Electrostatic Switches That Mediate the pH-Dependent Conformational Change of "Short" Recombinant Human Pseudocathepsin D

详细信息    查看全文

• 作者：Nathan E. Goldfarb ; Minh T. Lam ; Arjo K. Bose ; Ambar M. Patel ; Alexander J. Duckworth ; and Ben M. Dunn
• 刊名：Biochemistry
• 出版年：2005
• 出版时间：December 6, 2005
• 年：2005
• 卷：44
• 期：48
• 页码：15725 - 15733
• 全文大小：292K
• 年卷期：v.44,no.48(December 6, 2005)
• ISSN：1520-4995

文摘

Human cathepsin D (hCatD) is an aspartic peptidase with a low pH optimum. X-ray crystalstructures 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 suggestedthat ionizable switches involving the carboxylate side chains of E5, E180, and D187 may mediate thereversible 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 generatedsingle point mutants in "short" recombinant human pseudocathepsin D (srCatD), a model kinetically similarto hCatD [Beyer, B. M., and Dunn, B. M. (1996) J. Biol. Chem. 271, 15590-15596]. E180Q, Y10F, andD187N exhibit significantly higher k_cat/K_m values (2-, 3-, and 6-fold, respectively) at pH 3.7 and 4.75compared to srCatD, indicating that these residues are important in stabilizing the CatD_hi. E5Q exhibitsa 2-fold lower k_cat/K_m compared to srCatD at both pH values, indicating the importance of E5 in stabilizingthe CatD_lo. Accordingly, full time-course "pH-jump" (pH 5.5-4.75) studies of substrate hydrolysis indicatethat E180Q, D187N, and Y10F have shorter kinetic lag phases that represent the change from CatD_hi toCatD_lo compared to srCatD and E5Q. Intrinsic tryptophan fluorescence reveals that the variants have anative-like structure over the pH range of our assays. The results indicate that E180 and D187 participateas an electrostatic switch that initiates the conformational change of CatD_lo to CatD_hi and Y10 stabilizesCatD_hi by hydrogen bonding to the catalytic Asp 33. E5 appears to play a less significant role as an ionicswitch that stabilizes CatD_lo.