“…The formation of the enzyme−fDDF complex (E−fDDF) was followed by a time-dependent increase in the fluorescence signal at 400 nm (Figure ). The observed rate constant can be expressed in terms of the rate constants in Scheme under the near-equilibrium condition ( 22 ), and is a function of all four rate constants and the concentration of DDF and fDDF (eq 1) By varying the DDF concentration between 34 and 135 μM and fDDF between 39 and 230 μM, we obtained k obs values ranging from 380 to 670 s -1 .…”
Section: Resultsmentioning
confidence: 99%
“…This enzyme formylates glycinamide ribonucleotide (GAR) using the 10-formyltetrahydrofolate cofactor to make formylglycinamide ribonucleotide (fGAR) (Scheme 1). The GAR transformylase, especially the product of the Escherichia coli purN gene, 2 has been the subject of intensive structural studies owing to its small size (23 241 Da) (3,4) relative to the human trifunctional enzyme (5). An efficient overexpression system in an auxotropic E. coli (purN -, purT -) strain was developed recently (6) permitting either wild type or mutant enzyme production.…”
A kinetic scheme is presented for Escherichia coli glycinamide ribonucleotide transformylase (GAR transformylase, EC 2.1.2.2) based on a steady-state and pre-steady-state kinetic analysis of the reaction in both directions employing stopped-flow absorbance and fluorescence spectroscopy. Steady-state parameters showed that kcat for the reverse direction is about 10 times lower than that for the forward direction although the Km values for formyl dideazafolate and dideazafolate or for glycinamide ribonucleotide and formyl glycinamide ribonucleotide are similar. No pre-steady-state transient was observed in either direction, and the single-turnover rate constant under saturating levels of substrates in each direction was found to be very close to the respective steady-state kcat value. This indicates that steps involving ternary complexes are rate-determining for steady-state turnover in each direction. By conducting the single-turnover reactions under various preincubation and mixing conditions, a random sequential kinetic mechanism was implicated in which the enzyme binds glycinamide ribonucleotide or formyl dideazafolate productively in no obligatory order. The collective data provided a quantitative kinetic scheme to serve as a basis for the analysis of mutations.
“…The formation of the enzyme−fDDF complex (E−fDDF) was followed by a time-dependent increase in the fluorescence signal at 400 nm (Figure ). The observed rate constant can be expressed in terms of the rate constants in Scheme under the near-equilibrium condition ( 22 ), and is a function of all four rate constants and the concentration of DDF and fDDF (eq 1) By varying the DDF concentration between 34 and 135 μM and fDDF between 39 and 230 μM, we obtained k obs values ranging from 380 to 670 s -1 .…”
Section: Resultsmentioning
confidence: 99%
“…This enzyme formylates glycinamide ribonucleotide (GAR) using the 10-formyltetrahydrofolate cofactor to make formylglycinamide ribonucleotide (fGAR) (Scheme 1). The GAR transformylase, especially the product of the Escherichia coli purN gene, 2 has been the subject of intensive structural studies owing to its small size (23 241 Da) (3,4) relative to the human trifunctional enzyme (5). An efficient overexpression system in an auxotropic E. coli (purN -, purT -) strain was developed recently (6) permitting either wild type or mutant enzyme production.…”
A kinetic scheme is presented for Escherichia coli glycinamide ribonucleotide transformylase (GAR transformylase, EC 2.1.2.2) based on a steady-state and pre-steady-state kinetic analysis of the reaction in both directions employing stopped-flow absorbance and fluorescence spectroscopy. Steady-state parameters showed that kcat for the reverse direction is about 10 times lower than that for the forward direction although the Km values for formyl dideazafolate and dideazafolate or for glycinamide ribonucleotide and formyl glycinamide ribonucleotide are similar. No pre-steady-state transient was observed in either direction, and the single-turnover rate constant under saturating levels of substrates in each direction was found to be very close to the respective steady-state kcat value. This indicates that steps involving ternary complexes are rate-determining for steady-state turnover in each direction. By conducting the single-turnover reactions under various preincubation and mixing conditions, a random sequential kinetic mechanism was implicated in which the enzyme binds glycinamide ribonucleotide or formyl dideazafolate productively in no obligatory order. The collective data provided a quantitative kinetic scheme to serve as a basis for the analysis of mutations.
“…Glycinamide ribonucleotide transformylase (GAR transformylase, 1 EC 2.1.2.2) catalyzes the first of the two formyl transfers in the de novo purine biosynthetic pathway and forms the C-8 atom of the purine ring. The formyl group is transferred from N10-formyl tetrahydrofolate to the amino group of glycinamide ribonucleotide (GAR) to form formylglycinamide ribonucleotide (fGAR) (Figure 1).…”
mentioning
confidence: 99%
“…Because of its biological and pharmacological importance, the enzyme has been the subject of intensive studies on its structure and mode of action. These investigations have focused on the Escherichia coli enzyme which is a small monomer (23 kDa) ( , ) rather than a covalent constituent of the large trifunctional protein found in eukaryotes, yet exhibits a high degree of sequence homology and mechanistic similarity with respect to its eukaryotic counterpart ( , ). 1 Reaction catalyzed by GAR transformylase with a tetrahedral transition state.…”
Site-directed mutagenesis followed by studies of the pH dependence of the kinetic parameters of the mutants has been used to probe the role of the active site residues and loops in catalysis by glycinamide ribonucleotide transformylase (EC 2.1.2.2). The analysis of the mutants of the strictly conserved active site residues, His108 and Asp144, revealed that His108 acts in a salt bridge with Asp144 as a general acid catalyst with a pK(a) value of 9.7. Asp144 also plays a key role in the preparation of the active site geometry for catalysis. The rate-limiting step in the pH range of 6-10 appears to be the catalytic steps involving tetrahedral intermediates, supported by the observation of a pL (L being H or D)-independent solvent deuterium isotope effect of 2. The ionization of the amino group of glycinamide ribonucleotide both as a free and as a bound form dominates the kinetic behavior at low pH. The analysis of a mutation, H121Q, within the loop spanning amino acids 111-131 suggests the closure of the loop is involved in the binding of the substrate. The kinetic behavior parallels pH effects revealed by a series of X-ray crystallographic structures of the apoenzyme and inhibitor-bound enzyme [Su, Y., Yamashita, M. M., Greasley, S. E. , Mullen, C. A., Shim, J. H., Jennings, P. A., Benkovic, S. J., and Wilson, I. A. (1998) J. Mol. Biol. 281, 485-499], permitting a more exact formulation of the probable catalytic mechanism.
“…A comparison of the primary protein sequences of the four species has been made in Figure 1. These sequences were obtained from E. coli (Smith & Daum, 1987), Bacillus subtilis (Ebbole & Zalkin, 1987), and Drosophila melanogaster and Saccharomyces cerevisiae (Henikoff, 1986) by translation of the respective cDNA clones. Smith has noted regions of homology within these species.…”
The affinity reagent N10-(bromoacetyl)-5,8-dideazafolate has previously been shown to inactivate glycinamide ribonucleotide transformylase (EC 2.1.2.2) from Escherichia coli in an active-site-directed manner with a 1:1 stoichiometry [Inglese et al. (1990) Biochemistry 29, 1436-1443]. After a series of mild proteolytic digestions, the dideazafolate label was localized to an active-site peptide attached by an ester linkage to the highly conserved residue Asp 144. Subsequent site-specific mutagenesis of Asp 144 to Asn 144 resulted in a catalytically inactive enzyme that retained the ability to bind substrates and inhibitors. The Asn 144 mutant could be further labeled with the affinity reagent in an active-site-directed stoichiometric fashion; however, the site of modification in this case was His 119. These results imply that Asp 144 may function as a general base within the catalytic center of the transformylase and is in close proximity to His 119 in the folded protein.
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