A stopped flow transient kinetic analysis of substrate binding and catalysis in Escherichia coli D-3-phosphoglycerate dehydrogenase.

ABSTRACT: Pre-steady state, stopped flow analysis of Escherichia coli D-3-phosphoglycerate dehydrogenase was performed by following the fluorescence of protein tryptophan and the fluorescence resonance energy transfer from protein tryptophan to bound NADH. The results indicate that binding of substrates is ordered, with coenzyme, NADH, binding first. Furthermore, the analysis indicated that there are two sets of sites on the tetrameric enzyme that can be differentiated by their kinetic behavior. NADH binding was consistent with an initial binding event followed by a slow conformational change for each site. The slow conformational change is responsible for the apparent tight binding of NADH to the apoenzyme but is too slow to participate in the catalytic cycle when the enzyme is rapidly turning over

SUBMITTER: Burton RL 

PROVIDER: S-EPMC2573093 | biostudies-literature | 2008 Oct

REPOSITORIES: biostudies-literature