The Kinetics of Succinyl Coenzyme A Synthetase from Escherichia coli: A REACTION WITH A COVALENT ENZYME-SUBSTRATE INTERMEDIATE NOT EXHIBITING "PING-PONG" KINETICS

Abstract

Succinyl coenzyme A synthetase from Escherichia coli does not exhibit a steady state kinetic pattern indicative of kinetics, despite the fact that catalytic participation of a phosphorylated enzyme covalent intermediate has been established. Instead, kinetic patterns are consistent with the sequential addition of all substrates, to form a quaternary complex, before the release of any product. Double reciprocal plots are intersecting with ATP and succinate varied at nonsaturating concentration of the third substrate, CoA. These plots become parallel at higher CoA concentrations, suggesting that CoA can be the second substrate to add in the binding sequence. Analogous reciprocal plots with ATP and CoA varied at both nonsaturating and saturating succinate concentrations are also intersecting and parallel, respectively. The data may be reconciled with a binding sequence in which ATP binds to the enzyme first, followed by the random addition of CoA and succinate. The finding of a sequential mechanism for the addition of substrates to this enzyme, whose catalysis is known to involve a covalent intermediate, serves to illustrate a possible danger in the use of initial rate kinetics. It is not possible to discount chemical mechanisms involving covalent intermediates by failure to observe the ping-pong kinetic pattern which may be anticipated for these catalytic routes.