THE CARBOXYBIOTIN COMPLEX OF PYRUVATE-CARBOXYLASE - A KINETIC-ANALYSIS OF THE EFFECTS OF MG2+ IONS ON ITS STABILITY AND ON ITS REACTION WITH PYRUVATE

The enzyme-[14C]carboxybiotin complex of sheep liver pyruvate carboxylase was isolated and the reaction between this and pyruvate was studied by using the quenched-flow rapid-reaction technique. At 0.5.degree. C the reaction was 80% complete within 180 ms. The reaction was monophasic and obeyed pseudo-1st-order kinetics. Increasing concentrations of Mg2+ caused a decrease in the magnitude of the observed pseudo-1st-order rate constant. Throughout the carboxylation of pyruvate, the rate-limiting step of the reaction occurred after the dissociation of carboxybiotin from the 1st subsite, whereas in the slow phase of the reaction with 2-oxobutyrate this dissociation is the rate-limiting step. It is possible, from the reaction scheme proposed, that the inhibition of overall enzymic activity by high concentrations of Mg2+ could be caused by the transfer of the carboxy group from biotin to pyruvate becoming rate-limiting. The efficacy of a substrate as a signal for the movement of carboxybiotin from the 1st subsite is reflected by the amount that the effective affinity of the enzyme-carboxybiotin complex for Mg2+ is lowered. In the presence of the substrates tested, the affinities of the carboxybiotin complex can be arranged in order of increasing magnitude, i.e.: enzyme-biotin-CO2- .cntdot. pyruvate < enzyme-biotin-CO2- .cntdot. .alpha.-oxobutyrate < enzyme-biotin-CO2-. The kinetics of the decay of the enzyme-[14C]carboxybiotin complex at 0.degree. C in the absence of substrates are similar to the reaction with pyruvate, except that the carboxybiotin is also unstable in the 1st subsite to some degree. This similarity allows for the proposal of a general scheme for the decarboxylation of the enzyme-carboxybiotin complex in the presence or in the absence of substrates.