Major Increase in Endopeptidase Activity of Human Cathepsin B upon Removal of Occluding Loop Contacts † Dorit K. Na ¨gler, ‡ Andrew C. Storer, ‡ Fernanda C. V. Portaro, § Euridice Carmona, § Luiz Juliano, | and Robert Me ´nard* ,‡ Biotechnology Research Institute, National Research Council of Canada, Montreal, Quebec, H4P2R2 Canada, Lab. Farmacologia, Instituto Butantan, Sa ˜ o Paulo 05504, Brazil, and Department of Biophysics, Escola Paulista de Medicina, Sa ˜ o Paulo 04044-020, Brazil ReceiVed May 28, 1997; ReVised Manuscript ReceiVed August 7, 1997 X ABSTRACT: The main feature distinguishing cathepsin B from other cysteine proteases of the papain family is the presence of a large insertion loop, termed the occluding loop, which occupies the S′ subsites of the enzyme. The loop is held in place mainly by two contacts with the rest of the enzyme, involving residues His110 and Arg116 on the loop that form salt bridges with Asp22 and Asp224, respectively. The influence of this loop on the endopeptidase activity of cathepsin B has been investigated using site-directed mutagenesis and internally quenched fluorogenic (IQF) substrates. Wild-type cathepsin B displays poor activity against the substrates Abz-AFRSAAQ-EDDnp and Abz-QVVAGA-EDDnp as compared to cathepsin L and papain. Appreciable increases in k cat /K M were observed for cathepsin B containing the single mutations D22A, H110A, R116A, and D224A. The highest activity however is observed for mutants where both loop to enzyme contacts are disrupted. For the triple-mutant D22A/H110A/R116A, an optimum k cat /K M value of 12 × 10 5 M -1 s -1 was obtained for hydrolysis of Abz-AFRSAAQ-EDDnp, which corresponds to a 600-fold increase relative to wild-type cathepsin B and approaches the level of activity observed with cathepsin L or papain. By comparison, the mutations have little effect on the hydrolysis of Cbz-FR-MCA. The influence of the mutations on the pH dependency of activity also indicates that the complexity of pH activity profiles normally observed for cathepsin B is related to the presence of the occluding loop. The major increase in endopeptidase activity is attributed to an increase in loop “flexibility” and suggests that the occluding loop might move when an endopeptidase substrate binds to the enzyme. The possible contribution of these interactions in regulating endopeptidase activity and the implications for cathepsin B activity in physiological or pathological conditions are discussed. Cathepsin B is a lysosomal cysteine protease involved in intracellular protein turnover (Kirschke & Barrett, 1987). The enzyme has also been implicated in pathological conditions such as arthritis (Mort et al., 1984; Trabandt et al., 1991) and tumor metastasis [reviewed by Sloane (1990)]. Cathe- psin B is the best characterized mammalian member of the papain family of cysteine proteases. A significant amount of functional (kinetic) data for the purified enzyme has been gathered since the late 1970s and the crystal structures of human and rat cathepsin B (Musil et al., 1991; Jia et al., 1995) as well as of complexes with inhibitors (Jia et al., 1995; Turk et al., 1995) are now available. The crystal structure of procathepsin B, the first structure of a cysteine protease zymogen, has been solved recently (Cygler et al., 1996; Turk et al., 1996). In addition, numerous functional properties of cathepsin B have been investigated by site-directed mutagenesis [e.g., Hasnain et al. (1992, 1993), Mach et al. (1993), Taralp et al. (1995), Fox et al. (1995), and Illy et al. (1997)]. The enzyme shows close structural and functional homology to other cysteine proteases of the papain family. One feature that distinguishes cathepsin B from other cysteine proteases is the position of the peptide bond being cleaved. While most cysteine proteases of the papain family are exclusively endopeptidases, cathepsin B has both exo- and endopeptidase activities (Barrett & Kirschke, 1981). Despite the impressive amount of work done on cathepsin B, very few studies have dealt with the molecular determi- nants of the endo- and exopeptidase activities. The crystal structure of human cathepsin B (Musil et al., 1991) offered an explanation for the dipeptidyl carboxypeptidase activity of the enzyme. The presence of a large insertion loop found only in cathepsin B and positioned in the vicinity of the active site seems to favor dipeptidyl carboxypeptidase activity; two histidine residues (His110 and His111) in the loop provide positively charged anchors for the C-terminal carboxylate group of substrates. Accordingly, it was shown recently using site-directed mutagenesis that the presence of this loop is essential for the exopeptidase activity of the enzyme (Illy et al., 1997). The influence of the loop on the endopeptidase activity of cathepsin B however has not been investigated. It was determined many years ago that cysteine proteases of the papain family possess an extended binding site able to accommodate up to seven residues of a substrate [P 4 to P 3 ′, Schechter & Berger (1967)]. For cathepsin B, the large insertion loop occupies the S′ region of the enzyme (S 2 ′- S 3 ′) and should be detrimental to endopeptidase activity by † NRCC Publication Number 39977. Supported in part by the Protein Engineering Network of Centres of Excellence. * Address correspondence to this author at the Biotechnology Research Institute, 6100 Royalmount Avenue, Montreal, Canada H4P2R2. E-mail: robert.menard@nrc.ca. ‡ National Research Council of Canada. § Instituto Butantan. | Escola Paulista de Medicina. X Abstract published in AdVance ACS Abstracts, October 1, 1997. 12608 Biochemistry 1997, 36, 12608-12615 S0006-2960(97)01264-6 CCC: $14.00 © 1997 American Chemical Society