The Application of Insolubilized a-Chymotrypsin to Kinetic Studies on the Effect of Aprotic Dipolar Organic Solvents*
نویسندگان
چکیده
Insolubilized cr-chymotrypsin bound to porous glass gel has been prepared. The kinetic behavior of this fixed enzyme proved to be the same as that of the free enzyme. The effects of aprotic dipolar organic solvents on the kinetics of cr-chymotrypsin-catalyzed hydrolysis were successfully studied using this insolubilized enzyme at concentrations up to 95% v/v dioxane, taking advantage of the inability of the enzyme to aggregate. This is a considerably higher organic solvent concentration than has been achieved in homogeneous solution. The kinetic parameters of the insolubilized cr-chymotrypsin-catalyzed hydrolysis of N-acetyl-L-tryptophan p-nitrophenyl ester and N-benzoyl-L-tyrosine p-nitroanilide in aqueous organic solvent medium were determined. The specific rate constant of the acylation step, kz, does not depend on organic solvent concentration. However, the apparent Michaelis constant, K,, and deacylation rate constant, k3, depend strongly on the organic solvent concentration. The data are discussed in terms of microscopic reversibility proposed previously for the mechanism of cr-chymotrypsin action.
منابع مشابه
The application of insolubilized alpha-chymotrypsin to kinetic studies on the effect of aprotic dipolar organic solvents.
Insolubilized cr-chymotrypsin bound to porous glass gel has been prepared. The kinetic behavior of this fixed enzyme proved to be the same as that of the free enzyme. The effects of aprotic dipolar organic solvents on the kinetics of cr-chymotrypsin-catalyzed hydrolysis were successfully studied using this insolubilized enzyme at concentrations up to 95% v/v dioxane, taking advantage of the ina...
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