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. 1977 Feb;74(2):510–514. doi: 10.1073/pnas.74.2.510

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

B A Chibber, J M Tomich, E T Mertz, T Viswanatha
PMCID: PMC392319  PMID: 265516

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 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.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Baggott J. E., Klapper M. H. Rate enhancement specificity with alpha-chymotrypsin: temperature dependence of deacylation. Biochemistry. 1976 Apr 6;15(7):1473–1481. doi: 10.1021/bi00652a018. [DOI] [PubMed] [Google Scholar]
  2. Balls A. K., Aldrich F. L. ACETYL-CHYMOTRYPSIN. Proc Natl Acad Sci U S A. 1955 Apr 15;41(4):190–196. doi: 10.1073/pnas.41.4.190. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bernhard S. A., Lau S. J., Noller H. Spectrophotometric identification of acyl enzyme intermediates. Biochemistry. 1965 Jun;4(6):1108–1118. doi: 10.1021/bi00882a020. [DOI] [PubMed] [Google Scholar]
  4. DIXON G. H., NEURATH H. Acylation of the enzymatic site of delta-chymotrypsin by esters, acid anhydrides, and acid chlorides. J Biol Chem. 1957 Apr;225(2):1049–1059. [PubMed] [Google Scholar]
  5. Edelhoch H. Spectroscopic determination of tryptophan and tyrosine in proteins. Biochemistry. 1967 Jul;6(7):1948–1954. doi: 10.1021/bi00859a010. [DOI] [PubMed] [Google Scholar]
  6. GUTFREUND H., STURTEVANT J. M. The mechanism of the reaction of chymotrypsin with p-nitrophenyl acetate. Biochem J. 1956 Aug;63(4):656–661. doi: 10.1042/bj0630656. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. KEZDY F. J., BENDER M. L. The kinetics of the alpha-chymotrypsin-catalyzed hydrolysis of p-nitrophenyl acetate. Biochemistry. 1962 Nov;1:1097–1106. doi: 10.1021/bi00912a021. [DOI] [PubMed] [Google Scholar]
  8. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  9. VISWANATHA T., LAWSON W. B. The action of N-bromosuc-cinimide on chymotrypsin. Arch Biochem Biophys. 1961 Apr;93:128–134. doi: 10.1016/0003-9861(61)90324-1. [DOI] [PubMed] [Google Scholar]

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