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

Polymalatase from Physarum polycephalum calalysed the hydrolysis of beta-poly[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-methylester. Cyclic beta-tri(L-malate), cyclic beta-tetra(L-malate), and D-malate beta-methylester were not cleaved, but were competitive inhibitors. The O-terminal acetate of beta-tetra(L-malate) was neither a substrate nor an inhibitor. L-Malate was liberated; the Km, Ki and Vmax values were measured. The appearance of comparable amounts of beta-tri(L-malate), and beta-di(L-malate) during the cleavage of beta-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 beta-poly(L-malate) was processive. The results indicate that beta-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 beta-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.

Specificity and direction of depolymerization of beta-poly(L-malate) catalysed by polymalatase from Physarum polycephalum--fluorescence labeling at the carboxy-terminus of beta-poly(L-malate).

Beta-poly(L-malate), a major constituent of nuclei in plasmodia of Physarum polycephalum, is enzymatically degraded to L-malate after secretion into the culture medium. This depolymerization is specifically catalysed by an endogenous polymalatase. The mode of action and the specificity criteria have been investigated by employing various chemical derivatives of beta-poly(L-malate), including substitution at the hydroxy-terminus and carboxy-terminus of the polymer, esterification of the pending alpha-carboxylate, and beta-poly(DL-malate). The results of the investigation were summarized in a specificity model that involved recognition of the hydroxy-terminus and of the alpha-carboxylate as substituents of the asymmetric carbon in the malic acid unit. Depolymerization proceeded from the hydroxy-terminus towards the carboxy-terminus, thereby degrading the polymer to L-malate. When the terminal beta-carboxylate had been amidated with the fluorescent N-(1-naphthyl)ethylenediamine, degradation was normal but was arrested at the level of the terminal beta-carboxy-substituted dimer. It should be possible to employ polymalatase as a tool for the detection of branching and other modifications of beta-poly(L-malate).