S′2 substrate specificity and the role of His110 and His111 in the exopeptidase activity of human cathepsin B

The ability of the lysosomal cysteine protease cathepsin B to function as a peptidyldipeptidase (removing C-terminal dipeptides) has been attributed to the presence of two histidine residues (His 1 1 0 and His 1 1 1 ) present in the occluding loop, an extra peptide segment located in the primed side of the active-site cleft. Whereas His 1 1 1 is unpaired, His 1 1 0 is present as an ion pair with Asp 2 2 on the main body of the protease. This ion pair appears to act as a latch to hold the loop in a closed position. The exopeptidase activity of cathepsin B, examined using quenched fluorescence substrates, was shown to have a 20-fold preference for aromatic side chains in the P' 2 position relative to glutamic acid as the least favourable residue. Site-directed mutagenesis demonstrated that His 1 1 1 makes a positive 10-fold contribution to the exopeptidase activity, whereas His 1 1 0 is critical for this action with the Asp 2 2 -His 1 1 0 ion pair stabilizing the electrostatic interaction by a maximum of 13.9 kJ/mol (3.3 kcal/mol). These studies showed that cathepsin B is optimized to act as an exopeptidase, cleaving dipeptides from protein substrates in a successive manner, because of its relaxed specificity in P'2 and its other subsites.