The cysteine proteinase superfamily is a source of natural structural variants of value in the investigation of mechanism. It has long been considered axiomatic that catalytic competence of these enzymes mirrors the generation of the ubiquitous catalytic site imidazolium-thiolate ion pair. We here report definitive evidence from kinetic studies supported by electrostatic potential calculations, however, that at least for some of these enzymes the ion pair state which provides the nucleophilic and acid-base chemistry is essentially fully developed at low pH where the enzymes are inactive. Catalytic competence requires an additional protonic dissociation with a common pKa value close to 4 possibly from the Glu50 cluster to control ion pair geometry. The pH dependence of the second-order rate constant (k) for the reactions of the catalytic site thiol groups with 4,4'-dipyrimidyl disulfide is shown to provide the pKa values for the formation and deprotonation of the (Cys)-S-/(His)-Im+H ion pair state. Analogous study of the reactions with 2,2'-dipyridyl disulfide reveals other kinetically influential ionizations, and all of these pKa values are compared with those observed in the pH dependence of kcat/Km for the catalyzed hydrolysis of N-acetylphenylalanylglycine 4-nitroanilide. The discrepancy between the pKa value for ion pair formation and the common pKa value close to 4 related to generation of catalytic activity is particularly marked for ficin (pKa 2.49 +/- 0.02) and caricain (pKa 2.88 +/- 0.02) but exists also for papain (pKa 3.32 +/- 0.01).

The acylation and deacylation stages of the hydrolysis of N -acetyl-Phe-Gly methyl thionoester catalysed by papain and actinidin were investigated by stopped-flow spectral analysis. Differences in the forms of pH-dependence of the steady-state and pre-steady-state kinetic parameters support the hypothesis that, whereas for papain, in accord with the traditional view, the rate-determining step is the base-catalysed reaction of the acyl-enzyme intermediate with water, for actinidin it is a post-acylation conformational change required to permit release of the alcohol product and its replacement in the catalytic site by the key water molecule. Possible assignments of the kinetically influential p K (a) values, guided by the results of modelling, including electrostatic-potential calculations, and of the mechanistic roles of the ionizing groups, are discussed. It is concluded that Asp(161) is the source of a key electrostatic modulator (p K (a) 5.0+/-0.1) in actinidin, analogous to Asp(158) in papain, whose influence is not detected kinetically; it is always in the 'on' state because of its low p K (a) value (2.8+/-0.06).

The possibility of a slow post-acylation conformational change during catalysis by cysteine proteinases was investigated by using a new chromogenic substrate, N-acetyl-Phe-Gly methyl thionoester, four natural variants (papain, caricain, actinidin and ficin), and stopped-flow spectral analysis to monitor the pre-steady state formation of the dithioacylenzyme intermediates and their steady state hydrolysis. The predicted reversibility of acylation was demonstrated kinetically for actinidin and ficin, but not for papain or caricain. This difference between actinidin and papain was investigated by modelling using QUANTA and CHARMM. The weaker binding of hydrophobic substrates, including the new thionoester, by actinidin than by papain may not be due to the well-known difference in their S2-subsites, whereby that of actinidin in the free enzyme is shorter due to the presence of Met211. Molecular dynamics simulation suggests that during substrate binding the sidechain of Met211 moves to allow full access of a Phe sidechain to the S2-subsite. The highly anionic surface of actinidin may contribute to the specificity difference between papain and actinidin. During subsequent molecular dynamics simulations the P1 product, methanol, diffuses rapidly (over<8 ps) out of papain and caricain but 'lingers' around the active centre of actinidin. Uniquely in actinidin, an Asp142-Lys145 salt bridge allows formation of a cavity which appears to constrain diffusion of the methanol away from the catalytic site. The cavity then undergoes large scale movements (over 4.8 A) in a highly correlated manner, thus controlling the motions of the methanol molecule. The changes in this cavity that release the methanol might be those deduced kinetically.

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To investigate the hypothesis that decreased hapten flexibility may lead to increased catalytic antibody activity, we used two closely related immunogens differing only in the flexibility of the atomic framework around the structural motif of the haptens, analogous to the reaction centre of the corresponding substrates. Identical leaving-group determinants in the haptens and identical leaving groups in the substrates removed the ambiguity inherent in some data reported in the literature. Anti-phosphate and anti-phosphonate kinetically homogeneous polyclonal catalytic antibody preparations were compared by using carbonate and ester substrates respectively, each containing a 4-nitrophenolate leaving group. Synthetic routes to a new phosphonate hapten and new ester substrate were developed. The kinetic advantage of the more rigid anti-phosphonate/ester system was demonstrated at pH 8.0 by a 13-fold advantage in k(cat)/k(non-cat) and a 100-fold advantage in the proficiency constant, k(cat)/k (non-cat) x K(m). Despite these differences, the pH-dependences of the kinetic and binding characteristics and the results of chemical modification studies suggest closely similar catalytic mechanisms. The possible origin of the kinetic advantage of the more rigid hapten/substrate system is discussed.

As it is known that the understanding of the basic properties of the enzyme/inhibitor complex leads directly to enhancing the capability in drug designing and drug discovery. Molecular dynamics simulations have been performed to examine detailed information on the structure and dynamical properties of the HIV-1 PR complexed with saquinavir in the three protonated states, monoprotonates at Asp25 (Mono-25) and Asp25'(Mono-25') and diprotonate (Di-Pro) at both Asp25 and Asp25'. The obtained results support clinical data which reveal that Ile84 and Gly48 are two of the most frequent residues where mutation toward a protease inhibitor takes place. In contrast to the Ile84 mutation due to high displacement of Ile84 in the presence of saquinavir, source of the Gly48 mutation was observed to be due to the limited space in the HIV-1 PR pocket. The Gly48 was, on one side, found to form strong hydrogen bonds with saquinavir, while on the other side this residue was repelled by the hydrophobic Phe53 residue. In terms of inhibitor/enzyme binding, interactions between saquinavir and a catalytic triad of the HIV-1 PR were calculated using the ab initio method. The results show an order of the binding energy of Mono25

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