Mechanism of inactivation of chymotrypsin by 3-benzyl-6-chloro-2-pyrone

Abstract

The mechanism of inactivation of chymotrypsin by 3-benzyl-6-chloro-2-pyrone was studied. Chloride analysis of the inactivated enzyme suggests that the complex does not contain intact chloropyrone or an acid chloride. 13C NMR studies of the enzyme inactivated with 13C-enriched chloropyrones show that the pyrone ring is no longer intact, C-6 becomes a carboxylate group and C-2 becomes esterified to the enzyme, probably to serine-195, and a double bond is present adjacent to the serine ester. The inactivated enzyme slowly regains catalytic activity with the concomitant release of (E)-4-benzyl-2-pentenedioic acid. Double bond migration probably occurs during reactivation since the position of the double bond in the released diacid product is different than in the inactivator-enzyme complex. When the reactivation is carried out in [18O]H2O-enriched water, a single oxygen-18 is incorporated into the released product and is further evidence that the inactivator is bound to the enzyme only through a single ester linkage. A deuterium isotope effect on reactivation is observed when a chloropyrone deuterated at C-5 is used. Removal of a proton from C-5 is probably required for reactivation and isomerization of the double bond and not hydrolysis of the acyl-enzyme is rate determining. A variety of amines accelerate the rate of reactivation by functioning as general bases and not as nucleophiles. A reaction scheme is presented that accounts for the formation of the stable inactivator-enzyme complex as well as the production of 2 products derived from enzymatic hydrolysis of the chloropyrone. The importance of a C-6-derived carboxylate group in the stabilization of the acyl-enzyme is discussed.