Isolation and characterization of the carbon-phosphorus bond-forming enzyme phosphoenolpyruvate mutase from the mollusk Mytilus edulis

Abstract

The enzyme phosphoenolpyruvate mutase was purified to homogeneity from the mollusk Mytilus edulis. The subunit size of the native homotetramer was determined to be 34,000 Da. The steady-state kinetic constants for catalysis of the conversion of phosphonopyruvate to phosphoenolpyruvate at pH 7.5 and 25°C were measured at k(cat) = 34 s-1, phosphonopyruvate K(m) = 3 μM, and Mg2+ K(m) = 4 μM. The enzyme displayed a bread specificity for divalent metal ion activation; Co2+, Mn2+, Zn2+, and Ni2+ are activators, whereas Ca2+ is not. Analysis of the pH dependence of the Mg2+-activated mutase-catalyzed reaction of phosphonopyruvate revealed one residue that must be protonated (apparent pKα= 8.3) and a second residue that must be unprotonated (apparent pKα = 7.7) for maximal catalytic activity.