Energetics of alpha-chymotrypsin-mediated hydrolysis of a strained cyclic ester.

Abstract

Hydrolysis of omicron-hydroxy-alpha-toluenesulfonic acid sultone (sultone II) is mediated by alpha-chymotrypsin. Sultone II is a highly strained cyclic ester substrate that forms a covalent intermediate with the enzyme and is therefore expected to release ring-strain energy upon formation of the sulfonyl enzyme species. It is found that the equilibrium constant for forming the covalent intermediate from the Michaelis complex is quite modest (K2 = 16.4), suggesting that perhaps the strain energy is not released in the ring-cleavage event. The implied retention of chemical (strain) energy by the sulfonyl enzyme species raises the question of the means by which the enzyme avoids expression of strain energy and the implications of this effect in the catalytic sequence. High-pressure liquid chromatography (HPLC) rate data demonstrate facile reversion of sulfonyl enzyme to the Michaelis complex, and that reversion is preferred over hydrolysis of the covalent intermediate. pH-independent rate and equilibrium constants are derived for the alpha-chymotrypsin-mediated hydrolysis of sultone II, and pKa values for groups on the enzyme are reported that are consistent with literature values obtained from analysis of nonspecific substrate hydrolysis by the enzyme.