Human cathepsin X: A cysteine protease with unique carboxypeptidase activity

Cathepsin X is a novel cysteine protease which was identified recently from the EST (expressed sequence tags) database. In a homology model of the mat ure cathepsin X, a unique three residue insertion between the Gln22 of the oxyanion hole and the active site Cys31 was found to be located in the prim ed region of the binding cleft as part of a surface loop corresponding to r esidues His23 to Tyr27, which we have termed the "mini-loop". From the mode l, it became apparent that this distinctive structural feature might confer exopeptidase activity to the enzyme. To verify this hypothesis, human proc athepsin X was expressed in Pichia pastoris and converted to mature catheps in X using small amounts of human cathepsin L. Cathepsin X was found to dis play excellent carboxypeptidase activity against the substrate Abz-FRF-(4NO (2)), with a k(cat)/K-M value of 1.23 x 10(5) M-1 s(-1) at the optimal pH o f 5.0. However, the activity of cathepsin X against the substrates Cbz-FR-M CA and Abz-AFRSAAQ-EDDnp was found to be extremely low, with k(cat)/K-M val ues lower than 70 M-1 s(-1). Therefore, cathepsin X displays a stricter exo peptidase activity than cathepsin B. No inhibition of cathepsin X by cystat in C could be detected up to a concentration of 4 mu M of inhibitor. From a model of the protease complexed with Cbz-FRF, the bound carboxypeptidase s ubstrate is predicted to establish a number of favorable contacts within th e cathepsin X binding site, in particular with residues His23 and Tyr27 fro m the mini-loop. The presence of the mini-loop restricts the accessibility of cystatin C as well as of the endopeptidase and MCA substrates in the pri med subsites of the protease. The marked structural and functional differen ces of cathepsin X relative to other members of the papain family of cystei ne proteases will be of great value in designing specific inhibitors useful as research tools to investigate the physiological and potential pathologi cal roles of this novel enzyme.