Effect of pH on the activities of penicillopepsin and Rhizopus pepsin and a proposal for the productive substrate binding mode in penicillopepsin

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Abstract
The pH dependence of kinetic parameters for penicillopepsin and Rhizopus pepsin acting on acetylalanylalanyllysyl-p-nitrophenylalanylalanylalanine amide was determined. The velocity constants, Kcat, show optima between pH 4 and pH 4.5. The Michaelis-Menten constants, Km, show a strong pH dependence for both enzymes and rise from low values at pH 6.0 (0.08 mM for penicillopepsin and 0.23 mM for Rhizopus pepsin) to .apprx. 8 and 1.1 mM, respectively, at pH 2.0. This dependence strongly suggests that for this substrate, with lysine in the P1 position, binding is controlled by negatively charged carboxyl group(s) on the enzyme. These groups were tentatively identified in penicillopepsin as aspartic acid-115(114) and glutamic acid-16(13) on the basis of model building and by comparison with the binding of a pepstatin analog. The S1 binding site also has hydrophobic character which shows itself in the low Km (0.004 mM) for the substrate leucylseryl-p-nitrophenylalanylnorleucylalanylleucine methyl ester. Tyrosine-75(75), phenylalanine-112(111) and leucine-121(120) are the most likely residues involved in the hydrophobic binding. The binding site for P1'' residues is also hydrophobic and probably involves phenylalanine-190(189), isoleucine-211(213), phenylalanine-295(299) and isoleucine-297(301). In light of the structure of penicillopepsin, now refined at 1.8 .ANG. resolution, the detailed binding mode of a pepstatin analog also studied at 1.8 .ANG. resolution, and model-building studies, a productive binding mode for the scissile bond to aspartyl proteinases is proposed. Although physiologically the aspartic proteinases show a spread of pH optima from below pH 2 to over pH 7, their pH optima (as expressed in terms of kcat/Km) lie in a much narrower range when the enzymes act on defined good substrates.