Pneumococcal phosphorylcholine esterase, Pce, contains a metal binuclear center that is essential for substrate binding and catalysis.

The phosphorylcholine esterase from Streptococcus pneumoniae, Pce, catalyzes the hydrolysis of phosphorylcholine residues from teichoic and lipoteichoic acids attached to the bacterial envelope and comprises a globular N-terminal catalytic module containing a zinc binuclear center and an elongated C-terminal choline-binding module. The dependence of Pce activity on the metal/enzyme stoichiometry shows that the two equivalents of zinc are essential for the catalysis, and stabilize the catalytic module through a complex metal-ligand coordination network. The pH dependence of Pce activity toward the alternative substrate p-nitrophenylphosphorylcholine (NPPC) shows that k(cat) and k(cat)/K(m) depend on the protonation state of two protein residues that can be tentatively assigned to the ionization of the metal-bound water (hydrogen bonded to D89) and to H228. Maximum activity requires deprotonation of both groups, although the catalytic efficiency is optimum for the single deprotonated form. The drastic reduction of activity in the H90A mutant, which still binds two Zn2+ ions at neutral pH, indicates that Pce activity also depends on the geometry of the metallic cluster. The denaturation heat capacity profile of Pce exhibits two peaks with T(m) values of 39.6 degrees C (choline-binding module) and 60.8 degrees C (catalytic module). The H90A mutation reduces the high-temperature peak by about 10 degrees C. Pce is inhibited in the presence of 1 mM zinc, but this inhibition depends on pH, buffer, and substrate species. A reaction mechanism is proposed on the basis of kinetic data, the structural model of the Pce:NPPC complex, and the currently accepted mechanism for other Zn-metallophosphoesterases.