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. 1966 Nov;101(2):402–410. doi: 10.1042/bj1010402

The kinetics of papain- and ficin-catalysed hydrolyses in the presence of alcohols

A W Lake 1, G Lowe 1
PMCID: PMC1270121  PMID: 5966277

Abstract

1. The maximum rate of production of p-nitrophenol (Vmax.) for both papain- and ficin-catalysed hydrolyses of p-nitrophenyl hippurate is independent of methanol concentration up to 2m for papain and 1·5m for ficin. 2. The observed catalytic constant (k0) for the production of hippuric acid for both papain- and ficin-catalysed hydrolyses of methyl hippurate decreases with increasing methanol concentration, 1/k0 being linearly dependent on the methanol concentration. The kMeOH/kH2O ratio is determined. 3. These results provide strong evidence against general base catalysis for the rate-determining step in the deacylation of hippuryl-papain and hippuryl-ficin and probably for other specific acyl-papains and acyl-ficins. 4. The rate-determining step for the deacylation of the non-specific trans-cinnamoyl-papain appears to be different from that for the specific hippuryl-papain, and is probably subject to general base catalysis. It is possible, however, to accommodate all these observations in a single four-step reaction pathway. 5. Propan-2-ol did not influence the rate of production of hippuric acid for the papain-catalysed hydrolysis of methyl hippurate. A similar result has previously been reported for the ficin-catalysed hydrolysis of methyl hippurate. Ethanol and of course methanol (see 2) decrease the rate of production of hippuric acid for both papain- and ficin-catalysed hydrolyses of methyl hippurate. It is suggested that the secondary alcohol is incapable for structural reasons of approaching the bond to be hydrolysed.

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Selected References

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

  1. BALLS A. K., MCDONALD C. E. Transesterification reactions catalyzed by chymotrypsin. J Biol Chem. 1956 Aug;221(2):993–1003. [PubMed] [Google Scholar]
  2. BERNHARD S. A., GUTFREUND H. Ficincatalysed reactions: the affinity of ficin for some arginine derivatives. Biochem J. 1956 May;63(1):61–64. doi: 10.1042/bj0630061. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Glazer A. N. Transesterification reactions catalyzed by subtilisins. J Biol Chem. 1966 Feb 10;241(3):635–638. [PubMed] [Google Scholar]
  4. HAMMOND B. R., GUTFREUND H. The mechanism of ficin-catalysed reactions. Biochem J. 1959 Jun;72(2):349–357. doi: 10.1042/bj0720349. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Kirsch J. F., Igelström M. The kinetics of the papain-catalyzed hydrolysis of esters of carbobenzoxyglycine. Evidence for an acyl-enzyme intermediate. Biochemistry. 1966 Feb;5(2):783–791. doi: 10.1021/bi00866a053. [DOI] [PubMed] [Google Scholar]
  6. LIGHT A., FRATER R., KIMMEL J. R., SMITH E. L. CURRENT STATUS OF THE STRUCTURE OF PAPAIN: THE LINEAR SEQUENCE, ACTIVE SULFHYDRYL GROUP, AND THE DISULFIDE BRIDGES. Proc Natl Acad Sci U S A. 1964 Nov;52:1276–1283. doi: 10.1073/pnas.52.5.1276. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. LOWE G., WILLIAMS A. A STUDY OF SOME THIOL ESTER HYDROLYSES AS MODELS FOR THE DEACYLATION STEP OF PAPAIN-CATALYSED HYDROLYSES. Biochem J. 1965 Jul;96:194–198. doi: 10.1042/bj0960194. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. LOWE G., WILLIAMS A. DIRECT EVIDENCE FOR AN ACYLATED THIOL AS AN INTERMEDIATE IN PAPAIN- AND FICIN-CATALYSED HYDROLYSES. Biochem J. 1965 Jul;96:189–193. doi: 10.1042/bj0960189. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. LOWE G., WILLIAMS A. PAPAIN-CATALYSED HYDROLYSIS OF SOME HIPPURIC ESTERS. A NEW MECHANISM FOR PAPAIN-CATALYSED HYDROLYSIS. Biochem J. 1965 Jul;96:199–204. doi: 10.1042/bj0960199. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. SMITH E. L. Active site of papain and covalent high-energy bonds of proteins. J Biol Chem. 1958 Dec;233(6):1392–1397. [PubMed] [Google Scholar]
  11. WHITAKER J. R., BENDER M. L. KINETICS OF PAPAIN-CATALYZED HYDROLYSIS OF ALPHA-N-BENZOYL-L-ARGININE ETHYL ESTER AND ALPHA-N-BENZOYL-L-ARGININAMIDE. J Am Chem Soc. 1965 Jun 20;87:2728–2737. doi: 10.1021/ja01090a034. [DOI] [PubMed] [Google Scholar]

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