A model to explain the pH-dependent specificity of cathepsin B-catalysed hydrolyses

Abstract

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...

Highly Specific Protease-Based Approach for Detection of Porphyromonas gingivalis in Diagnosis of Periodontitis

“…Potentially, a diversity of hydrolases present in saliva can be responsible for this cleavage. Cathepsins, for instance, are known to recognize and cleave a wide range of peptide bonds, among which are Arg-Arg, Lys-Lys, and Phe-Arg (6,10,15,22,30,32,42). As well as the cathepsins, other salivary proteases, like alanine aminopeptidase and dipeptidyl peptidase IV, may play a role in the cleavage of these three substrates (2).…”

Papain-like peptidases: structure, function, and evolution

“…The enzyme has been found to be active and relatively stable under such conditions that are usually considered unfavorable for lysosomal enzymes (41) . It has, in fact, been shown that the activity of cathepsin B on small synthetic endoproteolytic substrates steadily increases with increasing pH, reaching maximal values between pH 7 and 8 (42) . In vitro , cathepsin B has been found to degrade various extracellular matrix components, including the basal lamina components laminin and type IV collagen, fibronectin, and tenascin C (43 -45) , which is probably the molecular mechanism underlying the contribution of cathepsin B to cell invasion (46) .…”

Identification and Characterization of Cathepsin B as the Cellular MARCKS Cleaving Enzyme

“…Although one such study used soluble, denatured proteins as substrates (65), most studies have employed synthetic peptides to analyze the interactions between the active site of the enzyme with the substrate cleavage site (21,64,66). From these studies it has been suggested that the endopeptidase activity of cathepsin B prefers, but is not limited to, a basic and hydrophobic amino acid at the P1 and P2 substrate sites, respectively (65).…”

“…8). It has been reported that Glu 245 in human and rat cathepsin B plays a significant role in the interaction of the enzyme with its substrates (64,66). It is tempting to speculate that PKC-mediated phosphorylation of the second serine within the PSD is responsible for inhibiting the cleavage at the site represented by Peptide A (Fig.…”