The carboxybiotin complex of chicken liver pyruvate carboxylase. A kinetic analysis of the effects of acetyl-CoA, Mg2+ ions and temperature on its stability and on its reaction with 2-oxobutyrate

Abstract

The enzyme-[14C]carboxybiotin complex of chicken liver pyruvate carboxylase has been isolated and shown to be relatively stable, with a half-life at 0 degree C of 342 min. The kinetic properties of the decay of this complex, in both the presence and the absence of the substrate analogue, 2-oxobutyrate, have been examined. The data for the reaction with 2-oxobutyrate at 0 degree C fitted a biphasic exponential decay curve, enabling the calculation of rate constants for both the fast and slow phases of the reaction at this temperature. The effect of temperature on the observed pseudo-first-order rate constant for the slow phase of the reaction with 2-oxobutyrate, and that for the decay of the enzyme-[14C]carboxybiotin complex alone, have been examined. Arrhenius plots of these data revealed that the processes being studied in each type of experiment were single reactions represented by one rate constant in each case. For the decay of the enzyme-[14C]carboxybiotin complex in the absence of 2-oxobutyrate, the rate-determining process may be the movement of carboxybiotin from the site of the first partial reaction to the site of the second. The calculated thermodynamic activation parameters indicate that this reaction is accompanied by a large change in protein conformation. With 2-oxobutyrate present, the observed process in the slow phase of the reaction was probably the dissociation of the carboxybiotin from the first subsite. Here, the activation parameters suggest that a much smaller change in protein conformation accompanies this reaction. Both sets of experiments were also performed in the presence of acetyl-CoA, but this activator had little effect on the measured thermodynamic activation parameters. However, in both cases the observed pseudo-first-order rate constants in the presence of acetyl-CoA were about 75% of those in its absence. The effects of Mg2+ on the reaction kinetics of the enzyme-[14C]carboxybiotin complex with 2-oxobutyrate were similar to those observed with the sheep enzyme by Goodall, Baldwin, Wallace & Keech [(1981) Biochem. J. 199, 603-609].