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 degrees C the reaction was 80% complete within 180 ms. The reaction was monophasic and obeyed pseudo-first-order kinetics. Increasing concentrations of Mg2+ caused a decrease in the magnitude of the observed pseudo-first-order rate constant. Throughout the carboxylation of pyruvate, the rate-limiting step of the reaction occurred after the dissociation of carboxybiotin from the first sub-site, 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 first sub-site 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.: (formula; see text). 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 first sub-site, 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.
D001710:Biotin / Q000031:analogs & derivatives* / Q000378:metabolism; D007700:Kinetics; D046911:Macromolecular Substances; D008274:Magnesium / Q000494:pharmacology*; D011766:Pyruvate Carboxylase / Q000378:metabolism*; D011773:Pyruvates / Q000378:metabolism; D019289:Pyruvic Acid
D046911:Macromolecular Substances; D011773:Pyruvates; C024582:N1'-carboxybiotin; D001710:Biotin; D019289:Pyruvic Acid; D011766:Pyruvate Carboxylase; D008274:Magnesium