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.

Temperature dependence of internal friction in enzyme reactions

“…The activation process occurs naturally when the N-terminal peptide of the zymogen form of trypsin is proteolytically cleaved by another protease. Recently, we showed that activation slowed by replacing this glycine with alanine or bulkier sidechains (11); however, the conformation of trypsin is affected slightly and only locally at this sidechain as it is indicated by the small activity change and also confirmed by molecular modeling (18). 16, 17).…”

“…We found that the reciprocal of the rate constant (i.e., the relaxation time) of the domain movement of trypsin depends linearly on solvent viscosity with a nonzero limit at zero viscosity (11). We wanted to learn about the energetic aspects of this internal viscosity by separating it from the external viscosity and determining its temperature dependence.…”

“…Trypsin activation is a domain rearrangement that occurs by the rotation of a separate domain called activation domain around 5 glycine hinges (residues 19, 142, 184, 193, 216; refs. We also found that the apparent activation energies of the activation of these mutants did not change, but the slower processes caused by the mutations with bulky sidechains at position 193 were the consequences of the increased apparent internal viscosities (11). The role of these glycines in the activation process and the proteolytic activity of trypsin were deeply investigated recently (17).…”

“…1). The reaction can be followed by the change of the intrinsic tryptophan fluorescence in a stoppedflow apparatus (11,15). At high pH, the active trypsin acquires the zymogen conformation, which is transformed to the active form on pH reduction.…”

A Laboratory Study of the Effect of Temperature on Densities and Viscosities of Binary and Ternary Blends of Soybean Oil, Soy Biodiesel and Petroleum Diesel Oil

“…Studies on viscosity have been performed on pineapple juice, vegetable oil, etc. [26]. However, there is a dearth of information on the effect of temperature on the viscosity and density of binary and ternary blends of biodiesel oil.…”