The active-site residue Cys-29 is responsible for the neutral-pH inactivation and the refolding barrier of human cathepsin B

Human cathepsin B, the most abundant lysosomal cysteine protease, has been implicated in a variety of important physiological and pathological process es, It has been known for a long time that like other lysosomal cysteine pr oteases, cathepsin B becomes inactivated and undergoes irreversible denatur ation at neutral or alkaline pH, However, the mechanism of this denaturatio n process remains mostly unknown up to this day. In the present work, nucle ar magnetic resonance spectroscopy was used to characterize the molecular o rigin of the neutral-pH inactivation and the refolding barrier of human cat hepsin B, Two forms of human cathepsin B, the native form with Cys-29 at th e active site and a mutant with Cys-29 replaced by Ala, were shown to have well-folded structures at the active and slightly acidic condition of pH 5, Surprisingly, while the native cathepsin B irreversibly unfolds at pH 7.5, the C29A mutant was found to maintain a stable three-dimensional structure at neutral pH conditions. In addition, replacement of Cys-29 by Ala render s the process of the urea denaturation of human cathepsin B completely reve rsible, in contrast to the opposite behavior of the wild-type cathepsin B, These results are very surprising in that replacement of one single residue , the active-site Cys-29, can eliminate the neutral-pH denaturation and the refolding barrier, We speculate that this finding may have important impli cations in understanding the process of pi-I-triggered inactivation commonl y observed for most lysosomal cysteine proteases, (C) 2000 Federation of Eu ropean Biochemical Societies, Published by Elsevier Science B.V. All rights reserved.