Rate-determining processes and the number of simultaneously active sties of D-glyceraldehyde 3-phosphate dehydrogenase.
Transient kinetic studies of the reversible oxidative phosphorylation of d-glyceraldehyde 3-phosphate catalysed by d-glyceraldehyde 3-phosphate dehydrogenase show that all four sites of the tetrameric lobster enzyme are simultaneously active, apparently with equal reactivity. The rate-determining step of the oxidative phosphorylation is NADH release at high pH and phosphorolysis of the acyl-enzyme at low pH. For the reverse reaction the rate-determining step is a process associated with NADH binding, probably a conformation change, at high pH and d-glyceraldehyde 3-phosphate release at low pH. NADH has previously been shown to be a competitive inhibitor of the enzyme with respect to d-glyceraldehyde 3-phosphate and vice versa. This is consistent with the mechanism deduced from transient experiments given the additional proviso that 1-arseno-3-phosphoglycerate has a half-life of about 1min or longer at pH7. The dissociation constants of d-glyceraldehyde 3-phosphate and 1,3-diphosphoglycerate to the NAD(+)-bound enzyme are too large to measure but are nevertheless consistent with the low K(m) values of these substrates.
D001665:Binding Sites; D005985:Glyceraldehyde; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008956:Models, Chemical; D009132:Muscles / Q000201:enzymology; D009243:NAD; D008121:Nephropidae; D010085:Oxidative Phosphorylation; D013053:Spectrophotometry
D009243:NAD; D005985:Glyceraldehyde; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases