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.

Effects of acetyl CoA on the pre-steady-state kinetics of the biotin carboxylation reaction of pyruvate carboxylase.

The approach to steady-state for the formation of the enzyme-carboxybiotin complex obeys first-order kinetics, with the proportion of the total enzyme present as the enzyme-carboxybiotin complex in the steady-state being about 60%. The approach to steady-state for ATP cleavage also obeys first-order kinetics. The apparent first-order rate constants for the approach to steady-state, in the presence and absence of acetyl CoA, respectively, are 6.6 and 0.028 s(-1) for ATP cleavage and 6.1 and 0.028 s(-1) for enzyme-carboxybiotin formation. The similarities of the values of the rate constants for the two reactions indicates that there is a common rate-limiting step. The large enhancement of these rate constants in the presence of acetyl CoA suggests that a major effect of acetyl CoA in the reaction is to enhance the rate of the step in which the putative carboxyphosphate complex is formed and in which ATP is cleaved. In addition, in the presence of acetyl CoA, the formation of the enzyme-carboxybiotin complex is much more tightly coupled to ATP cleavage in the presence of acetyl CoA than in its absence. Modeling studies were performed, and reaction schemes are proposed which give simulations similar to the experimental data. In the reaction schemes, the carboxyphosphate intermediate is able to undergo abortive decomposition without carboxylating biotin. The rate of this abortive reaction is greatly reduced in the presence of acetyl CoA.

Kinetics of nucleotide binding to pyruvate carboxylase.

The kinetics of nucleotide binding to pyruvate carboxylase have been studied by measuring the fluorescence changes that occur on the binding and release of FTP and FDP, which are fluorescent formycin analogues of ATP and ADP. The rate constants and equilibrium binding constants for both MgFTP and MgFDP binding to pyruvate carboxylase have been determined. From the kinetics of displacement of MgFTP by MgATP and binding of MgFTP in the presence of MgATP, the rate constants of MgATP binding were estimated. A slow component to the fluorescence changes was seen to occur after the initial rapid, bimolecular binding step, when formycin nucleotides were mixed with the enzyme. HCO3- and pyruvate were shown to quench the fluorescence of enzyme-bound MgFTP, but did not affect the affinity of the enzyme for the nucleotide. Acetyl CoA reduced the affinity of the enzyme for both MgFDP and MgFTP by about 3-fold by decreasing the association rate constants (by 25%) and increasing the dissociation rate constants (by 2-fold). In the absence of Mg2+ a very rapid component to FTP binding was observed that was complete within about 3 ms, but no fast component was observed comparable to that seen in the presence of 4.5 mM MgCl2. Increasing the [Mg2+] gradually abolished this very fast component of the binding, while the amplitude of the fast component increased, although the rate constant for this component did not appear to be strongly dependent on [Mg2+]. The rate constants of the slow component of Mg.formycin nucleotide binding did not appear to be dependent on nucleotide concentration.(ABSTRACT TRUNCATED AT 250 WORDS)