Ficin: a cysteine proteinase with binding site-catalytic site signalling characteristics intermediate between those of papain and actinidin

Abstract

To understand molecular recognition in enzyme-substrate and enzyme-time-dependent inhibitor systems, it is necessary to investigate not only binding per se but also the possibility that binding may control covalency changes that take place in the catalytic site. The cysteine proteinase family of enzymes (reviewed in [l]) provides valuable opportunities for investigation of the coupling of different binding interactions (electrostatic, hydrogen bonding, van der Waals and hydrophobic) with each other and with catalytic site chemistry [2,3]. Such studies are facilitated by the existence of (a) significant differences in both structure and chemical behaviour throughout members of the enzyme family and (b) the common catalytic site (Cys)-S-/(His)-Im+H ion pair. The latter is the device used to provide the nucleophilic and general acid or hydrogen bonding/electrostatic characteristics used in the transformation of the reaction centre of substrate or inhibitor to products.We are continuing to develop 2-pyridyl disulphide reactivity probes (R-S-S-2-Py), in which R may provide a variety of binding opportunities, for the study of the binding site-catalytic site coupling in the cysteine proteinases. In the absence of signalling to the catalytic site occasioned by binding of R, the pHk profile contains a bell-shaped component with k maximal at pH3-4. This characteristic profile shape results from (a) the coexistence in acidic media of significant concentrations of Cys-S~ /His-Im+H ion pair and the formally hydronated (protonated) form of the probe (R-S-S-2-Py+H) and (b) the lack of substantial association of the 2-pyridyl N atom with the His-Im+H component of the ion pair in neutral media. In reactions of papain, binding of the P,-P, amide bond of 2-(acetamido)-ethyIL2'-pyridyl disulphide [I] and particularly of the L-Phe side chain and the (PJ > C = O of 2-(N'-acetyl-L-phenylalanylamino)-ethyl-2'-pyridyldisulphide (11) results in substantial reaction via an imidazolium-ion assisted transition state (111), recognised by marked changes in the shape of the pH-k profile with k maximal at pH6-7 instead of at pH3-4. It is a striking observation that these marked changes in profile shape do not occur also for the analogous reactions of actinidin [2,3]. This was unexpected because the crystal structures of papain Table 1. Second-order raie Constanis (8 for lhe reactbns o! ficin %i!h ihrec Fpyridyl disulphides at 25%. 10.1M a1 (a) pH3.0 (b) pH4.0 (c) pti6 0 and (01 pH1O.O Prdbe k(u-'s-')

Variation in aspects of cysteine proteinase catalytic mechanism deduced by spectroscopic observation of dithioester intermediates, kinetic analysis and molecular dynamics simulations

“…Indeed of the members of the papain family investigated by such methods, papain and actinidin display the most disparate reactivity characteristics, with caricain and ficin occupying intermediate positions in the series. Examples include the responses to anionic reactivity probes [37], binding site-catalytic site signalling in reactions of substrate-derived 2pyridyl disulphide reactivity probes [11,38,39] and its interplay with electrostatic fields [13,14], and P # -S # stereochemical selectivity [15,40]. The MD simulations suggest a structural interpretation of the difference in the catalytic characteristics of papain and actinidin demonstrated in the present work.…”

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.…”

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.…”