Electrostatic switches that mediate the pH-dependent conformational change of "short" recombinant human pseudocathepsin D.
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, E180, 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 kcat/Km 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 CatD(hi). E5Q exhibits a 2-fold lower kcat/Km 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 CatD(lo) 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 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).
Goldfarb
Nathan E
NE
Department of Biochemistry and Molecular Biology, University of Florida College of Medicine, 1600 Archer Road, P.O. Box 100245, Gainesville, Florida 32610, USA.
Lam
Minh T
MT
Bose
Arjo K
AK
Patel
Ambar M
AM
Duckworth
Alexander J
AJ
Dunn
Ben M
BM
D000595:Amino Acid Sequence; D002402:Cathepsin D / Q000737:chemistry* / Q000235:genetics; D004795:Enzyme Stability; D005453:Fluorescence; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D017354:Point Mutation; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry* / Q000235:genetics; D016415:Sequence Alignment; D014364:Tryptophan / Q000737:chemistry
D011994:Recombinant Proteins; D014364:Tryptophan; D002402:Cathepsin D; C507414:short recombinant human pseudocathepsin D