The Enzymatic Carboxylation of Phosphoenolpyruvate

The results of the kinetic studies described in this report, of the binding studies in the accompanying paper, and of earlier investigations have led to the formulation of a kinetic mechanism for the carboxylation reaction catalyzed by phosphoenolpyruvate carboxykinase (PEP carboxykinase).Because of the complexity of this mechanism, no attempt was made to write an over-all rate equation for the initial velocity from which the experimental data might be interpreted.It proved more feasible to convert the pathway from free enzyme to the central complex (E-Mn2+-PEP-IDP-C02) from a random one to an ordered, sequential one by designing experiments in which the concentrations of some species of the substrate were negligible relative to the concentrations of other species, allowing simplified rate equations to be written.It was possible, with this approach, to compile kinetic data which are consistent with the mechanism.Attempts to interpret the data on the basis of other mechanisms have led to inconsistencies.The most significant features of this mechanism are as follows.(a) The formation of a central complex consisting of enzyme, Mn2+, PEP, IDP, and CO2 proceeds by what may be described as a mixed ordered-random addition of the components.(b) PEP must bind to the enzyme before either IDP or CO, in order to yield a kinetically active form of the enzyme.Although IDP and CO2 may bind without prior binding of PEP, they form inactive complexes that do not lead to the central complex, and they may be considered to be inhibitors with respect to PEP.(c) The binding of PEP proceeds in a random manner with respect to Mn2f to form the ternary enzyme-Mn2+-PEP complex.Both free