Dipole Formation and Solvent Electrostriction in Subtilisin Catalysis

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• 作者：Peter C. Michels ; Jonathan S. Dordick ; and Douglas S. Clark
• 刊名：Journal of the American Chemical Society
• 出版年：1997
• 出版时间：October 8, 1997
• 年：1997
• 卷：119
• 期：40
• 页码：9331 - 9335
• 全文大小：222K
• 年卷期：v.119,no.40(October 8, 1997)
• ISSN：1520-5126

文摘

The transition state for subtilisin-catalyzed transesterificationwas probed by high-pressure kinetic studiesin solvents spanning a wide range of dielectric constants. Theelectrostatic model of Kirkwood described the solventeffects and gave a lower limit of 31 ± 1.5 Debye for the dipolemoment of the transition state. This value remainedconstant in a wide range of polar and apolar solvents, indicating thatthe catalytic triad of subtilisin is remarkablyrobust. Despite the highly polar transition state, substantialrate enhancements relative to the uncatalyzed reactionwere measured in highly apolar solvents such as hexane; this is thefirst report of such an extreme disparity betweentransition-state and solvent polarities. Moreover, the solventdependence of the activation volume implies a loweffective dielectric of the polypeptide chain in the active site andsubstantial penetration of the active site by solvent.Kirkwood's model was also used to quantify the effect of anactive-site mutation on the transition-state dipolemoment.These results illustrate that the electrostatic model combinedwith high-pressure kinetics can provide unique informationon the basic properties of enzyme reaction processes and can be usefulin predicting solvent effects on enzymereaction rates.