Synthetic substrates and inhibitors of β-poly(L-malate)-hydrolase (polymalatase)

Abstract

Polymalatase from Physarum polycephalum calalysed the hydrolysis of β-poly[L-malate] and of the synthetic compounds β-di(L-malate), β-tetra(L-malate), β-tetra(L-malate) β-propylester, and L-malate β-methylester. Cyclic β-tri(L-malate), cyclic β-tetra(L-malate), and D-malate β-methylester were not cleaved, but were competitive inhibitors. The O-terminal acetate of β-tetra(L-malate) was neither a substrate nor an inhibitor. L-Malate was liberated; the K(m), K(i) and V(max) values were measured. The appearance of comparable amounts of β-tri(L-malate), and β-di(L-malate) during the cleavage of β-tetra(L-malate) indicated a distributive mechanism for small substrates. The accumulation of a series of oligomers, peaking with the 11-mer and 12-mer in the absence of higher intermediates, indicated that the depolymerization of β-poly(L-malate) was processive. The results indicate that β-poly(L-malate) is anchored at its OH-terminus by the highly specific binding of the penultimate malyl residue. The malyl moieties beyond 12 residues downstream from the OH-terminus extend into a diffuse second, electrostatic binding site. The catalytic site joins the first binding site, accounting for the cleavage of the polymer into malate residues. It is proposed that the enzyme does not dissociate from β-poly(L-malate) during hydrolysis, when both sites are filled with the polymer. When only the first binding site is filled, the reaction partitions at each oligomer between hydrolysis and dissociation.