| Abstract
| - 3-Phosphoglycerate kinase (PGK) is the first ATP-producing enzyme in glycolysis: ADP +1,3-bisphosphoglycerate (bPG) ↔ ATP + 3-phosphoglycerate (PG). Whereas extensive studies have beencarried out on its structure, there is less information about its reaction pathway, which is usually studiedin the reverse direction because of the instability of bPG. We studied the transients of the PGK reactionby chemical sampling in a rapid quench flow apparatus, using [γ-32P]ATP, in 30% methanol at 4 °C todecrease kcat. There were two types of experiment, both at low PG concentrations to prevent bPG release.In the first, reaction mixtures were quenched in acid at different times (from 4 ms) and the bPGconcentrations were determined. This type gave information about the ATP binding and phospho-transfersteps. In the second, PGK reaction mixtures at equilibrium were perturbed by the injection of ADP, thenew mixtures aged for different times and quenched in acid, and the bPG concentrations were determined.This gave information about the kinetics of the binding of ADP to a PGK intermediate. The data from thetwo types of experiments were fitted to simple schemes and then treated together by a global fittingprocedure using a five-step pathway, deduced from previous structural studies. Under our conditions, itappears that (1) a binary PGK·bPG complex is an important intermediate on the reaction pathway, i.e.,that ADP is released before bPG, (2) ADP binds to a “closed” conformation in the PGK·bPG complex,and (3) the PGK reaction can be studied in the physiologically important direction without having tohandle bPG.
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