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.
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, k(compound III)/k(2,2'-dipyridyl disulphide) approximately 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 S(2)-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.
D015082:2,2'-Dipyridyl / Q000031:analogs & derivatives; D000097:Acetonitriles; D001583:Benzoxazoles; D001665:Binding Sites; D002384:Catalysis; D055598:Chemical Phenomena; D002621:Chemistry; D003545:Cysteine; D004220:Disulfides; D004789:Enzyme Activation; D006639:Histidine; D006860:Hydrogen Bonding; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008956:Models, Chemical; D010206:Papain; D011522:Protons; D011725:Pyridines / Q000031:analogs & derivatives; D013438:Sulfhydryl Compounds
D000097:Acetonitriles; D001583:Benzoxazoles; D004220:Disulfides; D011522:Protons; D011725:Pyridines; D013438:Sulfhydryl Compounds; D006639:Histidine; D015082:2,2'-Dipyridyl; D010206:Papain; D003545:Cysteine