Transcarboxylase: VI. KINETIC ANALYSIS OF THE REACTION MECHANISM

Abstract

Abstract The forward and reverse reactions catalyzed by transcarboxylase have been studied in the presence and absence of products and substrate analogues. The kinetic patterns of initial velocities at low substrate concentrations support the conclusion that the transcarboxylase reaction proceeds through two half-reactions in a mechanism with an intermediate formation of an enzyme-biotin∼CO2 complex. However, kinetic patterns with inhibitors were found to be inconsistent with the predicted patterns for a ping-pong mechanism since the inhibition was competitive when the inhibitor and the varied substrate were similar, that is, both coenzyme A compounds or both keto acids. Dissimilar combinations of inhibitors and varied substrates resulted in noncompetitive inhibition when the inhibitor was a product of the reaction, and uncompetitive inhibition when the inhibitor was an inactive analogue of the fixed substrate. These findings are interpreted as indicating that transcarboxylase has two types of separate and independent binding sites, one for binding each of two groups of substrates and inhibitors; coenzyme A compounds constitute one group of reactants, and keto acids constitute the other group. Consequently, the mechanism constructed for the oxalacetate transcarboxylase reaction may be described as a hybrid between a ping-pong mechanism and a random, ternary complex mechanism, since it involves an intermediate formation of a substituted form of the enzyme and allows independent binding of substrates to two distinct sites on the enzyme. Rate equations that establish a consistency between the new hybrid ping-pong mechanism and the observed kinetic data have been derived with the use of the combined assumptions of rapid equilibrium and steady state kinetic theory.