Skip to main content
PMC home page
Biochemical Journal logoLink to Biochemical Journal
. 1988 Mar 15;250(3):761–772. doi: 10.1042/bj2500761

Consequences of molecular recognition in the S1-S2 intersubsite region of papain for catalytic-site chemistry. Change in pH-dependence characteristics and generation of an inverse solvent kinetic isotope effect by introduction of a P1-P2 amide bond into a two-protonic-state reactivity probe.

K Brocklehurst 1, D Kowlessur 1, G Patel 1, W Templeton 1, K Quigley 1, E W Thomas 1, C W Wharton 1, F Willenbrock 1, R J Szawelski 1
PMCID: PMC1148922  PMID: 2839145

Abstract

1. The pH-dependences of the second-order rate constant (k) for the reactions of papain (EC 3.4.22.2) with 2-(acetamido)ethyl 2'-pyridyl disulphide and with ethyl 2-pyridyl disulphide and of k for the reaction of benzimidazol-2-ylmethanethiol (as a minimal model of cysteine proteinase catalytic sites) with the former disulphide were determined in aqueous buffers at 25 degrees C at I 0.1. 2. Of these three pH-k profiles only that for the reaction of papain with 2-(acetamido)ethyl 2'-pyridyl disulphide has a rate maximum at pH approx. 6; the others each have a rate minimum in this pH region and a rate maximum at pH 4, which is characteristic of reactions of papain with other 2-pyridyl disulphides that do not contain a P1-P2 amide bond in the non-pyridyl part of the molecule. 3. The marked change in the form of the pH-k profile consequent upon introduction of a P1-P2 amide bond into the probe molecule for the reaction with papain but not for that with the minimal catalytic-site model is interpreted in terms of the induction by binding of the probe in the S1-S2 intersubsite region of the enzyme of a transition-state geometry in which nucleophilic attack by the -S- component of the catalytic site is assisted by association of the imidazolium ion component with the leaving group. 4. The greater definition of the rate maximum in the pH-k profile for the reaction of papain with an analogous 2-pyridyl disulphide reactivity probe containing both a P1-P2 amide bond and a potential occupant for the S2 subsite [2-(N'-acetyl-L-phenylalanylamino)ethyl 2'-pyridyl disulphide [Brocklehurst, Kowlessur, O'Driscoll, Patel, Quenby, Salih, Templeton, Thomas & Willenbrock (1987) Biochem. J. 244, 173-181]) suggests that a P2-S2 interaction substantially increases the population of transition states for the imidazolium ion-assisted reaction. 5. The overall kinetic solvent 2H-isotope effect at pL 6.0 was determined to be: for the reaction of papain with 2,2'-dipyridyl disulphide, 0.96 (i.e. no kinetic isotope effect), for its reaction with the probe containing only the P1-P2 amide bond, 0.75, for its reaction with the probe containing both the P1-P2 amide bond and the occupant for the S2 subsite, 0.61, and for kcat./Km for its catalysis of the hydrolysis of N-methoxycarbonylglycine 4-nitrophenyl ester, 0.67.(ABSTRACT TRUNCATED AT 400 WORDS)

Full text

PDF
761

Selected References

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

  1. Asbóth B., Polgár L. On the enhanced catalytic activity of papain towards amide substrates. Acta Biochim Biophys Acad Sci Hung. 1977;12(3):223–230. [PubMed] [Google Scholar]
  2. Asbóth B., Polgár L. Transition-state stabilization at the oxyanion binding sites of serine and thiol proteinases: hydrolyses of thiono and oxygen esters. Biochemistry. 1983 Jan 4;22(1):117–122. doi: 10.1021/bi00270a017. [DOI] [PubMed] [Google Scholar]
  3. Ascenzi P., Aducci P., Torroni A., Amiconi G., Ballio A., Menegatti E., Guarneri M. The pH dependence of pre-steady-state and steady-state kinetics for the papain-catalyzed hydrolysis of N-alpha-carbobenzoxyglycine p-nitrophenyl ester. Biochim Biophys Acta. 1987 Apr 8;912(2):203–210. doi: 10.1016/0167-4838(87)90090-2. [DOI] [PubMed] [Google Scholar]
  4. Berger A., Schechter I. Mapping the active site of papain with the aid of peptide substrates and inhibitors. Philos Trans R Soc Lond B Biol Sci. 1970 Feb 12;257(813):249–264. doi: 10.1098/rstb.1970.0024. [DOI] [PubMed] [Google Scholar]
  5. Brocklehurst K., Dixon H. B. PH-dependence of the steady-state rate of a two-step enzymic reaction. Biochem J. 1976 Apr 1;155(1):61–70. doi: 10.1042/bj1550061. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Brocklehurst K., Dixon H. B. The pH-dependence of second-order rate constants of enzyme modification may provide free-reactant pKa values. Biochem J. 1977 Dec 1;167(3):859–862. doi: 10.1042/bj1670859. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Brocklehurst K., Herbert J. A., Norris R., Suschitzky H. Evidence for association-activation effects in reactions of papain from studies on its reactivity towards isomeric two-protonic-state reactivity probes. Biochem J. 1979 Nov 1;183(2):369–373. doi: 10.1042/bj1830369. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Brocklehurst K., Kowlessur D., O'Driscoll M., Patel G., Quenby S., Salih E., Templeton W., Thomas E. W., Willenbrock F. Substrate-derived two-protonic-state electrophiles as sensitive kinetic specificity probes for cysteine proteinases. Activation of 2-pyridyl disulphides by hydrogen-bonding. Biochem J. 1987 May 15;244(1):173–181. doi: 10.1042/bj2440173. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Brocklehurst K., Little G. Reactions of papain and of low-molecular-weight thiols with some aromatic disulphides. 2,2'-Dipyridyl disulphide as a convenient active-site titrant for papain even in the presence of other thiols. Biochem J. 1973 May;133(1):67–80. doi: 10.1042/bj1330067. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. 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]
  11. Brocklehurst K., Malthouse J. P., Shipton M. Evidence that binding to the s2-subsite of papain may be coupled with catalytically relevant structural change involving the cysteine-25-histidine-159 diad. Kinetics of the reaction of papain with a two-protonic-state reactivity probe containing a hydrophobic side chain. Biochem J. 1979 Nov 1;183(2):223–231. doi: 10.1042/bj1830223. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Brocklehurst K., Mushiri S. M., Patel G., Willenbrock F. Evidence for a close similarity in the catalytic sites of papain and ficin in near-neutral media despite differences in acidic and alkaline media. Kinetics of the reactions of papain and ficin with chloroacetate. Biochem J. 1982 Jan 1;201(1):101–104. doi: 10.1042/bj2010101. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Brocklehurst K. The equilibrium assumption is valid for the kinetic treatment of most time-dependent protein-modification reactions. Biochem J. 1979 Sep 1;181(3):775–778. doi: 10.1042/bj1810775. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Brocklehurst K. Two-protonic-state electrophiles as probes of enzyme mechanisms. Methods Enzymol. 1982;87:427–469. doi: 10.1016/s0076-6879(82)87026-2. [DOI] [PubMed] [Google Scholar]
  15. Chaiken I. M., Smith E. L. Reaction of chloroacetamide with the sulfhydryl group of papain. J Biol Chem. 1969 Oct 10;244(19):5087–5094. [PubMed] [Google Scholar]
  16. Chaiken I. M., Smith E. L. Reaction of the sulfhydryl group of papain with chloroacetic acid. J Biol Chem. 1969 Oct 10;244(19):5095–5099. [PubMed] [Google Scholar]
  17. Creighton D. J., Gessouroun M. S., Heapes J. M. Is the thiolate--imidazolium ion pair the catalytically important form of papain? FEBS Lett. 1980 Feb 11;110(2):319–322. doi: 10.1016/0014-5793(80)80101-3. [DOI] [PubMed] [Google Scholar]
  18. Creighton D. J., Schamp D. J. Solvent isotope effects on tautomerization equilibria of papain and model thiolamines. FEBS Lett. 1980 Feb 11;110(2):313–318. doi: 10.1016/0014-5793(80)80100-1. [DOI] [PubMed] [Google Scholar]
  19. Dixon H. B. The unreliability of estimates of group dissociation constants. Biochem J. 1976 Mar 1;153(3):627–629. doi: 10.1042/bj1530627. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. 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]
  21. Fruton J. S. Proteinase-catalyzed synthesis of peptide bonds. Adv Enzymol Relat Areas Mol Biol. 1982;53:239–306. doi: 10.1002/9780470122983.ch7. [DOI] [PubMed] [Google Scholar]
  22. Huber C. P., Ozaki Y., Pliura D. H., Storer A. C., Carey P. R. Precise structural information for transient enzyme-substrate complexes by a combined X-ray crystallographic-resonance Raman spectroscopic approach. Biochemistry. 1982 Jun 22;21(13):3109–3115. doi: 10.1021/bi00256a012. [DOI] [PubMed] [Google Scholar]
  23. Jencks W. P. The utilization of binding energy in coupled vectorial processes. Adv Enzymol Relat Areas Mol Biol. 1980;51:75–106. doi: 10.1002/9780470122969.ch2. [DOI] [PubMed] [Google Scholar]
  24. Johnson F. A., Lewis S. D., Shafer J. A. Determination of a low pK for histidine-159 in the S-methylthio derivative of papain by proton nuclear magnetic resonance spectroscopy. Biochemistry. 1981 Jan 6;20(1):44–48. doi: 10.1021/bi00504a008. [DOI] [PubMed] [Google Scholar]
  25. Jolley C. J., Yankeelov J. A., Jr Reaction of papain with -bromo- -(5-imidazolyl)propionic acid. Biochemistry. 1972 Jan 18;11(2):164–169. doi: 10.1021/bi00752a005. [DOI] [PubMed] [Google Scholar]
  26. Kamphuis I. G., Drenth J., Baker E. N. Thiol proteases. Comparative studies based on the high-resolution structures of papain and actinidin, and on amino acid sequence information for cathepsins B and H, and stem bromelain. J Mol Biol. 1985 Mar 20;182(2):317–329. doi: 10.1016/0022-2836(85)90348-1. [DOI] [PubMed] [Google Scholar]
  27. Kamphuis I. G., Kalk K. H., Swarte M. B., Drenth J. Structure of papain refined at 1.65 A resolution. J Mol Biol. 1984 Oct 25;179(2):233–256. doi: 10.1016/0022-2836(84)90467-4. [DOI] [PubMed] [Google Scholar]
  28. Lewis S. D., Johnson F. A., Shafer J. A. Effect of cysteine-25 on the ionization of histidine-159 in papain as determined by proton nuclear magnetic resonance spectroscopy. Evidence for a his-159--Cys-25 ion pair and its possible role in catalysis. Biochemistry. 1981 Jan 6;20(1):48–51. doi: 10.1021/bi00504a009. [DOI] [PubMed] [Google Scholar]
  29. Lowe G., Yuthavong Y. Kinetic specificity in papain-catalysed hydrolyses. Biochem J. 1971 Aug;124(1):107–115. doi: 10.1042/bj1240107. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Lowe G., Yuthavong Y. pH-dependence and structure-activity relationships in the papain-catalysed hydrolysis of anilides. Biochem J. 1971 Aug;124(1):117–122. doi: 10.1042/bj1240117. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Mackenzie N. E., Malthouse J. P., Scott A. I. Chemical synthesis and papain-catalysed hydrolysis of N-alpha-benzyloxycarbonyl-L-lysine p-nitroanilide. Biochem J. 1985 Mar 1;226(2):601–606. doi: 10.1042/bj2260601. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Malthouse J. P., Brocklehurst K. Preparation of fully active ficin from Ficus glabrata by covalent chromatography and characterization of its active centre by using 2,2'-depyridyl disulphide as a reactivity probe. Biochem J. 1976 Nov;159(2):221–234. doi: 10.1042/bj1590221. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Migliorini M., Creighton D. J. Active-site ionizations of papain. An evaluation of the potentiometric difference titration method. Eur J Biochem. 1986 Apr 1;156(1):189–192. doi: 10.1111/j.1432-1033.1986.tb09566.x. [DOI] [PubMed] [Google Scholar]
  34. Mole J. E., Horton H. R. Kinetics of papain-catalyzed hydrolysis of -N-benzoyl-L-arginine-p-nitroanilide. Biochemistry. 1973 Feb 27;12(5):816–822. doi: 10.1021/bi00729a005. [DOI] [PubMed] [Google Scholar]
  35. Polgár L., Asbóth B. The basic difference in catalyses by serine and cysteine proteinases resides in charge stabilization in the transition state. J Theor Biol. 1986 Aug 7;121(3):323–326. doi: 10.1016/s0022-5193(86)80111-4. [DOI] [PubMed] [Google Scholar]
  36. Polgár L. Deuterium isotope effects on papain acylation. Evidence for lack of general base catalysis and for enzyme--leaving-group interaction. Eur J Biochem. 1979 Aug 1;98(2):369–374. doi: 10.1111/j.1432-1033.1979.tb13196.x. [DOI] [PubMed] [Google Scholar]
  37. 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]
  38. Polgár L., Halász P. Evidence for multiple reactive forms of papain. Eur J Biochem. 1978 Aug 1;88(2):513–521. doi: 10.1111/j.1432-1033.1978.tb12477.x. [DOI] [PubMed] [Google Scholar]
  39. Polgár L. Mercaptide-imidazolium ion-pair: the reactive nucleophile in papain catalysis. FEBS Lett. 1974 Oct 1;47(1):15–18. doi: 10.1016/0014-5793(74)80415-1. [DOI] [PubMed] [Google Scholar]
  40. Polgár L. On the mode of activation of the catalytically essential sulfhydryl group of papain. Eur J Biochem. 1973 Feb 15;33(1):104–109. doi: 10.1111/j.1432-1033.1973.tb02660.x. [DOI] [PubMed] [Google Scholar]
  41. Salih E., Malthouse J. P., Kowlessur D., Jarvis M., O'Driscoll M., Brocklehurst K. Differences in the chemical and catalytic characteristics of two crystallographically 'identical' enzyme catalytic sites. Characterization of actinidin and papain by a combination of pH-dependent substrate catalysis kinetics and reactivity probe studies targeted on the catalytic-site thiol group and its immediate microenvironment. Biochem J. 1987 Oct 1;247(1):181–193. doi: 10.1042/bj2470181. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Schechter I., Berger A. On the active site of proteases. 3. Mapping the active site of papain; specific peptide inhibitors of papain. Biochem Biophys Res Commun. 1968 Sep 6;32(5):898–902. doi: 10.1016/0006-291x(68)90326-4. [DOI] [PubMed] [Google Scholar]
  43. Schechter I., Berger A. On the size of the active site in proteases. I. Papain. Biochem Biophys Res Commun. 1967 Apr 20;27(2):157–162. doi: 10.1016/s0006-291x(67)80055-x. [DOI] [PubMed] [Google Scholar]
  44. Shipton M., Brochlehurst K. Characterization of the papain active centre by using two-protonic-state electrophiles as reactivity probes. Evidence for nucleophilic reactivity in the un-interrupted cysteine-25-histidine-159 interactive system. Biochem J. 1978 May 1;171(2):385–401. doi: 10.1042/bj1710385. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Shipton M., Brocklehurst K. Benzofuroxan as a thiol-specific reactivity probe. Kinetics of its reactions with papain, ficin, bromelain and low-molecular-weight thiols. Biochem J. 1977 Dec 1;167(3):799–810. doi: 10.1042/bj1670799. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Shipton M., Kierstan M. P., Malthouse J. P., Stuchbury T., Brocklehurst K. The case for assigning a value of approximately 4 to pKa-i of the essential histidine-cysteine interactive systems of papain, bromelain and ficin. FEBS Lett. 1975 Feb 15;50(3):365–368. doi: 10.1016/0014-5793(75)80529-1. [DOI] [PubMed] [Google Scholar]
  47. Smith D. J., Maggio E. T., Kenyon G. L. Simple alkanethiol groups for temporary blocking of sulfhydryl groups of enzymes. Biochemistry. 1975 Feb 25;14(4):766–771. doi: 10.1021/bi00675a019. [DOI] [PubMed] [Google Scholar]
  48. Storer A. C., Carey P. R. Comparison of the kinetics and mechanism of the papain-catalyzed hydrolysis of esters and thiono esters. Biochemistry. 1985 Nov 19;24(24):6808–6818. doi: 10.1021/bi00345a012. [DOI] [PubMed] [Google Scholar]
  49. Storer A. C., Lee H., Carey P. R. Relaxed and perturbed substrate conformations in enzyme active sites: evidence from multichannel resonance raman spectra. Biochemistry. 1983 Sep 27;22(20):4789–4796. doi: 10.1021/bi00289a027. [DOI] [PubMed] [Google Scholar]
  50. Stuchbury T., Shipton M., Norris R., Malthouse J. P., Brocklehurst K., Herbert J. A., Suschitzky H. A reporter group delivery system with both absolute and selective specificity for thiol groups and an improved fluorescent probe containing the 7-nitrobenzo-2-oxa-1,3-diazole moiety. Biochem J. 1975 Nov;151(2):417–432. doi: 10.1042/bj1510417. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Wallenfels K., Eisele B. Stereospecific alkylation with asymmetric reagents. Eur J Biochem. 1968 Jan;3(3):267–275. doi: 10.1111/j.1432-1033.1968.tb19526.x. [DOI] [PubMed] [Google Scholar]
  52. Wandinger A., Creighton D. J. Solvent isotope effects on the rates of alkylation of thiolamine models of papain. FEBS Lett. 1980 Jul 11;116(1):116–121. doi: 10.1016/0014-5793(80)80541-2. [DOI] [PubMed] [Google Scholar]
  53. Wharton C. W., Szawelski R. J. Half-time analysis of the integrated Michaelis equation. Simulation and use of the half-time plot and its direct linear variant in the analysis of some alpha-chymotrypsin, papain- and fumarase-catalysed reactions. Biochem J. 1982 May 1;203(2):351–360. doi: 10.1042/bj2030351. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES