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

Polymalatase from Physarum polycephalum calalysed the hydrolysis of beta-po ly[L-malate] and of the synthetic compounds beta-di(L-malate), beta-tetra(L -malate), beta-tetra(L-malate) beta-propylester, and L-malate beta-methyles ter. Cyclic beta-tri(L-malate), cyclic p-tetra(L-malate), and D-malate beta -methylester were not cleaved, but were competitive inhibitors. The O-termi nal acetate of p-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 ap pearance of comparable amounts of beta-tri(L-malate), and beta-di(L-malate) during the cleavage of beta-tetra(L-malate) indicated a distributive mecha nism for small substrates. The accumulation of a series of oligomers, peaki ng with the 11-mer and 12-mer in the absence of higher intermediates, indic ated that the depolymerization of beta-poly(L-malate) was processive. The r esults indicate that beta-poly(L-malate) is anchored at its OH-terminus by the highly specific binding of the penultimate malyl residue. The malyl moi eties beyond 12 residues downstream from the OH-terminus extend into a diff use second, electrostatic binding site. The catalytic site joins the first binding site, accounting for the cleavage of the polymer into malate residu es. It is proposed that the enzyme does not dissociate from beta-poly(L-mal ate) during hydrolysis, when both sites are filled with the polymer. When o nly the first binding site is filled, the reaction partitions at each oligo mer between hydrolysis and dissociation.