Reactivities of the various protonic states in the reactions of papain and of L-cysteine with 2,2′- and with 4,4′- dipyridyl disulphide: evidence for nucleophilic reactivity in the un-ionized thiol group of the cysteine-25 residue of papain occasioned by its interaction with the histidine-159-asparagine-175 hydrogen-bonded system

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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.

Section: -mentioning

confidence: 88%

Identification of signalling and non-signalling binding contributions to enzyme reactivity. Alternative combinations of binding interactions provide for change in transition-state geometry in reactions of papain

Kowlessur

Topham

Thomas

et al. 1989

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“…values close to 4, we described the pH-dependence of k in terms of Scheme 2(a) by eqn. (1) (Brocklehurst & Little, 1972;Brocklehurst, 1974;Shipton & Brocklehurst, 1978), which contains molecular acid dissociation constants associated with only the enzyme molecule:…”

Section: Methodsmentioning

confidence: 99%

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

Brocklehurst¹,

Malthouse²

1980

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The kinetics of the reactions of the active-centre thiol groups of papain (EC 3.4.22.2) and ficin (EC 3.4.22.3) with the two-protonic-state reactivity probes 2,2'-dipyridyl disulphide, n-propyl 2-pyridyl disulphide and 4-(N-aminoethyl 2'-pyridyl disulphide)- 7-nitrobenzo-2-oxa-1,3-diazole (compound I) were studied over a wide range of pH. Differences between the reactivities of ficin and papain towards the cationic forms of the alkyl 2-pyridyl disulphide probes suggest that ficin contains a cationic site without exact analogue in papain, and the striking difference in the shapes of the pH-rate profiles for the reactions of the two enzymes with compound (1) suggests differences in the mobilities or dispositions of the active-centre histidine imidazole groups with respect to relevant hydrophobic binding areas. The evidence from reactivity-probe studies that the papain catalytic mechanism involves substantial repositioning of the active-centre imidazole group during the catalytic act does not apply also to ficin. If ficin contains an aspartic acid residue analogous to aspartic acid-158 in papain, the pKa of its carboxy group is probably significantly lower than the pKa of the analogous group in papain.

“…Papain was shown to be free of any contaminating chymopapains by equal thiol titrations at pH 4 and pH 8 [17,19]. Its identity was confirmed by the pH dependence of its reactivity towards 2,2h-dipyridyl disulphide [20] and its catalytic activity (k cat \K m ) towards N-benzoyl--arginine-p-nitroanilide [21]. For NMR experiments with Z-Phe-[2-"$C]Ala-glyoxal, papain was prepared by the method of Baines and Brocklehurst [17] and it had 0.38 mol of thiol\mol of protein.…”

Section: Isolation and Quantification Of Fully Active Papainmentioning

confidence: 99%

A 13C-NMR study of the inhibition of papain by a dipeptide-glyoxal inhibitor

Lowther

Djurdjevic-Pahl

Hewage

et al. 2002

Z-Phe-Ala-glyoxal (where Z is benzyloxycarbonyl) has been synthesized and shown to be a competitive inhibitor of papain with a Ki = 3.30±0.25nM. 13C-NMR has been used to show that in aqueous media, Z-Phe-[2-13C]Ala-glyoxal gives signals at 207.7p.p.m. and 96.3p.p.m. showing that both the α-keto carbon and its hydrate are present. When this inhibitor is bound to papain a single signal at 209.7p.p.m. is observed due to the 13C-enriched carbon. This demonstrates that the glyoxal α-keto carbon is not hydrated when it is bound to papain and that it does not form a thiohemiketal with the thiol group of Cys-25. Z-Phe-[1-13C]Ala-glyoxal has also been synthesized and its aldehyde carbon is fully hydrated in aqueous solution giving signals at 88.7p.p.m. and 90.2p.p.m. when the α-keto carbon and its hydrate are present respectively. When this inhibitor is bound to papain a single signal at 71.04p.p.m. was observed due to the 13C-enriched carbon showing that the 13C-enriched aldehyde carbon forms a thiohemiacetal with Cys-25.