Skip to main content
PMC home page
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1973 Feb;70(2):517–520. doi: 10.1073/pnas.70.2.517

Influence of Leaving-Group Electronic Effect on α-Chymotrypsin: Catalytic Constants of Specific Substrates

Manfred Philipp *, Ralph M Pollack †, Myron L Bender *
PMCID: PMC433295  PMID: 16592059

Abstract

Rate constants and binding constants for the α-chymotrypsin-catalyzed hydrolysis of N-acetyltyrosine, tryptophan, and phenylalanine anilides are presented. Both kcat and Km are independent of electronic effects in the substrate over a range of 9.8 orders of magnitude (as measured by pK of the leaving group). Similarly, Km is independent of charge and orientation about the α-carbon for various substrates and pseudo-substrates. These results are not consistent with the pretransition state protonation hypothesis; instead, they are discussed in terms of a tetrahedral intermediate that is thermodynamically less stable than the Michaelis complex.

Keywords: enzyme, protease, kinetics, intermediates

Full text

PDF
517

Selected References

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

  1. BENDER M. L., KEZDY J. MECHANISM OF ACTION OF PROTEOLYTIC ENZYMES. Annu Rev Biochem. 1965;34:49–76. doi: 10.1146/annurev.bi.34.070165.000405. [DOI] [PubMed] [Google Scholar]
  2. Baumann W. K., Bizzozero S. A., Dutler H. Specificity of alpha-chymotrypsin. Dipeptide substrates. FEBS Lett. 1970 Jun 27;8(5):257–260. doi: 10.1016/0014-5793(70)80280-0. [DOI] [PubMed] [Google Scholar]
  3. Bender M. L., Begué-Cantón M. L., Blakeley R. L., Brubacher L. J., Feder J., Gunter C. R., Kézdy F. J., Killheffer J. V., Jr, Marshall T. H., Miller C. G. The determination of the concentration of hydrolytic enzyme solutions: alpha-chymotrypsin, trypsin, papain, elastase, subtilisin, and acetylcholinesterase. J Am Chem Soc. 1966 Dec 20;88(24):5890–5913. doi: 10.1021/ja00976a034. [DOI] [PubMed] [Google Scholar]
  4. Bender M. L., Gibian M. J., Whelan D. J. The alkaline pH dependence of chymotrypsin reactions: postulation of a pH-dependent intramolecular competitive inhibition. Proc Natl Acad Sci U S A. 1966 Sep;56(3):833–839. doi: 10.1073/pnas.56.3.833. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bundy H. F., Moore C. L. Chymotrypsin-catalyzed hydrolysis of m-, p-, and o-nitroanilides of N-benzoyl-L-tyrosine. Biochemistry. 1966 Feb;5(2):808–811. doi: 10.1021/bi00866a056. [DOI] [PubMed] [Google Scholar]
  6. CUNNINGHAM L. W., BROWN C. S. The influence of pH on the kinetic constants of alpha-chymotrypsin-catalyzed esterolysis. J Biol Chem. 1956 Jul;221(1):287–299. [PubMed] [Google Scholar]
  7. Caplow M. Chymotrypsin catalysis. Evidence for a new intermediate. J Am Chem Soc. 1969 Jun 18;91(13):3639–3645. doi: 10.1021/ja01041a037. [DOI] [PubMed] [Google Scholar]
  8. Fersht A. R. Mechanism of the -chymotrypsin-catalyzed hydrolysis of specific amide substrates. J Am Chem Soc. 1972 Jan 12;94(1):293–295. doi: 10.1021/ja00756a061. [DOI] [PubMed] [Google Scholar]
  9. Fersht A. R., Requena Y. Mechanism of the -chymotrypsin-catalyzed hydrolysis of amides. pH dependence of k c and K m . Kinetic detection of an intermediate. J Am Chem Soc. 1971 Dec 15;93(25):7079–7087. doi: 10.1021/ja00754a066. [DOI] [PubMed] [Google Scholar]
  10. HEIN G., NIEMANN C. An interpretation of the kinetic behavior of model substrates of alpha-chymotrypsin. Proc Natl Acad Sci U S A. 1961 Sep 15;47:1341–1355. doi: 10.1073/pnas.47.9.1341. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Himoe A., Brandt K. G., DeSa R. J., Hess G. P. Investigations of the chymotrypsin-catalyzed hydrolysis of specific substrates. IV. Pre-steady state kinetic approaches to the investigation of the catalytic hydrolysis of esters. J Biol Chem. 1969 Jul 10;244(13):3483–3493. [PubMed] [Google Scholar]
  12. INWARD P. W., JENCKS W. P. THE REACTIVITY OF NUCLEOPHILIC REAGENTS WITH FUROYL-CHYMOTRYPSIN. J Biol Chem. 1965 May;240:1986–1996. [PubMed] [Google Scholar]
  13. Inagami T., Patchornik A., York S. S. Participation of an acidic group in the chymotrypsin catalysis. J Biochem. 1969 May;65(5):809–819. doi: 10.1093/oxfordjournals.jbchem.a129081. [DOI] [PubMed] [Google Scholar]
  14. Inagami T., York S. S., Patchornik A. An electrophilic mechanism in the chymotrypsin-catalyzed hydrolysis of anilide substrates. J Am Chem Soc. 1965 Jan 5;87(1):126–127. doi: 10.1021/ja01079a027. [DOI] [PubMed] [Google Scholar]
  15. Johnson C. H., Knowles J. R. The binding of inhibitors to alpha-chymotrypsin. Biochem J. 1966 Oct;101(1):56–62. doi: 10.1042/bj1010056. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Lucas E. C., Caplow M. Chymotrypsin catalysis. Evidence for a new intermediate. J Am Chem Soc. 1972 Feb 9;94(3):960–963. doi: 10.1021/ja00758a039. [DOI] [PubMed] [Google Scholar]
  17. McConn J., Ku E., Himoe A., Brandt K. G., Hess G. P. Investigations of the chymotrypsin-catalyzed hydrolysis of specific substrates. V. Determination of pre-steady state kinetic parameters for specific substrate esters by stopped flow techniques. J Biol Chem. 1971 May 10;246(9):2918–2925. [PubMed] [Google Scholar]
  18. Morihara K., Oka T., Tsuzuki H. Comparison of alpha-chymotrypsin and subtilisin BPN': size and specificity of the active site. Biochem Biophys Res Commun. 1969 Apr 29;35(2):210–214. doi: 10.1016/0006-291x(69)90269-1. [DOI] [PubMed] [Google Scholar]
  19. O'Leary M. H., Kluetz M. D. Identification of the rate-limiting step in the chymotrypsin-catalyzed hydrolysis of N-acetyl-L-tryptophanamide. J Am Chem Soc. 1970 Oct 7;92(20):6089–6090. doi: 10.1021/ja00723a062. [DOI] [PubMed] [Google Scholar]
  20. Parker L., Wang J. H. On the mechanism of action at the acylation step of the alpha-chymotrypsin-catalyzed hydrolysis of anilides. J Biol Chem. 1968 Jul 10;243(13):3729–3734. [PubMed] [Google Scholar]
  21. Wang J. H. Facilitated proton transfer in enzyme catalysis. It may have a crucial role in determining the efficiency and specificity of enzymes. Science. 1968 Jul 26;161(3839):328–334. doi: 10.1126/science.161.3839.328. [DOI] [PubMed] [Google Scholar]
  22. Williams A. Chymotrypsin-catalyzed phenyl ester hydrolysis. Evidence for electrophilic assistance on carbonyl oxygen. Biochemistry. 1970 Aug 18;9(17):3383–3390. doi: 10.1021/bi00819a014. [DOI] [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES