The occluding loop in cathepsin B defines the pH dependence of inhibition by its propeptide

Papain-like proenzymes are prone to autoprocess under acidic pH conditions. Similarly, peptides derived from the proregion of cathepsin B are potent p H-dependent inhibitors of that enzyme; i.e., at pH 6.0 the inhibition of hu man cathepsin B by its propeptide is defined by slow binding kinetics with a K-i of 3.7 nM and at pH 4.0 by classical kinetics with a K-i of 82 nM. Th is pH dependency is essentially eliminated either by the removal of a porti on of the enzyme's occluding loop through deletion mutagenesis or by the mu tation of either residue Asp22 or His110 to alanine; e.g., the mutant enzym e His110Ala is inhibited by its propeptide with K-i's of 2.0 +/- 0.3 nM at pH 4.0 and 1.1 +/- 0.2 nM at pH 6.0. For the His 10Ala mutant the inhibitio n also displays slow binding kinetics at both pH 4.0 and pH 6.0. As shown b y the crystal structure of mature cathepsin B [Musil, D., et al. (1991) EMB O J. 10, 2321-2330] Asp22 and His110 form a salt bridge in the mature enzym e, and it has been shown that this bridge stabilizes the occluding loop in its closed position I[Nagler, D. K., et al. (1997) Biochemistry 36, 12608-1 2615]. Thus the pH dependency of propeptide binding can be explained on the basis of a competitive binding between the occluding loop and the propepti de. At low pH, when the Asp22-His110 pair forms a salt bridge stabilizing t he occluding loop in its closed conformation, the loop more effectively com petes with the propeptide than at higher pH where deprotonation of His110 a nd the concomitant destruction of the Asp22-His110 salt bridge results in a destabilization of the closed form of the loop. The rate of autocatalytic processing of procathepsin B to cathepsin B correlates with the affinity of the enzyme for its propeptide rather than with its catalytic activity, thu s suggesting a possible influence of occluding loop stability on the rate o f processing.