase and IMP cyclohydrolase, catalyzes reactions 9 and 10 of the pathway (AICAR — FAICAR— IMP Fig. 15-16). Human IMP synthase has a subunit molecular weight of 62.1 kDa and associates as a dimer. A... 

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. 

Human IMP synthase has a subunit molecular weight of 62.1 kDa and associates as a dimer. A trifunctional enzyme, Cj-THF synthase, containing (V °-methenyl tetrahydrofolate (5,10-CH-THF) cyclohydrolase and A °-formyl tetrahydrofolate (lO-CHO-THF) synthetase, catalyzes the reactions 5,10-CH2-THF 5,10-CH-THF and THF —> lO-CHO-THF. The A °-CH-THF produced is a substrate for GAR and AICAR transformylases catalyzing reactions 3 and 9 of the pathway. In higher eukaryotes, the dehydrogenase and cyclohydrolase activities are located in one domain of the protein, which is fused to a larger synthetase domain, forming a trifunctional enzyme. 

F ormyltransferases 5,10-Methenyltetra-hydrofolate, 10-formyltetrahy dro-folate ( activated formic acid ) Glycineamide ribonucleotide transformylase (D 10.4) 5-Aminoimidazole-4-carboxamide ribonucleotide transformylase (D 10.4)... 

To decide between the remaining two possibilities an experiment was performed b ed on some recent observations concerning the optical specificity of tetrahydrofolate in the phenylalanine hydroxylating reaction. It has been found that only one isomer of tetrahydrofolate is active in the hydroxylating system the isomer which is synthesized enzymically in either the dihydrofolic reductase (Osborn and Huennekens, 1958) or the glutamic transformylase system (Silverman et al., 1957) is almost completely inactive (Kaufman, unpublished). 

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