Kinetic evidence for an intermediate in the deacetylation of monoacetyl-chymotrypsin.

Mono[14C]acetyl-chymotrypsin was prepared by treating alpha-chymotrypsin with a 10-fold molar excess of p-nitrophenyl[14C]acetate at pH 5, and the acetylated enzyme was isolated free of excess reagents by gel filtration. Deacetylation at pH 6.0 was followed by observing the decrease in acid-precipitable radioactivity and provided a first-order rate constant of 0.02 +/- 0.008 min-1. Reactivation of the acetylated protein was followed by continuously monitoring the appearance of esterolytic activity towards alpha-N-acetyltyrosine ethyl ester. Reactivation at pH 6.0 occurred exponentially with a first-order rate constant of 0.2 +/- 0.015 min-1, the reactivated enzyme exhibiting an apparent catalytic contant (k' cat) of 1200 +/- 60 min-1, which decreased to a value of 945 +/- 15 min-1 by an apparent first-order process with a rate constant of 0.025 +/- 0.006 min-1. These results are interpreted in terms of a two-step deacetylation of monoacetyl-chymotrypsin involving an acetylated intermediate with esterase activity.