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

Abstract

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.

Evaluation of hydrogen-bonding and enantiomeric P2-S2 hydrophobic contacts in dynamic aspects of molecular recognition by papain

“…Transition-state geometry is the fundamental characteristic on which catalytic ability depends, and the reactivity of a catalytic-site nucleophile that plays a central role in the catalytic act is a prime target for investigation of factors that contribute to the control of transition-state geometry. The cysteine proteinase papain (EC 3.4.22.2) provides valuable opportunities for the study of the interdependence of binding interactions with each other and with catalytic-site chemistry (Kowlessur et al, 1989b). The reactivity of its catalytic site responds to combinations of hydrogenbonding and hydrophobic effects in its extended binding site (Brocklehurst et al, 1987a(Brocklehurst et al, , 1988aKowlessur et al, 1989a Kowlessur et al, ,b, 1990Templeton et al, 1990;Berti et al, 1991;Hanzlik et al, 1991;Patel et al, 1992).…”

Substrate Recognition

“…There is considerable variation among members of the papain family, however, in substrate specificity and catalytic-site reactivity [81,[153][154][155][156][157][158], which reveals that this view is correct only for low-resolution aspects of the mechanism, such as the roles of the cysteine and histidine components of the catalytic-site ion pair. The results of detailed mechanistic investigations on papain and some of its natural variants (discussed in [44,45]) have revealed that papain exhibits characteristics at one end of a spectrum of chemical behavior with actinidin at the other, and with caricain and ficin occupying intermediate positions.…”

“…Clear evidence for this phenomenon [141,[155][156][157] is provided by the nature of the pH-k profiles for reactions of papain with substrate-derived 2-pyridyl disulfides used as thiol-specific, two-protonic-state reactivity probes [41]. These probes contain in one part of the molecule various potential binding sites and, in the other, the 2-mercaptopyridine leaving group to serve as a detector of the relative disposition of the thiolate and imidazolium components of the Cys25/His159 ion pair.…”

“…Non-signaling binding interactions can be distinguished from signaling binding interactions by detailed analysis of the family of pH-k profiles [157]. A central role in the binding-site catalytic-site communication in papain is provided by the hydrogen bond between the carbonyl component of the P 1 -P 2 amide bond and Gly66.…”

Ionization characteristics and chemical influences of aspartic acid residue 158 of papain and caricain determined by structure-related kinetic and computational techniques: multiple electrostatic modulators of active-centre chemistry

“…One of the least well understood aspects of enzyme catalysis and active-centre chemistry is the range and nature of the kinetic consequences of electrostatic effects initiated by ionizations at various distances from and orientations to the catalytic site [6][7][8][9] ; evidence continues to accumulate that electrostatic effects [10] are a major factor in determining the behaviour of cysteine proteinases [4,[11][12][13][14][15][16][17][18][19][20][21][22]. The carboxy group of Asp"&) is the ionizable group nearest to the catalytic sites in both enzymes obeing separated from the (Cys#&)-S − \(His"&*)-Im + H (in which Im represents imidazole) ion pairs by approx.…”