Mammalian Glycinamide Ribonucleotide Transformylase

Tight Binding of Folate Substrates and Inhibitors to Recombinant Mouse Glycinamide Ribonucleotide Formyltransferase

“…DDATHF was found to bind to rmGARFT equally well in the presence or absence of saturating concentrations of GAR, and GAR was found to bind equally well to enzyme in the presence or absence of 2 µM (>1000 K d s) DDATHF, leading to the conclusion that rmGARFT binds these ligands in a random order. The previous experiments (26), which suggested an ordered sequential binding mechanism, were performed at high concentrations of 10-CHO-5,8-dideazafolate, relative to the K m reported in this paper, so that binding reactions involving folate substrate would become essentially irreversible (heavy arrows) and the contribution of the competing reactions shown in the dotted box would be negligible. Under such conditons, an enzyme operating by a random addition pathway will behave like an ordered sequential system.…”

“…If one superimposes the crystal structures for the ternary complex of E. coli GARFT, GAR, and 5-deazatetrahydrofolate with that of the apo enzyme (31), the shape of the GAR binding pocket does not change, suggesting that the binding of folates does not alter the binding site for GAR. It is noted that the previously reported steady-state kinetic patterns suggesting an ordered sequential binding order (26) would have been performed under uniformly saturating folate conditions, under which condition any binding step which competed with binding of folate would have a negligible impact on the applicable kinetic equations (Figure 7). We have not been able to pursue steady-state experiments to examine the order of binding by a parallel approach due to the low signal at K m levels of substrate even with 10 cm cuvettes and the level of product accumulation at higher substrate concentrations in the 25 mL volume of these cuvettes.…”

“…The binding constant calculated from the data in Figure 3A for the interaction of rmGARFT and DDATHF hexaglutamate was 0.4 nM, consistent with the results obtained for direct binding of labeled DDATHF hexaglutamate to the enzyme (0.2 nM) as well as the K i values obtained from steady-state kinetics [0.5 nM (13), and 0.11 nM (see below)]. It was noted that the K d calculated 4 for 10-CHO-5,8-dideazafolic acid from these data, 30 nM, was much lower than the K m of this substrate for mouse GARFT [reported to be 1.3 µM (13) or higher (25,26)).…”

“…Binding of GAR to rmGARFT. Previous kinetic investigations on vertebrate GARFT (26), based on the use of dead-end inhibitors, have supported an ordered sequential pathway for the addition of substrates with folate binding preceding that of GAR. The K m values found in the present studies would predict that the concentrations of the folate substrate would be saturating at all concentrations used in previous studies, a condition which could mask a random order binding mechanism, which would appear to be ordered sequential.…”

“…Kinetic ReeValuation of the Interaction of DDATHF with rmGARFT. Although it was clearly possible that the concentration range at which 10-CHO-5,8-dideazafolic acid competed for DDATHF binding to rmGARFT (Figure 3B) and the K m for this compound as a substrate for GARFT (13,(25)(26)(27) be different, the magnitude of this difference prompted us to reexamine the kinetics of the GARFT reaction. When the dependency of the rate of the rmGARFT reaction on 10-CHO-5,8-dideazafolate concentration was studied spectrophotometrically using 10 cm cuvettes to amplify the signal and measuring the rate of the reaction during the first 10-15 s as an estimate of the initial velocity and to minimize the effects of product accumulation (see below), the K m was found to be 75 ( 14 nM.…”