Crystal structure of human procathepsin X: A cysteine protease with the proregion covalently linked to the active site cysteine

Human cathepsin X is one of many proteins discovered in recent years throug h the mining of sequence databases. Its sequence shows clear homology to cy steine proteases from the papain family, containing the characteristic resi due patterns, including the active site. However, the proregion of cathepsi n X is only 38 residues long, the shortest among papain-like enzymes, and t he cathepsin X sequence has an atypical insertion in the regions proximal t o the active site. This protein was recently expressed and partially charac terized biochemically. Unlike most other cysteine proteases from the papain family, procathepsin X is incapable of autoprocessing in vitro but can be processed under reducing conditions by exogenous cathepsin L. Atypically, t he mature enzyme is primarily a carboxypeptidase and has extremely poor end opeptidase activity. We have determined the three-dimensional structure of the procathepsin X at 1.7 Angstrom resolution. The overall structure of the mature enzyme is characteristic for enzymes of the papain superfamily, but contains several novel features. Most interestingly, the short proregion b inds to the enzyme with the aid of a covalent bond between the cysteine res idue in the proregion (Cys10p) and the active site cysteine residue (Cys31) . This is the first example of a zymogen in which the inhibition of enzyme' s proteolytic activity by the proregion is achieved through a reversible co valent modification of the active site nucleophile. Such mode of binding re quires less contact area between the proregion and the enzyme than observed in other procathepsins, and no auxiliary binding site on the enzyme surfac e is used. A three-residue insertion in a highly conserved region, just pri or to the active site cysteine residue, confers a significantly different s hape on the S' subsites, compared to other proteases from papain family. Th e 3D structure provides an explanation for the rather unusual carboxypeptid ase activity of this enzyme and confirms the predictions based on homology modeling. Another long insertion in the cathepsin X amino acid sequence for ms a P-hairpin pointing away from the active site. This insertion, thought to be an equivalent of cathepsin B occluding loop, is located on the side o f the protein, distant from the substrate binding site. (C) 2000 Academic P ress.