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Evidence for association-activation effects in reactions of papain from studies on its reactivity towards isomeric two-protonic-state reactivity probes.

ABSTRACT: 4-(N-Aminoethyl 4-pyridyl disulphide)-7-nitrobenzo-2-oxa-1,3-diazole was synthesized and evaluted as a two-protonic-state reactivity probe by kinetic study of its reactions with papain (EC 3.4.22.2) and with benzimidazol-2-ylmethanethiol. Evidence is presented to suggest that: (i) both this probe molecule and its 2-pyridyl isomer bind to papain; (ii) the binding is followed by a change in the environment of the thiol group of cysteine-25; (iii) the striking rate maximum in neutral media observed in the reaction of papain with the 2-pyridyl isomer but not with the 4-pyridyl isomer arises from association of the 2-pyridyl leaving group with the imidazolium ion of histidine-159.

SUBMITTER: Brocklehurst K

PROVIDER: S-EPMC1161567 | biostudies-other | 1979 Nov

REPOSITORIES: biostudies-other

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Project description:1.2,2'-Dipyridyl disulphide (2-Py-S-S-2-Py) and n-propyl 2-pyridyl disulphide (propyl-S-S-2-Py) were used as two-protonic-state reactivity probes to investigate the active centre of papain (EC 3.4.22.2).2. The existence of a striking rate optimum at pH approx. 4 in the reaction of papain not only with the symmetrical probe but also with the unsymmetrical probe is shown to constitute compelling evidence that the thiolate ion component of the cysteine-25-histidine-159 interactive system of papain possesses appreciable nucleophilic character. It is not a necessary requirement that the probe reagent should engage the imidazolium ion of histidine-159 in hydrogen-bonding for the sulphur atom of the interactive system to display nucleophilic character. The single proton-binding site of propyl-S-S-2-Py cannot simultaneously interrupt the active-centre ion pair and provide for rate enhancement as the pH is lowered towards 4. The possible implication of this for the mechanism of papain-catalysed hydrolysis is discussed. 3. The suspected difference in the active centres of papain and ficin (EC 3.4.22.3), which could be a lack in ficin of a carboxy group conformationally equivalent to that of aspartic acid-158 of papain is confirmed. The reactivity of the papain thiol group towards both probe reagents is controlled by two ionizations with pKa close to 4 that are positively co-operative. 4. In the reaction of papain with 2-Py-S-S-2-Py. the reactivity appears to be controlled also by an addition ionization with pKa approx. 5. Possible origins of this additional ionization are discussed. K. The spectral and ionization characteristics of propyl-S-S-2-Py are reported. 6. The reagent reacts rapidly with thiol groups at the sulphur atom distal from the pyridyl ring to provide, at pH values below 9, stoicheiometric release of 2-thiopyridone. This property, together with the ability of the reagent markedly to increase its electrophilicity consequent on protonation, suggests alkyl-2-pyridyl disulphides in general as valuable two-protonic-state reactivity probes with exceptional specificity for thiol groups.

Project description:1. 2-(N'-Acetyl-L-phenylalanyl)hydroxyethyl 2'-pyridyl disulphide (compound V) was synthesized, and a study of the pH-dependence of the second-order rate constant (k) for its reaction with the catalytic-site thiol group of papain (EC 3.4.22.2) was used to evaluate the consequences for transition-state geometry of the presence of a hydrophobic occupant for the S2 subsite of the enzyme in the absence of the N-H component of the P1-P2 amide bond. 2. Comparison of the pH-dependences of K for reactions of compound (V), 2-(acetamido)ethyl 2'-pyridyl disulphide (compound I) and 2-(acetoxy)ethyl 2'-pyridyl disulphide (compound III) with the cysteine-proteinase minimal catalytic-site model, benzimidazol-2-ylmethanethiol, established the activation of all of these pyridyl disulphides by hydronation and that their reactivities are relatively insensitive to structural change in the non-pyridyl part of the molecule. The marked differences in their reactivities towards papain therefore derive from binding, either directly, or indirectly via signalling mechanisms. 3. Comparison of the kinetic data for the reaction of papain with compound (V) with those for analogous reactions with reactivity probes that provide opportunities for a variety of binding interactions in the S1-S2 intersubsite region and in the S2 subsite itself lead to the following conclusions: (a) the (Gly-66) N-H...O = C less than (P1-P2 ester) interaction of papain with compound (III) provides for better binding relative to that for a probe with a simple hydrocarbon side chain, but no signalling to the catalytic site to provide a (His-159)-ImH+-assisted transition state; (b) when this interaction is augmented either by a (P1-P2 amide) N-H...O = C less than (Asp-158) interaction (compound I) or hydrophobic P2/S2 contacts (compound V), signalling to the catalytic region occurs to provide the assisted transition state; (c) when both the P2/S2 contacts and the interaction involving Gly-66 exist, provision additionally of the (P1-P2 amide) N-H...O = C less than (Asp-158) interaction [as in 2-(N'-acetyl-L-phenylalanylamino)ethyl 2'-pyridyl disulphide] serves only to assist the binding without an additional signalling effect. 4. Such studies promise to allow binding interactions that merely locate substrates in appropriate enzyme loci to be distinguished from those that transmit signals with a chemical consequence to catalytic sites.

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Evidence for association-activation effects in reactions of papain from studies on its reactivity towards isomeric two-protonic-state reactivity probes.

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