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Diverse role of conformational dynamics in carboxypeptidase A-driven peptide and ester hydrolyses: Disclosing the “Perfect Induced Fit” and “Protein Local Unfolding” pathways by altering protein stability

Authors

  • Mikhael Shushanyan,

    1. Department of Chemistry and Pharmacy, University of Erlangen-Nürnberg, 91058 Erlangen, Germany
    2. Institute for Biophysics and Bionanosciences at the Department of Physics, I. Javakhishvili Tbilisi State University, 0128 Tbilisi, Georgia (Republic)
    3. Department of Biophysics, LEPL Life Science Research Center, 0160 Tbilisi, Georgia (Republic)
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  • Dimitri E. Khoshtariya,

    Corresponding author
    1. Department of Chemistry and Pharmacy, University of Erlangen-Nürnberg, 91058 Erlangen, Germany
    2. Institute for Biophysics and Bionanosciences at the Department of Physics, I. Javakhishvili Tbilisi State University, 0128 Tbilisi, Georgia (Republic)
    3. Department of Biophysics, LEPL Life Science Research Center, 0160 Tbilisi, Georgia (Republic)
    • Department of Chemistry and Pharmacy, University of Erlangen-Nürnberg, 91058 Erlangen, Germany
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  • Tatyana Tretyakova,

    1. Institute for Biophysics and Bionanosciences at the Department of Physics, I. Javakhishvili Tbilisi State University, 0128 Tbilisi, Georgia (Republic)
    2. Department of Biophysics, LEPL Life Science Research Center, 0160 Tbilisi, Georgia (Republic)
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  • Maya Makharadze,

    1. Institute for Biophysics and Bionanosciences at the Department of Physics, I. Javakhishvili Tbilisi State University, 0128 Tbilisi, Georgia (Republic)
    2. Department of Biophysics, LEPL Life Science Research Center, 0160 Tbilisi, Georgia (Republic)
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  • Rudi van Eldik

    Corresponding author
    1. Department of Chemistry and Pharmacy, University of Erlangen-Nürnberg, 91058 Erlangen, Germany
    • Department of Chemistry and Pharmacy, University of Erlangen-Nürnberg, 91058 Erlangen, Germany
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  • This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com

Abstract

Catalytic mechanisms of carboxypeptidase A (CPA) are well known for their diversity and the relative inaccessibility for a decisive comprehension. Recent encouraging attempts through modern computational techniques promoted new challenges for the complementary experimental endeavors. In this work, we have applied the stopped-flow technique and the method of reaction progress curve fitting to extract kinetic parameters for the CPA-catalyzed hydrolyses of smaller (typical) peptide and ester substrates, known for their strong activating/inhibiting impact, thus to which the traditional method of “initial rates” is not applicable. Our approach that innately implies the overall constancy of the affecter (substrate plus “active” product) concentration, made it possible to rigorously determine the physically meaningful “effective” values for the catalytic and Michaelis constants under diverse experimental conditions including variable temperature and urea or trimethylamine N-oxide concentrations. Analysis of the obtained results allowed for: (i) the further substantiation of diverse mechanistic patterns for archetypal specific peptide and ester substrates, (ii) testing and disclosure of intrinsic links between the stabilizing/destabilizing and activating/inhibiting effects for the important model enzyme, CPA, and (iii) tentative explanation of a distinct activating/inhibiting impact of these substrates through the strong specific interaction of their benzyl (Bz) moiety with the substrate binding S3 subsite of CPA. We have demonstrated that stabilization of CPA either through the interaction with an extra Bz moiety (belonging to another substrate or to the product) leads to the increase of its catalytic power with respect to the specific peptide substrate and to its decrease with respect to the counterpart ester substrate. We conjecture that the catalytic mechanisms operating in these two cases include: (a) the “promoted water” mechanism for the peptide substrate that, seemingly, provides the almost “perfect induced fit” (low-barrier conformational adaptation), and (b) presumably, the “anhydride intermediate” mechanism for the ester substrate that, anyway, requires substantial conformational rearrangement (in fact, “partial or local unfolding”) of the protein environment in the course of the rate-determining step. © 2011 Wiley Periodicals, Inc. Biopolymers 95: 852–870, 2011.

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