title: Evaluation of the kinetic mechanism of Escherichia coli glycinamide ribonucleotide transformylase.
abstract: 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.
authors.affiliation: Department of Chemistry, 152 Davey Laboratory, The Pennsylvania State University, University Park, Pennsylvania 16802, USA.
authors.forename: J H
authors.initials: JH
authors.lastname: Shim
authors.forename: S J
authors.initials: SJ
authors.lastname: Benkovic
mesh_terms: D001426:Bacterial Proteins / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology*; D019877:Hydroxymethyl and Formyl Transferases / Q000378:metabolism*; D007700:Kinetics; D051026:Phosphoribosylglycinamide Formyltransferase; D013379:Substrate Specificity
chemical_list: D001426:Bacterial Proteins; D019877:Hydroxymethyl and Formyl Transferases; D051026:Phosphoribosylglycinamide Formyltransferase