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Thermodynamics of -Chymotrypsin-Inhibitor Complex Formation

EFFECTS OF STRUCTURAL MODIFICATION OF THE INHIBITOR

From the ¹ From the Department of Biochemistry, West Virginia University Medical Center, Morgantown, West Virginia 26506

Temperature dependence studies, at a slightly acid pH, on the competitive water analogue inhibitors of -chymotrypsin show that complex formation is independent of temperature and that binding is primarily entropy-driven. The inhibitors of this type studied here were tertiary amyl alcohol and 3-pentanone.

In addition, studies on another class of inhibitor are presented. These competitive inhibitors consist of a benzene ring with different functional groups attached. The compounds are benzene, toluene, benzyl alcohol, benzaldehyde, and benzoate ion. Such inhibitors are similar to the substrate methyl hippurate in that the H⁰ values for complex formation are quite negative while S⁰ values may range from slightly positive to quite negative. Compounds of this type give rise to very large negative values for the change in heat capacity, C_p, for the complex-forming step, suggesting that considerable conformational change takes place in the enzyme during the process. This is in striking contrast to the first mentioned water analogue inhibitors where C_p is equal to zero. In addition it is shown that C_p for the formation of the ES complex from diethylglutarate which does not contain an aromatic nucleus is also very close to zero; this implies that the proposed conformational change brought about by the aromatic substrates and inhibitors does not take place in the absence of an aromatic ring, and suggests that a charge transfer complex may be involved in triggering the aforementioned conformational change. Examples are produced to show that comparison of association constants at a single temperature can be very misleading. Evidence is presented which indicates that there is no charge neutralization when complex formation with an anion such as benzoate takes place, but that there is a truly enormous change in heat capacity.

It is suggested that the aliphatic alcohol and ketone may well bind at a slightly different site or portion of the enzyme than that with which the aromatic inhibitors combine. It is further proposed that both of these sites may be used to help bind a substrate such as methyl hippurate by means of a two-point hydrophobic attachment. The hydrophobic binding of the aromatic portion of the substrate molecule is probably exothermic, while the binding of the aliphatic portion of the alcohol component may not make a significant contribution to the enthalpy change.

The ratio of inhibitor to enzyme in the enzyme-inhibitor complex is unity for all types of inhibitors studied.

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