Pyruvate Carboxylase

Abstract

Abstract Pyruvate carboxylase contains firmly bound manganese which affects the longitudinal relaxation rate of the protons of water as measured by pulsed nuclear magnetic resonance. The manganese shows an enhanced effect (ϵb = 4.2) on the relaxation rate when bound to the enzyme. Studies on the effect of added substrates and inhibitors on the proton relaxation rate have shown that components of the second partial reaction, e.g. the carboxylation of pyruvate by the enzyme-biotin ∼ CO2 complex, interact with the bound manganese of the enzyme and cause a reduction in the enhancement. In contrast, components of the first partial reaction, the formation of enzyme-biotin ∼ CO2 from adenosine triphosphate and HCO3-, do not significantly affect the enhancement. Examination of the enhancement of the bound manganese as a function of substrate or inhibitor concentration has permitted the determination of dissociation constants and enhancement values for the enzyme-substrate and enzyme-inhibitor complexes. The rate of inactivation of pyruvate carboxylase by avidin is increased by pyruvate and oxalacetate, and decreased by certain dissociable inhibitors, e.g. oxalate. Analysis of these data permits an independent determination of the dissociation constants of the enzyme-substrate and enzyme-inhibitor complexes. The dissociation constants derived from the proton relaxation rate measurements are in good agreement with those obtained from their effects on the rate of inactivation by avidin and also with inhibitor constants obtained from initial rate studies of the over-all reaction. These data strongly support the thesis that the bound manganese plays a functional role in the transcarboxylation portion of the pyruvate carboxylase reaction, i.e. the transfer of a carboxyl group from the enzyme-biotin ∼ CO2 intermediate to pyruvate. This locus of action differs from that of the added divalent metal ion which is required for this reaction and which participates in the formation of the enzyme-biotin ∼ CO2 intermediate from HCO3- and ATP. While dissociable inhibitors such as oxalate probably act by direct chelation of the bound manganese since the enhancement of the enzyme-inhibitor complex has a very low value (ϵc l 0.3), the corresponding values for the enzyme-substrate complexes are considerably higher (ϵc = 1.5 to 2.5), suggesting either that fewer metal-H2O ligands are displaced by substrates or that conformational changes in the protein contribute to the situation in a different way with the substrates and the inhibitors. Several possible mechanisms for the involvement of the bound manganese in this reaction are discussed, and a mechanism is suggested which is compatible with the kinetic and magnetic resonance data.