Studies on the mechanism and kinetics of the 2 oxoglutarate dehydrogenase system from pig heart

Abstract

The kinetic properties of the 2 oxoglutarate dehydrogenase system were investigated. To this end, initial velocity studies were carried out by the method of Fromm. Reciprocal plots of the results did not agree with those expected for the Hexa Uni Ping Pong mechanism previously proposed for the system. The measured initial velocities were fitted to initial rate equations corresponding to several possible mechanisms by using a computer optimization technique. Statistical analyses performed on the results of the optimization studies indicated that one mechanism was a significantly better fit to the experimental data than the other mechanisms tested. This mechanism is one in which there is a random order of binding of NAD+ and CoA and release of succinyl CoA, although the binding of 2 oxoglutarate and release of CO2 is still given a Ping Pong mechanism, which precedes the binding of the other substrates. These conclusions were supported by NADH inhibition studies. The usefulness of the method of fitting initial rate data to rate equations and the applicability of the proposed enzymic mechanism to the enzyme complex are discussed.