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Site directed mutagenesis at position 193 of human trypsin 4 alters the rate of conformational change during activation: Role of local internal viscosity in protein dynamics

Authors

  • Júlia Tóth,

    1. Department of Biochemistry, Institute of Biology, Eötvös Loránd University, H-1117 Budapest, Hungary
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    • The first two authors contributed equally to this paper.

  • Zoltán Simon,

    1. Department of Biochemistry, Institute of Biology, Eötvös Loránd University, H-1117 Budapest, Hungary
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    • The first two authors contributed equally to this paper.

  • Péter Medveczky,

    1. Department of Biochemistry, Institute of Biology, Eötvös Loránd University, H-1117 Budapest, Hungary
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  • Linda Gombos,

    1. Department of Biochemistry, Institute of Biology, Eötvös Loránd University, H-1117 Budapest, Hungary
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  • Balázs Jelinek,

    1. Department of Biochemistry, Institute of Biology, Eötvös Loránd University, H-1117 Budapest, Hungary
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  • László Szilágyi,

    1. Department of Biochemistry, Institute of Biology, Eötvös Loránd University, H-1117 Budapest, Hungary
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  • László Gráf,

    Corresponding author
    1. Department of Biochemistry, Institute of Biology, Eötvös Loránd University, H-1117 Budapest, Hungary
    • Department of Biochemistry, Institute of Biology, Eötvös Loránd University, Pázmány Péter sétány 1/C, H-1117 Budapest, Hungary
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  • András Málnási-Csizmadia

    Corresponding author
    1. Department of Biochemistry, Institute of Biology, Eötvös Loránd University, H-1117 Budapest, Hungary
    • Department of Biochemistry, Institute of Biology, Eötvös Loránd University, Pázmány Péter sétány 1/C, H-1117 Budapest, Hungary
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Abstract

Upon activation of trypsinogen four peptide segments flanked by hinge glycine residues undergo conformational changes. To test whether the degree of conformational freedom of hinge regions affects the rate of activation, we introduced amino acid side chains of different characters at one of the hinges (position 193) and studied their effects on the rate constant of the conformational change. This structural rearrangement leading to activation was triggered by a pH-jump and monitored by intrinsic fluorescence change in the stopped-flow apparatus. We found that an increase in the size of the side chain at position 193 is associated with the decrease of the reaction rate constant. To analyze the thermodynamics of the reaction, temperature dependence of the reaction rate constants was examined in a wide temperature range (5–60°C) using a novel temperature-jump/stopped-flow apparatus developed in our laboratory. Our data show that the mutations do not affect the activation energy (the exponential term) of the reaction, but they significantly alter the preexponential term of the Arrhenius equation. The effect of solvent viscosity on the rate constants of the conformational change during activation of the wild type enzyme and its R193G and R193A mutants was determined and evaluated on the basis of Kramers' theory. Based on this we propose that the reaction rate of this conformational transition is regulated by the internal molecular friction, which can be specifically modulated by mutagenesis in the hinge region. Proteins 2007. © 2007 Wiley-Liss, Inc.

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