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In a previous study, it was shown that replacing Asp158 in papain by Asn had little effect on activity and that the negatively charged carboxylate of Asp158 does not significantly stabilize the active site thiolate-imidazolium ion pair of papain (Ménard et al., 1990). In this paper, we report the kinetic characterization of three more mutants at this position: Asp158Gly, Asp158Ala, and Asp158Glu. From the pH-activity profiles of these and other mutants of papain, it has been possible to develop a model that enables us to dissect out the contribution of the various mutations toward (i) intrinsic activity, (ii) ion pair stability, and (iii) the electrostatic potential at the active site. Results obtained with mutants that place either Gly or Ala at position 158 indicate that the hydrogen bonds involving the side chain of Asp158 in wild-type papain are indirectly important for enzyme activity. When CBZ-Phe-Arg-MCA is used as a substrate, the (kcat/KM)obs values at pH 6.5 are 3650 and 494 M-1 s-1 for Asp158Gly and Asp158Ala, respectively, as compared to 119,000 M-1 s-1 for papain. Results with the Asp158Glu mutant suggest that the side chain of Glu moves closer to the active site and cannot form hydrogen bonds similar to those involving Asp158 in papain. From the four mutations introduced at position 158 in papain, we can conclude that it is not the charge but the hydrogen-bonding interactions involving the side chain of Asp158 that contribute the most to the stabilization of the thiolate-imidazolium ion pair in papain. However, the charge and the hydrogen bonds of Asp158 both contribute to the intrinsic activity of the enzyme.

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1. Three synthetic substrates of cathepsin B (EC 3.4.22.1) with various amino acid residues at the P2 position (Cbz-PheArg-NH-Mec, Cbz-Arg-Arg-NH-Mec and Cbz-Cit-Arg-NH-Mec, where Cbz represents benzyloxycarbonyl and NH-Mec represents 4-methylcoumarin-7-ylamide) were used to investigate the pH-dependency of cathepsin B-catalysed hydrolyses and to obtain information on the nature of enzyme-substrate interactions. 2. Non-linear-regression analysis of pH-activity profiles for these substrates indicates that at least four ionizable groups on cathepsin B with pKa values of 3.3, 4.55, 5.46 and > 7.3 can affect the rate of substrate hydrolysis. 3. Ionization of the residue with a pKa of 5.46 has a strong effect on activity towards the substrate with an arginine in P2 (8.4-fold increase in activity) but has only a moderate effect on the rate of hydrolysis with Cbz-Cit-Arg-NH-Mec (2.3-fold increase in activity) and virtually no effect with Cbz-Phe-Arg-NH-Mec. The kinetic data are consistent with this group being an acid residue with a side chain able to interact with the side chains of an arginine or a citrulline in the P2 position of a substrate. Amino acid sequence alignment and model building with the related enzyme papain (EC 3.4.22.2) INTRODUCTIONCathepsin B (EC 3.4.22.1) is a lysosomal cysteine proteinase that has been isolated from many mammalian tissues, including human liver (Barrett, 1977). Much of the information on the mechanism of cathepsin B has been obtained through comparisons with the well characterized cysteine proteinase papain (EC 3.4.22.2). Alignment and comparison of the sequences of related cysteine proteinases suggest similar threedimensional structures for cathepsin B and papain (Kamphuis et al., 1985;Dufour, 1988). Even though the basic features of the mechanisms are similar, major differences exist between the two enzymes. Cathepsin B is both an endopeptidase and a dipeptidyl carboxydipeptidase, whereas papain works exclusively as an endopeptidase (Aronson & Barrett, 1978;Bond & Barrett, 1980;McKay et al., 1983;Mason, 1989). The specificities of the two enzymes are also somewhat different. Papain shows a marked preference for peptides with a bulky non-polar side chain, especially L-phenylalanine, on the N-terminal side of the amino acid containing the peptide bond being hydrolysed, i.e. at the P2 position (Berger & Schechter, 1970). This specificity is attributed to interaction of the phenylalanine side chain with hydrophobic residues at the S2 subsite of papain, mainly and (Drenth et al., 1976). (Cathepsin B numbering is used throughout the text except for residues of papain, where the position number for papain is followed in parentheses by the corresponding number for cathepsin B.) Cathepsin B activity towards substrates with a P2 phenylalanine residue is still very high, but the enzyme also has a strong affinity for substrates with an arginine residue at the P2 position, such as Cbz-Arg-Arg-NHNap and Cbz-Arg-Arg-NH-Mec (Barrett & Kirschke, 1981).Ionization of a group with a pKa of ab...