Abstract
The mini-chain of human cathepsin H has been identified as the major structural element determining the proteases substrate specificity. A genetically engineered mutant of human cathepsin H lacking the minichain, des[Glu(-18)-Thr(-11)]-cathepsin H, exhibits endopeptidase activity towards the synthetic substrate Z-Phe-Arg-NH-Mec (kcat = 0.4 s[-1], Km = 92 uM, kcat/Km = 4348 M[-1] s[-1]) which is not cleaved by r-wt cathepsin H. However, the mutant enzyme shows only minimal aminopeptidase activity for H-Arg-NH-Mec (kcat = 0.8 s[-1], Km = 3.6mM, kcat/Km = 222 M[-1] s[-1]) which is one of the best known substrates for native human cathepsin H (kcat = 2.5 s[-1], Km = 150 uM, kcat/Km = 16 666 M[-1] s[-1]). Inhibition studies with chicken egg white cystatin and E-64 suggest that the mini-chain normally restricts access of inhibitors to the active site. The kinetic data on substrates hydrolysis and enzyme inhibition point out the role of the mini-chain as a structural framework for transition state stabilization of free αamino groups of substrates and as a structural barrier for endopeptidase-like substrate cleavage.
Copyright © 2003 by Walter de Gruyter GmbH & Co. KG
