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. 1979 Nov 1;183(2):223–231. doi: 10.1042/bj1830223

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

Keith Brocklehurst 1,*, J Paul G Malthouse 1,†, Michael Shipton 1
PMCID: PMC1161550  PMID: 43129

Abstract

A method is proposed by which site-specific reactivity probes that exhibit different reactivities in two ionization states can be used to detect association–activation phenomena that involve repositioning of acid/base groups in enzyme active centres. The pH-dependences of the apparent second-order rate constants (k) for the reactions of the thiol group of papain (EC 3.4.22.2) with a series of two-protonic-state reactivity probes are compared. The short-chain probes, 2,2′-dipyridyl disulphide and n-propyl 2-pyridyl disulphide, react at pH6 in adsorptive complexes and/or transition states with geometries that do not permit hydrogen-bonding of the pyridyl nitrogen atom with the active-centre imidazolium ion, as evidenced by the rate minima at pH6 and the rate maxima at pH4 provided by reagent protonation. Only when the probe molecule, e.g. 4-(N-aminoethyl 2′-pyridyl disulphide)-7-nitrobenzo-2-oxa-1,3-diazole [compound(III)], contains a long hydrophobic side chain is the reaction characterized by maximal rates at about pH6, as in the acylation step of the catalytic act (at pH6, kcompound III/k2,2′-dipyridyl disulphide ≃ 100). It is proposed that this striking difference in profile shape may result from binding of the hydrophobic side chain of compound (III) possibly in the S2-subsite of papain, which promotes a change in catalytic-site geometry involving repositioning of the imidazolium ion of histidine-159 and hydrogen-bonding with the N atom of the leaving group, as has been postulated to occur in the acylation step of substate hydrolysis.

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Selected References

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