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. 1972 Feb;126(3):645–657. doi: 10.1042/bj1260645

Comparative studies of the specificities of α-chymotrypsin and subtilisin BPN′. Studies with flexible substrates

T N Pattabiraman 1,*, W B Lawson 1
PMCID: PMC1178422  PMID: 5075271

Abstract

A series of arylalkanoate esters and α-acetamidoarylalkanoate esters were tested as substrates for α-chymotrypsin and subtilisin BPN′. Chymotrypsin hydrolysed N-acetyl-l-phenylalanine methyl ester and methyl 4-phenylbutyrate faster than their respective higher and lower homologues, whereas methyl 2-acetamido-6-phenylhexanoate and methyl 6-phenylhexanoate were better substrates for subtilisin than their lower homologues. N-Acetyl-l-tryptophan methyl ester and its analogue, N-acetyl-3-(1-naphthyl)-alanine methyl ester, were hydrolysed 23 times faster by chymotrypsin than by subtilisin. These results indicate that the binding site of α-chymotrypsin is roughly 1.1nm (11Å) long and curved, whereas that of subtilisin is a longer system and less curved. The stereo-specificity during the hydrolysis of typical substrates by both enzymes was found to vary over a wide range. The enhancing effect of the α-acetamido group in the l-series of substrates and the detrimental effect in the d-series of substrates also varies considerably.

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Selected References

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

  1. ANDRAKO J., SMITH J. D., HARTUNG W. H. Substituted acetamidomalonic esters and DL-2-acetamido acids as antitumor agents. J Pharm Sci. 1961 Apr;50:337–340. doi: 10.1002/jps.2600500413. [DOI] [PubMed] [Google Scholar]
  2. Alden R. A., Wright C. S., Kraut J. A hydrogen-bond network at the active site of subtilisin BPN'. Philos Trans R Soc Lond B Biol Sci. 1970 Feb 12;257(813):119–124. doi: 10.1098/rstb.1970.0014. [DOI] [PubMed] [Google Scholar]
  3. BIRNBAUM S. M., LEVINTOW L., KINGSLEY R. B., GREENSTEIN J. P. Specificity of amino acid acylases. J Biol Chem. 1952 Jan;194(1):455–470. [PubMed] [Google Scholar]
  4. Barel A. O., Glazer A. N. Comparative studies of the enzymatic properties of Novo and Carlsberg subtilisins. J Biol Chem. 1968 Apr 10;243(7):1344–1348. [PubMed] [Google Scholar]
  5. 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]
  6. Birktoft J. J., Blow D. M., Henderson R., Steitz T. A. I. Serine proteinases. The structure of alpha-chymotrypsin. Philos Trans R Soc Lond B Biol Sci. 1970 Feb 12;257(813):67–76. doi: 10.1098/rstb.1970.0009. [DOI] [PubMed] [Google Scholar]
  7. CLEMENT G. E., BENDER M. L. THE EFFECT OF APROTIC DIPOLAR ORGANIC SOLVENTS ON THE KINETICS OF ALPHA-CHYMOTRYPSIN-CATALYZED HYDROLYSES. Biochemistry. 1963 Jul-Aug;2:836–843. doi: 10.1021/bi00904a036. [DOI] [PubMed] [Google Scholar]
  8. Cohen S. G., Lo L. W. On the active site of alpha-chymotrypsin. Cyclized and noncyclized substrates with tetrasubstituted alpha carbon atoms. J Biol Chem. 1970 Nov 10;245(21):5718–5727. [PubMed] [Google Scholar]
  9. Cohen S. G., Schultz R. M. The active site in alpha-chymotrypsin. Absolute configuration and kinetics of hydrolysis of methyl 3,4-dihydroisocoumarin-3-carboxylate. J Biol Chem. 1968 May 25;243(10):2607–2617. [PubMed] [Google Scholar]
  10. Cohen S. G., Schultz R. M., Weinstein S. Y. Stereospecificity in hydrolysis by alpha-chymotrypsin of esters of alpha, alpha-disubstituted acetic and beta, beta-disubstituted propionic acids. J Am Chem Soc. 1966 Nov 20;88(22):5315–5319. doi: 10.1021/ja00974a052. [DOI] [PubMed] [Google Scholar]
  11. Dupaix A., Béchet J. J., Roucous C. Separation of polar, steric and specific effects in the alpha-chymotrypsin-catalyzed hydrolysis of acyl-substituted p-nitrophenyl esters. Biochem Biophys Res Commun. 1970 Oct 23;41(2):464–470. doi: 10.1016/0006-291x(70)90528-0. [DOI] [PubMed] [Google Scholar]
  12. Enriquez P. M., Gerig J. T. Deacylation rates of several trifluoromethyl-substituted acylchymotrypsins. Biochemistry. 1969 Aug;8(8):3156–3161. doi: 10.1021/bi00836a004. [DOI] [PubMed] [Google Scholar]
  13. Freer S. T., Kraut J., Robertus J. D., Wright H. T., Xuong N. H. Chymotrypsinogen: 2.5-angstrom crystal structure, comparison with alpha-chymotrypsin, and implications for zymogen activation. Biochemistry. 1970 Apr 28;9(9):1997–2009. doi: 10.1021/bi00811a022. [DOI] [PubMed] [Google Scholar]
  14. HOFSTEE B. H. Fatty acid esters as substrates for trypsin and chymotrypsin. Biochim Biophys Acta. 1957 Apr;24(1):211–213. doi: 10.1016/0006-3002(57)90173-7. [DOI] [PubMed] [Google Scholar]
  15. Hayashi Y., Lawson W. B. Stereochemistry of the active site of alpha-chymotrypsin. The binding geometry of tryptophan derivatives. J Biol Chem. 1969 Aug 10;244(15):4158–4167. [PubMed] [Google Scholar]
  16. Ingles D. W., Knowles J. R. The stereospecificity of alpha-chymotrypsin. Biochem J. 1968 Jul;108(4):561–569. doi: 10.1042/bj1080561. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. JONES J. B., KUNITAKE T., NIEMANN C., HEIN G. E. THE PRIMARY SPECIFICITY OF ALPHA-CHYMOTRYPSIN. ACYLATED AMINO ACID ESTERS WITH NORMAL ALKYL SIDE CHAINS. J Am Chem Soc. 1965 Apr 20;87:1777–1781. doi: 10.1021/ja01086a029. [DOI] [PubMed] [Google Scholar]
  18. MATSUBARA H., KASPER C. B., BROWN D. M., SMITH E. L. SUBTILISIN BPN'. I. PHYSICAL PROPERTIES AND AMINO ACID COMPOSITION. J Biol Chem. 1965 Mar;240:1125–1130. [PubMed] [Google Scholar]
  19. Milstien J. B., Fife T. H. Steric effects in the acylation of alpha-chymotrypsin. Biochemistry. 1969 Feb;8(2):623–627. doi: 10.1021/bi00830a024. [DOI] [PubMed] [Google Scholar]
  20. Morihara K., Tsuzuki H. Comparison of the specificities of various serine proteinases from microorganisms. Arch Biochem Biophys. 1969 Feb;129(2):620–634. doi: 10.1016/0003-9861(69)90223-9. [DOI] [PubMed] [Google Scholar]
  21. Morihara K., Tsuzuki H., Oka T. Comparison of the specificities of various neutral proteinases from microorganisms. Arch Biochem Biophys. 1968 Mar 11;123(3):572–588. doi: 10.1016/0003-9861(68)90179-3. [DOI] [PubMed] [Google Scholar]
  22. Pattabiraman T. N., Lawson W. B. Comparative studies of the specificities of -chymotrypsin and subtilisin BPN'. Studies with flexible and 'locked' substrates. Biochem J. 1972 Feb;126(3):659–665. doi: 10.1042/bj1260659. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Rossi G. L., Bernhard S. A. On the relationship between the conformation and the catalyzed reactivity of acyl-chymotrypsin. J Mol Biol. 1971 Jan 28;55(2):215–230. doi: 10.1016/0022-2836(71)90193-8. [DOI] [PubMed] [Google Scholar]
  24. Rumsh L. D., Volkova L. I., Antonov V. K. Hydrolysis of L-3-benzyl-2,5-diketomorpholine by chymotrypsin. FEBS Lett. 1970 Jul 29;9(2):64–66. doi: 10.1016/0014-5793(70)80313-1. [DOI] [PubMed] [Google Scholar]
  25. Silver M. S., Stoddard M., Sone T., Matta M. S. Alpha-chymotrypsin. The use of substrates of restricted geometry to define the reactive conformation of methyl N-acetyl-l-phenylalaninate. J Am Chem Soc. 1970 May 20;92(10):3151–3160. doi: 10.1021/ja00713a038. [DOI] [PubMed] [Google Scholar]
  26. Steitz T. A., Henderson R., Blow D. M. Structure of crystalline alpha-chymotrypsin. 3. Crystallographic studies of substrates and inhibitors bound to the active site of alpha-chymotrypsin. J Mol Biol. 1969 Dec 14;46(2):337–348. doi: 10.1016/0022-2836(69)90426-4. [DOI] [PubMed] [Google Scholar]
  27. WALLACE R. A., KURTZ A. N., NIEMANN C. INTERACTION OF AROMATIC COMPOUNDS WITH ALPHA-CHYMOTRYPSIN. Biochemistry. 1963 Jul-Aug;2:824–836. doi: 10.1021/bi00904a035. [DOI] [PubMed] [Google Scholar]
  28. WU F. C., LASKOWSKI M. Action of the naturally occurring trypsin inhibitors against chymotrypsins alpha and beta. J Biol Chem. 1955 Apr;213(2):609–619. [PubMed] [Google Scholar]
  29. Wright C. S., Alden R. A., Kraut J. Structure of subtilisin BPN' at 2.5 angström resolution. Nature. 1969 Jan 18;221(5177):235–242. doi: 10.1038/221235a0. [DOI] [PubMed] [Google Scholar]

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