Role of the divalent metal cation in the pyruvate oxidase reaction.
Purified pyruvate oxidase requires a divalent metal cation for enzymatic activity. The function of the divalent metal cation was studied for unactivated, dodecyl sulfate-activated, and phosphatidylglycerol-activated oxidase. Assays performed in the presence of Mg2+, CA2+, Zn2+, Mn2+, Ba2+, Ni2+, Co2+, Cu2+, and Cr3+ in each of four different buffers, phosphate, 1,4-piperazinediethanesulfonic acid, imidazole, and citrate, indicate that any of these metal cations will fulfill the pyruvate oxidase requirement. Extensive steady state kinetics data were obtained with both Mg2+ and Mn2+. All the data are consistent with the proposition that the only role of the metal is to bind to the cofactor thiamin pyrophosphate (TPP) and that it is the Me2+-TPP complex which is the true cofactor. Values of the Mg2+ and Mn2+ dissociation constants with TPP were determined by EPR spectroscopy and these data were used to calculate the Michaelis constant for the Me2+-TPP complexes. The results show that the Michaelis constants for the Me2+-TPP complexes are independent of the metal cation in the complex. Fluorescence quenching experiments show that the Michaelis constant is equal to the dissociation constant of the Mn2+-TPP complex with the enzyme. It was also shown that Mn2+ will only bind to the enzyme in the presence of TPP and that one Mn2+ binds per subunit. Steady state kinetics experiments with Mn2+ were more complicated than those obtained with Mg2+ because of the formation of an abortive Mn2+-pyruvate complex. Both EPR and steady state kinetics data indicated complex formation with a dissociation constant of about 70 mM.
D002021:Buffers; D004578:Electron Spin Resonance Spectroscopy; D004926:Escherichia coli / Q000201:enzymology; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D008345:Manganese / Q000378:metabolism / Q000494:pharmacology; D011771:Pyruvate Oxidase / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013835:Thiamine Pyrophosphate / Q000378:metabolism
D002021:Buffers; D008345:Manganese; D011771:Pyruvate Oxidase; D008274:Magnesium; D013835:Thiamine Pyrophosphate