Polymer Effects under Pressure. IV. The Hydrolysis of Phenyl Esters Catalyzed by α-Chymotrypsin up to 2000 bar

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Abstract
The rates of the hydrolysis of 2-valeryloxy(C5)- and 2-heptanoyloxy(C7)-benzoic acids catalyzed by α-chymotrypsin (α-CHT) were measured at pressures up to 2000 bar at 30 °C and pH 7.8 in a 0.05 M Tris buffer solution. The apparent Michaelis constant, Kmapp, was estimated to vary from 5.9 to 9.9 mM, and kcat from 11×10−3 to 52×10−3 s−1, for the C5 ester, and Kmapp, from 4.7 to 10 mM, and kcat from 37×10−3 to 200×10−3 s−1, for the C7 ester, between 1 and 2000 bar. From the pressure dependences of Kmapp and kcat, the volume changes accompanying the dissociation of the enzyme–substrate complex and the activation volume for the process of the product formation were calculated to be −6.3±2 and −20±2 cm3/mol for the C5 ester and −9.5±2 and −21±2 cm3/mol for the C7 ester. The effects of the pressure on the hydrolysis of p-nitrophenyl acetate (PNPA) catalyzed by α-CHT have also been measured up to 3000 bar at pH 7.8 in a 0.05 M Tris buffer solution and at 25 °C and 35 °C. The activation volumes were −3 cm3/mol at 25 °C and −4 cm3/mol at 30 °C and 25 °C. These results were discussed on the basis of the X-ray study of the enzyme-substrate complex.