Crystalline Pyruvate, Phosphate Dikinase from Bacteroides symbiosus

Pyruvate, phosphate dikinase was purified and crystallized from Bacteroides symbiosus. The function of histidyl and cysteinyl residues of the enzyme were investigated by chemical modification with diethylpyrocarbonate and bromopyruvate. Diethylpyrocarbonate rapidly inactivated the enzyme by combination with histidyl residues. The modified enzyme loses the ability to form phosphoryl enzyme and evidence is presented that only 2 of the 18 histidyl residues form phosphoryl derivatives. The inhibition is reversed by hydroxylamine but not by dialysis or gel filtration. Relatively low concentrations of bromopyruvate irreversibly inactivated the enzyme. This compound is a competitive inhibitor with respect to P-enolpyruvate. The rate of inactivation of the enzyme followed pseudofirst order kinetics and a plot of the rate of inactivation against bromopyruvate concentration gave a typical saturation curve. These results are consistent with a two-step reaction mechanism in which an alkylation step follows a rapid formation of an enzymebromopyruvate complex. The maximum inactivation rate constant was 0.125 min-’ at pH 7 and 3O”C, and the concentration of bromopyruvate giving half-maximum rate of inactivation was 0.25 mM. Bromopyruvate inactivation was pHand temperature-dependent and partially prevented in the presence of ATP or P-enolpyruvate. With [3-3Hz]bromopyruvate, it was shown that complete inactivation occurs when 1 mol of radioactive residue is covalently bound per mol of enzyme. S-Carboxyhydroxyethylcysteine has been identified in acid hydrolysates of the bromopyruvate-inactivated enzyme. Bromopyruvate may specifically interact with a cationic group at or near the pyruvate, P-enolpyruvate site of the enzyme and then alkylate a nearby cysteinyl residue. The Pi, PPi site also was partially inhibited, but the ATP, AMP site was not affected.