The full time course of reduced coenzyme oxidation by glutamate dehydrogenase has been measured and the data have been compared with computer simulated progress curves. It is shown that the oxidation of TPNH, at saturating concentrations of α ketoglutarate and ammonium ion, follows a relatively simple mechanism but must include the formation of an abortive enzyme TPN substrate complex. The progress curve for the oxidation of DPNH is multiphasic and reflects the fact that DPNH may bind to a second site, inducing an isomerization of the enzyme which results in inhibition of the reaction. As a consequence of the fact that this isomerization is relatively slow, the kinetic properties using DPNH as coenzyme are dependent upon enzyme concentration. The rate constants for the induced isomerization and the dissociation constants for the abortive complex formation, for the reduced coenzyme binding to the active site, and for DPNH binding to the second site have been determined from fits of computer calculated simulation curves to the experimental data.
|Number of pages
|Journal of Biological Chemistry
|Published - 1973