Consequences ofmolecular recognition intheS1-S2 intersubsite region ofpapain forcatalytic-site chemistry Change inpH-dependence characteristics andgeneration ofaninverse solvent kinetic isotope effect byintroduction ofaP1-P2amidebondinto atwo-protonic-state reactivity probe

2'-pyridyl disulphide andwithethyl 2-pyridyl disulphide andofkforthereaction of benzimidazol-2-ylmethanethiol (asaminimal modelofcysteine proteinase catalytic sites) withtheformer disulphide weredetermined inaqueous buffers at25°CatI0.1. 2.Ofthese three pH-kprofiles onlythat forthereaction ofpapain with2-(acetamido)ethyl 2'-pyridyl disulphide hasarate maximumatpHapprox. 6;theothers eachhavearateminimuminthis pH region anda ratemaximumatpH4,whichis characteristic ofreactions ofpapain withother 2-pyridyl disulphides that donotcontain aP1-P2 amidebond inthenon-pyridyl part ofthemolecule. 3.Themarked change intheformofthepH-kprofile consequent uponintroduction ofaP1-P2 amidebondinto theprobe molecule forthereaction withpapain butnotfor that withtheminimal catalytic-site modelisinterpreted interms oftheinduction bybinding oftheprobe intheS,-intersubsite region oftheenzyme ofatransition-state geometry inwhichnucleophilic attack by the-S-component ofthecatalytic site isassisted byassociation oftheimidazolium ioncomponent withthe leaving group. 4.Thegreater definition oftherate maximuminthepH-kprofile forthereaction ofpapain withananalogous 2-pyridyl disulphide reactivity probe containing bothaP1-P2 amide bondandapotential occupant fortheS2subsite {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 aP2-S2 interaction substantially increases thepopulation oftransition states forthe imidazolium ion-assisted reaction. 5.Theoverall kinetic solvent 2H-isotope effect atpL6.0wasdetermined tobe:forthereaction ofpapain with2,2'-dipyridyl disulphide, 0.96(i.e. nokinetic isotope effect), forits reaction withtheprobe containing only theP1-P2 amide bond, 0.75, forits reaction with theprobe containing boththeP1-P2 amidebondandtheoccupant fortheS2subsite, 0.61, andforkcat./Km forits catalysis ofthe hydrolysis ofN-methoxycarbonylglycine 4-nitrophenyl ester, 0.67. 6.Thedevelopment ofarate maximum atpHapprox. 6appears tocorrelate inthese reactions ofpapain withthedevelopment ofaninverse kinetic solvent isotope effect (k'H20/k2H2O < 1). Possible interpretation ofthese dataandofdataforother reactions ofpapain intheliterature isdiscussed interms ofbreaking ofahydrogen bondwithin the-S-* - *H-Im+ion-pair. Ethyl2-pyridyl disulphide wasprepared by reaction ofpyridine-2-thion e