| Abstract
| - The effects of Mg2+ concentration on the kinetics of both ATP cleavage and carboxyenzymeformation in the approach to steady state of the biotin carboxylation reaction of pyruvate carboxylasehave been studied. It was found that the enzyme underwent dilution inactivation at low Mg2+ concentrationsand that this occurred at higher enzyme concentrations than had been previously observed. At 10 mMMg2+, dilution inactivation was prevented and activation of the enzyme also occurred. When the enzymewas mixed with an ATP solution to initiate the carboxylation reaction, dilution inactivation was reversedand further enzyme activation was induced to a final level that was dependent on Mg2+ concentration.With the exception of the reaction at 10 mM Mg2+ in the presence of acetyl CoA, the experimental datacould be adequately described as first-order exponential approaches to steady state. At 10 mM Mg2+ inthe presence of acetyl CoA, both ATP cleavage and carboxyenzyme formation data were best describedas a biexponential process, in which there was little ATP turnover at steady state. Modeling studies havebeen performed which produced simulated data that were similar to the experimental data, using a reactionscheme modified from one proposed previously [Legge, G. B., et al. (1996) Biochemistry 35, 3849−3856]. These studies indicate that the major foci of action of Mg2+ are in the decarboxylation of theenzyme−carboxybiotin complex, the return of the biotin to the site of the biotin carboxylation reaction,and the coupling of ATP cleavage to biotin carboxylation.
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