MAJOR INCREASE IN ENDOPEPTIDASE ACTIVITY OF HUMAN CATHEPSIN-B UPON REMOVAL OF OCCLUDING LOOP CONTACTS

The main feature distinguishing cathepsin B from other cysteine protea ses of the papain family is the presence of a large insertion loop, te rmed 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 sa lt bridges with Asp22 and Asp224, respectively. The influence of this loop on the endopeptidase activity of cathepsin B has been investigate d 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 obser ved for cathepsin B containing the single mutations D22A, H110A, R116A , and D224A. The highest activity however is observed for mutants wher e both loop to enzyme contacts are disrupted. For the triple-mutant D2 2A/H110A/R116A, an optimum k(cat)/K-M value of 12 x 10(5) M-1 s(-1) wa s obtained for hydrolysis of Abz-AFRSAAQ-EDDnp, which corresponds to a 600-fold increase relative to wild-type cathepsin B and approaches th e level of activity observed with cathepsin L or papain. By comparison , the mutations have little effect on the hydrolysis of Cbz-FR-MCA. Th e influence of the mutations on the pH dependency of activity also ind icates that the complexity of pH activity profiles normally observed f or cathepsin B is related to the presence of the occluding loop. The m ajor increase in endopeptidase activity is attributed to an increase i n loop ''flexibility'' and suggests that the occluding loop might move when an endopeptidase substrate binds to the enzyme, The possible con tribution of these interactions in regulating endopeptidase activity a nd the implications for cathepsin B activity in physiological or patho logical conditions are discussed.