Effects of Mg(2+) on the pre-steady-state kinetics of the biotin carboxylation reaction of pyruvate carboxylase.
The effects of Mg(2+) concentration on the kinetics of both ATP cleavage and carboxyenzyme formation in the approach to steady state of the biotin carboxylation reaction of pyruvate carboxylase have been studied. It was found that the enzyme underwent dilution inactivation at low Mg(2+) concentrations and that this occurred at higher enzyme concentrations than had been previously observed. At 10 mM Mg(2+), dilution inactivation was prevented and activation of the enzyme also occurred. When the enzyme was mixed with an ATP solution to initiate the carboxylation reaction, dilution inactivation was reversed and further enzyme activation was induced to a final level that was dependent on Mg(2+) concentration. With the exception of the reaction at 10 mM Mg(2+) in the presence of acetyl CoA, the experimental data could be adequately described as first-order exponential approaches to steady state. At 10 mM Mg(2+) in the presence of acetyl CoA, both ATP cleavage and carboxyenzyme formation data were best described as a biexponential process, in which there was little ATP turnover at steady state. Modeling studies have been performed which produced simulated data that were similar to the experimental data, using a reaction scheme 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 Mg(2+) are in the decarboxylation of the enzyme-carboxybiotin complex, the return of the biotin to the site of the biotin carboxylation reaction, and the coupling of ATP cleavage to biotin carboxylation.
Branson
J P
JP
Department of Biochemistry, The University of Western Australia, Nedlands.
Attwood
P V
PV
D000105:Acetyl Coenzyme A / Q000378:metabolism; D000818:Animals; D001710:Biotin / Q000378:metabolism*; D002264:Carboxylic Acids / Q000378:metabolism*; D002645:Chickens; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D008274:Magnesium / Q000378:metabolism*; D011766:Pyruvate Carboxylase / Q000378:metabolism*
D002264:Carboxylic Acids; D001710:Biotin; D000105:Acetyl Coenzyme A; D011766:Pyruvate Carboxylase; D008274:Magnesium