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

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Abstract
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 tel 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 constant (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 2-step deacetylation of monoacetyl-chymotrypsin involving an acetylated intermediate with esterase activity.