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. 1984 Jun 1;220(2):609–612. doi: 10.1042/bj2200609

Differences between the electric fields of the catalytic sites of papain and actinidin detected by using the thiol-located nitrobenzofurazan label as a spectroscopic reporter group.

K Brocklehurst, E Salih, T S Lodwig
PMCID: PMC1153668  PMID: 6378189

Abstract

The catalytic-site thiol groups of papain (EC 3.4.22.2) and actinidin (EC 3.4.22.14) were each labelled with the nitrobenzofurazan (Nbf) chromophore by reaction with 4-chloro-7-nitrobenzofurazan at pH 4.4. The electronic-absorption spectra of both labelled enzymes were determined in aqueous solution, in the pH ranges approx. 2-5 for S-Nbf-papain and approx. 3.3-8 for S-Nbf-actinidin, and for the latter also in 6 M-guanidinium chloride. The spectrum of S-Nbf-papain is characterized by lambda max. = 402 nm at pH 5 and by lambda max. = 422 nm at pH 2.18. The pH-dependent shift in lambda max. accompanies a pH-dependent change in A 430, the nature of which is consistent with its dependence on a single ionizing group with pKa 3.7. The spectrum of S-Nbf-actinidin is pH-independent in the pH range approx. 3.3-8 and is characterized by lambda max. = 413 nm. This absorption maximum shifts to 425 nm in 6M-guanidinium chloride. These results are discussed and related to those reported previously from studies on papain and actinidin with various reactivity probes. Despite the close similarity in the catalytic sites of papain and actinidin deduced from X-ray-diffraction studies, the considerable differences in their reactivity characteristics are mirrored by differences in their electric fields detected by the Nbf spectroscopic label. The microenvironment in the catalytic site of actinidin appears to favour the existence of ions significantly more than in the corresponding region in papain.

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Selected References

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  1. Allen G., Lowe G. Investigation of the active site of papain with fluorescent probes. Biochem J. 1973 Aug;133(4):679–686. doi: 10.1042/bj1330679. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Baines B. S., Allen G., Brocklehurst K. The highly electrophilic character of 4-chloro-7-nitrobenzofurazan and possible consequences for its application as a protein-labelling reagent. Biochem J. 1977 Apr 1;163(1):189–192. doi: 10.1042/bj1630189. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Baines B. S., Brocklehurst K. A necessary modification to the preparation of papain from any high-quality latex of Carica papaya and evidence for the structural integrity of the enzyme produced by traditional methods. Biochem J. 1979 Feb 1;177(2):541–548. doi: 10.1042/bj1770541. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Baker E. N. Structure of actinidin, after refinement at 1.7 A resolution. J Mol Biol. 1980 Aug 25;141(4):441–484. doi: 10.1016/0022-2836(80)90255-7. [DOI] [PubMed] [Google Scholar]
  5. Baker E. N. Structure of actinidin: details of the polypeptide chain conformation and active site from an electron density map at 2-8 A resolution. J Mol Biol. 1977 Sep 25;115(3):263–277. doi: 10.1016/0022-2836(77)90154-1. [DOI] [PubMed] [Google Scholar]
  6. Bendall M. R., Lowe G. Co-operative ionisation of aspartic-acid-158 and histidine-159 in papain. Evidence from 19F nuclear-magnetic-resonance and fluorescence spectroscopy. Eur J Biochem. 1976 Jun 1;65(2):481–491. doi: 10.1111/j.1432-1033.1976.tb10364.x. [DOI] [PubMed] [Google Scholar]
  7. Birkett D. J., Price N. C., Radda G. K., Salmon A. G. The reactivity of SH groups with a fluorogenic reagent. FEBS Lett. 1970 Feb 25;6(4):346–348. doi: 10.1016/0014-5793(70)80095-3. [DOI] [PubMed] [Google Scholar]
  8. Brocklehurst K., Baines B. S., Malthouse J. P. Differences in the interaction of the catalytic groups of the active centres of actinidin and papain. Rapid purification of fully active actinidin by covalent chromatography and characterization of its active centre by use of two-protonic-state reactivity probes. Biochem J. 1981 Sep 1;197(3):739–746. doi: 10.1042/bj1970739. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Brocklehurst K., Malthouse J. P. Evidence for a two-state transition in papain that may have no close analogue in ficin. Differences in the disposition of cationic sites and hydrophobic binding areas in the active centres of papain and ficin. Biochem J. 1980 Dec 1;191(3):707–718. doi: 10.1042/bj1910707. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Brocklehurst K., Mushiri S. M., Patel G., Willenbrock F. A marked gradation in active-centre properties in the cysteine proteinases revealed by neutral and anionic reactivity probes. Reactivity characteristics of the thiol groups of actinidin, ficin, papain and papaya peptidase A towards 4,4'-dipyridyl disulphide and 5,5'-dithiobis-(2-nitrobenzoate) dianion. Biochem J. 1983 Mar 1;209(3):873–879. doi: 10.1042/bj2090873. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Drenth J., Jansonius J. N., Koekoek R., Wolthers B. G. The structure of papain. Adv Protein Chem. 1971;25:79–115. doi: 10.1016/s0065-3233(08)60279-x. [DOI] [PubMed] [Google Scholar]
  12. Drenth J., Kalk K. H., Swen H. M. Binding of chloromethyl ketone substrate analogues to crystalline papain. Biochemistry. 1976 Aug 24;15(17):3731–3738. doi: 10.1021/bi00662a014. [DOI] [PubMed] [Google Scholar]
  13. McDowall M. A. Anionic proteinase from Actinidia chinensis. Preparation and properties of the crystalline enzyme. Eur J Biochem. 1970 Jun;14(2):214–221. doi: 10.1111/j.1432-1033.1970.tb00280.x. [DOI] [PubMed] [Google Scholar]
  14. Polgár L., Halász P. Current problems in mechanistic studies of serine and cysteine proteinases. Biochem J. 1982 Oct 1;207(1):1–10. doi: 10.1042/bj2070001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Salih E., Brocklehurst K. Investigation of the catalytic site of actinidin by using benzofuroxan as a reactivity probe with selectivity for the thiolate-imidazolium ion-pair systems of cysteine proteinases. Evidence that the reaction of the ion-pair of actinidin (pKI 3.0, pKII 9.6) is modulated by the state of ionization of a group associated with a molecular pKa of 5.5. Biochem J. 1983 Sep 1;213(3):713–718. doi: 10.1042/bj2130713. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Shipton M., Stuchbury T., Brocklehurst K. 4-Chloro-7-nitrobenzo-2-oxa-1,3-diazole as a reactivity probe for the investigation of the thiol proteinases. evidence that ficin and bromelain may lack carboxyl groups conformationally equivalent to that of aspartic acid-158 of papain. Biochem J. 1976 Nov;159(2):235–244. doi: 10.1042/bj1590235. [DOI] [PMC free article] [PubMed] [Google Scholar]

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