Electrostatic switches that mediate the pH-dependent conformational change of short recombinant human pseudocathepsin D

摘要

Human Cathepsin D (hCatD) is an aspartic peptidase with a low pH optimum. X-ray crystal structures have been solved for an active, low pH (pH 5.1) form (CatDlo) [Baldwin ET, Bhat, T.N., Gulnik, S., Hosur, M.V., Sowder, R.C., Cachau, R.E., Collins, J., Silva, A.M. and Erickson J.W. (1993) Proc. Natl. Acad. Sci. 90:6796–6800] and an inactive, high pH (pH 7.5) form (CatDhi) [Lee AY, Gulnik SV, Erickson JW. (1998) Nat Struct Biol 5:866–871]. It has been suggested that ionizable switches involving the carboxylate side chains of E5, E180, and D187 may mediate the reversible interconversion between CatDhi and CatDlo and that Y10 stabilizes CatDhi [Lee AY, Gulnik SV, Erickson JW. (1998) Nat Struct Biol 5:866–871]. To test these hypotheses, we generated single point mutants in “short” recombinant human pseudocathepsin D (srCatD), a model kinetically similar to hCatD [Beyer BM and Dunn BM. (1996) J Biol Chem 271:15590–15596]. E180Q, Y10F, and D187N exhibit significantly higher kcat/Km values (2, 3, and 6-fold, respectively) at pH 3.7 and pH 4.75 compared to srCatD indicating that these residues are important in stabilizing the CatDhi. E5Q exhibits a 2-fold lower kcat/Km compared to srCatD at both pH values indicating the importance of E5 in stabilizing the CatDlo. Accordingly, full time-course “pH-jump” (pH 5.5 to 4.75) studies of substrate hydrolysis indicate that E180Q, D187N, and Y10F have shorter kinetic lag phases that represent the change from CatDhi to CatDlo compared to srCatD and E5Q. Intrinsic tryptophan fluorescence reveals that the variants have a native-like structure over the pH range of our assays. The results indicate that E180 and D187 participate as an electrostatic switch that initiates the conformational change of CatDlo to CatDhi and Y10 stabilizes CatDhi by hydrogen bonding to the catalytic Asp 33. E5 appears to play a less significant role as an ionic switch that stabilizes CatD_lo.