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

Abstract

Polymalatase from Physarum polycephalum calalysed the hydrolysis of b-poly [l-malate] and of the synthetic compounds b-di(l-malate), b-tetra(l-malate), b-tetra(l-malate) b-propylester, and l-malate b-methylester. Cyclic b-tri(l-malate), cyclic b-tetra(l-malate), and d-malate b-methylester were not cleaved, but were competitive inhibitors. The O-terminal acetate of b-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 b-tri(l-malate), and b-di(l-malate) during the cleavage of b-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 b-poly(l-malate) was processive. The results indicate that b-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 b-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.Keywords: polymalatase; polymalate; depolymerase; synthetic substrates; inhibitors.The plasmodium of Physarum polycephalum and of other myxomycetes produces b-poly(l-malate) (PMLA), an unbranched polyester of l-malic acid [1,2]. The highly charged polyanion binds the replicative DNA polymerases, histones, and other nuclear proteins forming higher-order protein complexes in the nuclei of only these types of cells [3,4±6]. Plasmodia are multinucleated cells typical for myxomycetes in the vegetative branch of their life cycle [7]. Because they develop into cells of extreme sizes and yet display a high degree of synchrony in the timing of cellular events, PMLA has been proposed to function as a storage and carrier matrix in the maintenance of an adequate supply of proteins for all nuclei. In high PMLA-producer strains, the polymer is secreted into the culture medium and depolymerized to l-malate. The hydrolase (polymalatase) has been identified and characterized [8]. The catalytic activity has its optimum at pH 3.5. The large quantity of polymalatase found in the extract of the plasmodium probably refers to the zymogen, which is spontaneously activated during cell lysis [9].Protein chemistry and inhibitor studies have suggested a catalytic mechanism that is unusual for esterases [8]. Structure±function correlation studies with PMLA-derivatives have indicated that the enzyme cleaves the ester bonds while moving in a downstream direction from the OH-terminus [10]. Because of their hydrolytic instability, the employed inhibitors and substrates were...

The polyion complex nano-prodrug of doxorubicin (DOX) with poly(lactic acid-co-malic acid)-block-polyethylene glycol: preparation and drug controlled release

“…The PIC has found wide applications in the delivery of DNA, proteins, and drugs (Nguyen et al, 2000;Tang et al, 2003). Poly(b-L-malic acid) (PMA) has been recognized as a promising carrier matrix of biopharmaceuticals because it possesses many attractive properties such as stable in the bloodstream, biodegradable and biocompatible, non-toxic in vitro and vivo, non-immunogenicity, and easy to cellular uptake (Lee et al, 2002;Gasslmaier et al, 2000;Braud et al, 1985;Cammas et al, 1999;Domurado et al, 2003). The availability of negatively charged free carboxyl groups is capable of reacting with a positively charged molecule such as DOX through complexation.…”

Preparation of Free and of Specifically Protected Oligo[β-Malic Acids] for Enzymatic Degradation Studies

“…[24] ) shown in Figure 2 were subjected to enzymatic degradation by the extracellular β-PMA hydrolase from P. polycephalum by Holler and Gasslmaier. [46] The C-terminally protected tetramer 22 was thus readily hydrolyzed to give fragments that were detected by RP-HPLC. In contrast, neither the acetate 21 nor the cyclic oligomer was degraded at all.…”

“…Complete and detailed results will be published elsewhere. [46] Conclusion Starting from (S)-malic acid, differently protected monomers (3 and 4) were prepared in such a manner that con- and of a cyclic tetramer (an X-ray crystal structure of the tetrabenzyl ester [24] of this tetramer was determined; the coordinates can be provided at request to the correspondence author) struction of oligomers of up to eight units (24) was possible by segment coupling. Acid chloride coupling proved to be the most reliable method for backbone ester formation and can, so far, be replaced by a condensation reagent only in the case of the dimer.…”

New promising biodegradable polyesters derived from poly((R,S)-3,3-dimethylmalic acid) for cardiovascular applications

“…These biodegradable polyesters were used either alone or as copolymers side chains to improve their mechanical properties, hydrolysis, long-term biodegradation, or biocompatibility behavior for the desired therapeutic applications (PLGA [9,10], PLMA [11][12][13][14][15], etc.). Among this class of polymers, the PMLA is known to present good biocompatibility, non-toxicity in vitro and in vivo, non--immunogenic properties, and stability in the bloodstream [5,[16][17][18][19]. The particularity of this family of polyesters is the presence of functionalized groups in their side chain which, furthermore, allow chemical modification for grafting, thus delivering drugs [20].…”

“…PMLA has many interesting properties, in addition to its functionalisable properties, such as non-toxicity in vitro and in vivo, non-immunogenicity, biodegradability, stability in the bloodstream, and cell affinity [39][40][41][42][43]. However, its application is limited because of the synthesis conditions (time and cost) and transfer reactions during its chemical synthesis and/or modification [18,[44][45][46][47][48][49][50][51][52][53].…”