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 (CatD_(lo)) [Baldwin,E.T.,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.U.S.A.90,6796-6800] and an inactive,high pH (pH 7.5) form (CatD_(hi)) [Lee,A.Y.,Gulnik,S.V.,and Erickson,J.W.(1998) Nat.Struct.Biol.5,866-871].It has been suggested that ionizable switches involving the carboxylate side chains of E5,El80,and D187 may mediate the reversible interconversion between CatD_(hi) and CatD_(lo) and that Y10 stabilizes CatD_(hi) [Lee,A.Y.,Gulnik,S.V.,and Erickson,J.W.(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,B.M.,and Dunn,B.M.(1996) J.Biol.Chem.271,15590-15596].E180Q,Y10F,and D187N exhibit significantly higher k_(cat)/K_m values (2-,3-,and 6-fold,respectively) at pH 3.7 and 4.75 compared to srCatD,indicating that these residues are important in stabilizing the CatDhi.E5Q exhibits a 2-fold lower k_(cat)/K_m compared to srCatD at both pH values,indicating the importance of E5 in stabilizing the CatD_(lo).Accordingly,full time-course "pH-jump" (pH 5.5-4.75) studies of substrate hydrolysis indicate that E180Q,D187N,and Y10F have shorter kinetic lag phases that represent the change from CatD_(hi) to CatDio 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 El80 and D187 participate as an electrostatic switch that initiates the conformational change of CatD_(lo) to CatD_(hi) and Y10 stabilizes CatD_(hi) by hydrogen bonding to the catalytic Asp 33.E5 appears to play a less significant role as an ionic switch that stabilizes CatD_(lo).