@entity1 @entity10815 (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 @entity3338 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 @entity1 pseudocathepsin D (srCatD), a model kinetically similar to hCatD [Beyer, B. M., and Dunn, B. M. (1996) J. Biol. Chem. 271, 15590-15596]. @entity69454 , @entity69455 , and @entity69456 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 @entity69454 , @entity69456 , and @entity69455 have shorter kinetic lag phases that represent the change from CatD(hi) to CatD(lo) compared to srCatD and E5Q. Intrinsic @entity2340 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 @entity3884 bonding to the catalytic @entity2574 33. E5 appears to play a less significant role as an ionic switch that stabilizes CatD(lo).

Electrostatic switches that mediate the pH-dependent conformational change of "short" recombinant XXXX pseudocathepsin D.

@entity1 :: ('9606', 'Species') :: ['human', 'Human'] @entity10815 :: ('1509', 'Gene') :: ['cathepsin D'] @entity3884 :: ('MESH:D006859', 'Chemical') :: ['hydrogen'] @entity69454 :: ('p|SUB|E|180|Q', 'ProteinMutation') :: ['E180Q'] @entity2340 :: ('CHEBI:27897', 'Chemical') :: ['tryptophan'] @entity69455 :: ('p|SUB|Y|10|F', 'ProteinMutation') :: ['Y10F'] @entity69456 :: ('p|SUB|D|187|N', 'ProteinMutation') :: ['D187N'] @entity3338 :: ('CHEBI:35757', 'Chemical') :: ['carboxylate'] @entity2574 :: ('CHEBI:22660', 'Chemical') :: ['Asp']
@entity1 :: (9606,Species) :: ['human']
