title: 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.
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 degrees 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.
authors: Department of Biochemistry, Institute of Biology, Eötvös Loránd University, H-1117 Budapest, Hungary.
authors: Júlia
authors: J
authors: Tóth
authors: Zoltán
authors: Z
authors: Simon
authors: Péter
authors: P
authors: Medveczky
authors: Linda
authors: L
authors: Gombos
authors: Balázs
authors: B
authors: Jelinek
authors: László
authors: L
authors: Szilágyi
authors: László
authors: L
authors: Gráf
authors: András
authors: A
authors: Málnási-Csizmadia
mesh_terms: D004789:Enzyme Activation; D006801:Humans; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D017434:Protein Structure, Tertiary; D013379:Substrate Specificity; D013816:Thermodynamics; D014357:Trypsin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D014783:Viscosity
chemical_list: C040749:PRSS3 protein, human; D014357:Trypsin