Absence of evidence for an intermediate in the deacetylation of acetylchymotrypsin.
A recent paper [Chibber, B. A. K., Tomich, J. M., Mertz, E. T. & Viswanatha, T. (1977) Proc. Natl. Acad. Sci. USA 74, 510-514] presented evidence that was taken to support the existence of an intermediate in the deacetylation of acetylchymotrypsin. It was observed that deacylation, as measured by following the decrease in [(14)C]acetylchymotrypsin (decrease in acid-precipitable radioactivity), occurred at 1/10 the rate of reactivation, as measured by return of activity toward N-acetyl-L-tyrosine ethyl ester. Our experiments have shown that, at pH 6, the deacylation rate constant (measured by the loss of [(14)C]acetylchymotrypsin and by the formation of [(14)C]acetate) is identical (within experimental error) with the rate constant for reactivation (measured by determining the activity of aliquots of reactivating enzyme against N-acetyl-L-tryptophan ethyl ester) and with K(cat) for the turnover of p-nitrophenyl acetate by alpha-chymotrypsin. Part of the 10-fold greater reactivation rate observed by Chibber et al. has been shown to be due to the presence of 10% (vol/vol) isopropanol in their reactivation mixture, and it is argued that the balance of the effect is a manifestation of the "indole effect" produced by the simultaneous presence of 10 mM N-acetyl-L-tyrosine ethyl ester throughout the reactivation experiments. The results presented are entirely consistent with the three-step mechanism of catalysis by alpha-chymotrypsin and negate the existence of the proposed additional acetyl-enzyme intermediate.
D000107:Acetylation; D002384:Catalysis; D002918:Chymotrypsin / Q000378:metabolism*; D007700:Kinetics
D002918:Chymotrypsin
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