Kinetic .alpha.-deuterium isotope effects for acylation of chymotrypsin by 4-methoxyphenyl formate and for deacylation of formylchymotrypsin

Abstract

The kinetic ..cap alpha..-deuterium isotope effect, (V/sub max//Km)/sub D//(V/sub max//Km)/sub H/, for acylation of ..cap alpha..-chymotrypsin by 4-methoxyphenyl formate in aqueous solution over the pH range 5.8-6.8 is 11.8 +- 0.02. For deacylation of the formylchymotrypsin generated in this reaction, the kinetic ..cap alpha..-deuterium isotope effect, (V/sub max/)/sub D//(V/sub max/)/sub H/, is 1.15 +- 0.03 over the pH range 6.2-6.8. These values strongly suggest that acylation of chymotrypsin by this reagent occurs at serine and that ..-->.. C-O-Enz bond formation and cleavage in the transition states for acylation and deacylation reactions are well advanced. Kinetic ..cap alpha..-deuterium isotope effects have proved to be useful criteria of mechanism for a growing number of enzymatic reactions. Two such isotope effects were reported, measured in an effort to obtain novel information concerning the transition state structure for ..cap alpha..-chymotrypsin-catalyzed hydrolysis of esters. The nature of the measurements required the utilization of formates; 4-methoxyphenyl formate, 4-methoxyphenyl (/sup 2/H) formate, 4- methoxyphenyl (/sup 3/H) formate and 4-methoxyphenyl (/sup 14/C) formate were synthesized. The ..cap alpha..-deuterium secondary isotope effect on V/sub max/ was measured by a direct spectrophotometric assay. The secondary deuterium isotope effect on V/sub max//K/sub m/ for acylation of chymotrypsin by 4-methoxyphenyl formate, 1.18more » +- 0.02 suggeststhat acylation occurs at serine and not at histidine. The secondary deuterium isotope effect for chymotrypsin deformylation, 1.15 +- 0.03 is consistent with the conclusions. The kinetic isotope effect for ph-independent nonenzymatic hydrolysis of phenyl formates is near 1.25. The limiting isotope effect must be at least this large. It follows that geometry at the formyl carbon atom in enzyme deacylation is intermediate between tetrahedral and triginal. 1 figure. (DP)« less