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).

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1-trans-Epoxysuccinyl-L-leucylamido(4-guanidino)butane (E-64) was shown to inhibit beta-trypsin by a reversible competitive mechanism; this contrasts with the widely held view that E-64 is a class-specific inhibitor of the cysteine proteinases and reports in the literature that it does not inhibit a number of other enzymes including, notably, trypsin. The K1, value (3 x 10(-5) M) determined by kinetic analysis of the hydrolysis of N alpha-benzoyl-L-arginine 4-nitroanilide in Tris/HCl buffer, pH 7.4, at 25 degrees C, I = 0.1, catalysed by beta-trypsin is comparable with those for the inhibition of trypsin by benzamidine and 4-aminobenzamidine, which are widely regarded as the most effective low Mr inhibitors of this enzyme. Computer modelling of the beta-trypsin-E64 adsorptive complex, by energy minimization, molecular dynamics simulation and Poisson-Boltzmann electrostatic-potential calculations, was used to define the probable binding mode of E-64; the ligand lies parallel to the active-centre cleft, anchored principally by the dominant electrostatic interaction of the guanidinium cation at one end of the E-64 molecule with the carboxylate anion of Asp-171 (beta-trypsin numbering from Ile-1) in the S1-subsite, and by the interaction of the carboxylate substituent on C-2 of the epoxide ring at the other end of the molecule with Lys-43; the epoxide ring of E-64 is remote from the catalytic site serine hydroxy group. The possibility that E-64 might bind to the cysteine proteinases clostripain (from Clostridium histolyticum) and alpha-gingivain (one of the extracellular enzymes from phyromonas gingivalis) in a manner analogous to that deduced for the beta-trypsin-E-64 complex is discussed.

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.

Lignocellulose is the most abundant biopolymer available on earth. Hydrolysis of lignocellulose into fermentable sugars, sugar acids and phenolics is the pre-requisite for its successful exploitation as substrates for the large scale production of industrially significant value added products. Though remarkable collection of lignocellulolytic microorganisms has been brought to limelight, only a few especially fungi have been studied extensively as they secrete copious lignocellulolytic enzymes extracellularly. Enzymatic hydrolysis of lignocelluloses is carried out by a group of enzymes viz., cellulases, hemicellulases, ligninases in unison or individually, and are collectively known as lignocellulolytic enzymes. In this light of this background, followed by a short introduction on lignocellulose, lignocellulolytic enzymes and mainly focused on fungal cellulase, this review discusses recent approaches on the production of cellulase by submerged and solid-state fermentation strategies; current knowledge on the purification, characterisation of cellulase; and finally concluded with detailed industrial applications of fungal cellulase.

1. Values of the kinetic specificity constant, kcat./Km, for the hydrolysis of N-acetyl-L-phenylalanylglycine 4-nitroanilide (I) and of its D-enantiomer (II) catalysed by ficin (EC 3.4.22.3) and by actinidin (EC 3.4.22.14) at pH 6.0, I 0.1 mol/l, 8.3% (v/v) NN-dimethylformamide and 25 degrees C were determined by using initial-rate data with [S] much less than Km and weighted nonlinear regression analysis as: for ficin, (kcat./Km)L = 271 +/- 6 M-1.s-1, (kcat./Km)D = 2.9 +/- 0.1 M-1.s-1, and for actinidin (kcat./Km)L = 13.3 +/- 0.7 M-1.s-1, (kcat/Km)D = 0.34 +/- 0.01 M-1.s-1.2. These data and analogous values for the corresponding reactions catalysed by papain (EC 3.4.22.2), (kcat./Km)L = 2064 +/- 31 M-1.s-1, (kcat./Km)D = 5.5 +/- 0.1 M-1.s-1, demonstrate marked variation in stereochemical selectivity for substrates (I) and (II) among the three cysteine proteinases with the following values for the index of stereochemical selectivity Iss = (kcat./Km)L/(kcat./Km)D: for papain, 375; for ficin 93; for actinidin 39. 3. Model building suggests ways in which, for the papain-catalysed reactions, binding interactions involving the extended acyl groups of the substrates may need to change as the reaction proceeds from adsorptive complex (ES) to tetrahedral intermediate (THI) before its rate-determining, general acid-catalysed collapse to acylenzyme intermediate. In particular, satisfactory alignment in the catalytic site at the THI stage of the acylation process appears to demand rotation of the substrate moiety about its long axis. 4. The different consequences of this rotation for the L- and D-enantiomers suggest that for closely related systems the greater the extent of this rotational adjustment the greater would be the value of Iss.5. For the actinidin-substrate combinations, model building suggests that even at the ES complex stage of catalysis it is not possible to approach optimized P2-S2 contacts and the three hydrogen-bonding interactions deduced for papain-ligand complexes in the absence of significant movement of protein conformation. Possible binding modes in which some of the interactions deduced for papain are relaxed are discussed. Consideration of postulated binding modes in the various transition states is shown to account for the order of reactivity reflected in values kcat./Km for the four reactions involving papain (Pap) and actinidin (Act) with the L- and D-enantiomeric substrates: Pap-L much greater than Act-L greater than Pap-D much greater than Act-D.(ABSTRACT TRUNCATED AT 400 WORDS)

Expression cloning of a dust mite cysteine proteinase, Drr pl. a major allergen associated with asthma and hypersensitivity reactionsThe house dust mite (HDM), Dermarophagoides pteronyssinus, is a major source of allergens, implicated in diseases such as asthma and atopic dermatitis. About 75% of