Alternate Substrate Inhibitors of an α-Chymotrypsin: Enantioselective Interaction of Aryl-Substituted Enol Lactones

Four enol lactones, bearing phenyl or 1-naphthyl substituents on the α or ß positions [3-phenyl-6-methylenetetrahydro-2-pyranone (αPh6H, IIe), 3-(1 -naphthyl)-6-methylenetetrahydro-2-pyranone (αNp6H, IId), 4-phenyl-6-methylenetetrahydro-2-pyranone (βPh6H, IIIe), and 4-(l-naphthyl)-6-methylenetetrahydro-2-pyranone (βNp6H, IIId)], available as pure R and S enantiomers, have been studied as alternate substrate inhibitors of chymotrypsin. Kinetic constants for substrate binding (K_s) and acylation (K_a) were determined by a competitive substrate assay, using succinyl-L-Ala-L-Ala-L-Pro-L-Phe p-nitroanilide; the deacylation rate constant (K_d) was determined by the proflavin displacement assay. All lactones undergo rapid acylation (K_a varies from 17 to 170 min^‒1) that shows little enantioselectivity; there is, however, pronounced enantioselectivity in substrate binding for three of the lactones [K_S(R/S) = 40–110]. In each case it is the enantiomer with the S configuration that has the higher affinity. In all cases, deacylation rates are slow, and in two cases, acyl enzymes with half-lives of 4.0 and 12.5 h at pH 7.2, 25 °C, are obtained (for βPh6H and αNp6H, respectively). In these cases, high deacylation enantioselectivity is observed [k_dS/R) = 60–70], and the lactone more weakly bound as a substrate (R enantiomer) gives the more stable acyl enzyme. Two hypotheses, involving hindrance of the attack of water or an exchange of the ester and ketone carbonyl groups in the acyl enzyme, are advanced as possible explanations for the high stability of these acyl enzymes.