Rate-determining processes and the number of simultaneously active sites of d-glyceraldehyde 3-phosphate dehydrogenase |
| |
Authors: | D R Trentham |
| |
Institution: | Molecular Enzymology Laboratory, Department of Biochemistry, University of Bristol, University Walk, Bristol BS8 1TD, U.K. |
| |
Abstract: | 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. |
| |
Keywords: | |
|
|