Brownian dynamics simulation of substrate motion near active site of enzyme entrapped inside reverse micelle

Brownian dynamics simulation has been applied to analyze the influence of the electrostatic field of a reverse micelle on the enzyme-substrate complex formation inside a micelle. The probability that the enzyme-substrate complex will form from serine protease (trypsin) and the specific hydrophilic cationic substrate Nα-benzoyl-l-arginine ethyl ester has been studied within the framework of the encounter complex formation theory. It has been shown that surfactant charge, dipole moments created by charged surfactant molecules and counterions, and permittivity of the inner core of reverse micelles can all be used as regulatory parameters to alter the substrate orientation near the active site of the enzyme and to change the probability that the enzyme-substrate complex will form.     

Dynamic aspect of kinetics of reaction catalyzed by lactate dehydrogenase

The influence of solvent viscosity on the kinetic parameters of the pyruvate reduction reaction catalyzed by lactate dehydrogenase has been investigated. The viscosity was adjusted by sucrose and glycerol solutions at concentrations from 0 to 44% and from 0 to 63%, respectively. The reaction rate decreased abruptly with an increase in viscosity. The study of different reaction stages (enzyme-substrate complex formation, catalysis, inhibitory complex decomposition, competitive inhibition by chlorine ions) revealed that the catalysis (and the related conformational changes) is the only stage (of the above mentioned) that depends markedly on the solvent viscosity. The reaction is insensitive to the changes in the dielectric properties of the solution induced by the addition of alcohols and dioxane. The observed power dependence of the rate constant on viscosity is explained in terms of Kramer's theory which considers the proton transition through the activation barrier to be a diffusion in the field of random forces. The influence of solvent viscosity on enzymic kinetics indicates a direct relation between solvent dynamics and relevant protein conformational movements.     

Kinetic characteristics of thiamine diphosphate biosynthesis by thiamine pyrophosphokinase from rat liver]

The kinetic analysis of bisubstrate enzymatic reaction catalysed by electrophoretically homogenous thiamine pyrophosphokinase (EC 2.7.6.2), isolated from rat liver has been carried out. Kinetic studies of the initial rates in the absence of the products and inhibition by the reaction products as well as the data from the equilibrium dialysis suggest that the reaction proceeds through the formation of a ternary enzyme-substrate complex. The combination with substrates and release of the products appears to be highly ordered. A possible scheme of the reaction mechanism is discussed.     

Hydrolysis of aryl beta-D-glucopyranosides and beta-D-xylopyranosides by an induced beta-D-glucosidase from Stachybotrys atra.

The induced beta-D-glucosidase from Stachybotrys atra hydrolyzes aryl beta-D-glucopyranosides and aryl beta-D-xylopyranosides by the same basic two-step mechanism. In the first step the aglycon group is split of with simultaneous formation of an enzyme-glycosyl complex. In the second step this intermediate complex reacts with water yeilding beta-D-glucose or beta-D-xylose. For beta-D-xyloside hydrolysis each of the two steps is partially rate-controlling, whereas for beta-D-glucoside hydrolysis the second step is rate-limiting. The enzyme is inhibited by high concentrations of substrate and the exact rate-concentration equation is a second-order equation. 1-Thio-beta-D-glycopyranosides with an aromatic aglycon inhibit the reaction in both a competitive and non-competitive way. A tentative mechanism is proposed to explain all types of inhibition. In this mechanism substrates and inhibitors with an aromatic aglycon group bind through hydrophobic forces to the 'aglycon subsite' of the intermediate enzyme-glycosyl complex. Binding of the second substrate molecule or of the inhibitor to this complex does not prevent the reaction of the glycosyl moiety with water, it only decreases the rate of the second step.     

Differential effects of metal-binding agents on the D-glucose-6-phosphate phosphohydrolase activity of the rat liver.

The metal-binding agents (citrate, oxalate, bicarbonate, EDTA) exert dual effects on D-glucose-6-phosphate phosphohydrolase activity in the homogenate as well as in the subcellular fractions. The important differences of the effects are associated with the concentration of the chelator and with time of its addition. The small (appropriate) concentrations of the metal-binding agents stimulate and stabilize the enzyme activity. However, chelators used in higher concentrations exert the inhibitory influence on the activity of glucose-6-phosphatase. Stimulation of the reaction was observed only if the chelator was added before the enzyme-substrate complex formation has been started. The formation of the ternary complex: the enzyme(metal)-chelator substrate exerting a protective influence on the active centre has been suggested. The hypothesis of a similar action of the metal-binding agents and Pi on the glucose-6-phosphatase as a metaloproteid has been proposed.     

Analysis of secretory immunoglobulin A in human saliva by laser-induced fluorescence capillary electrophoresis

The utility of capillary electrophoresis (CE) has been demonstrated for the analysis of secretory immunoglobulin A (sIgA) in human saliva. The amount of sIgA in saliva correlates with immune status. For detecting salivary sIgA, laser-induced fluorescence was conducted in this report for signal amplification. sIgA and anti-sIgA antibody were labeled with cyanine fluorescence (Cy5) for competitive immunoassay and non-competitive analysis, respectively. Cy5 was excited by He-Ne laser with a wavelength of 635 nm, with maximum emission at 670 nm. Migration time during electrophoresis depended on whether sIgA-Cy5 was mixed with antibody or anti-sIgA-Cy5 mixed with sIgA to form Ag-Ab complex. The results indicated that CE competitive immunoassay was effective for analyzing serum sIgA, but not for salivary sIgA. However, salivary sIgA can be analyzed by complex formation assay. The peak area of the complex was proportional to the amount of sIgA added. A standard linear regression curve was generated using purified sIgA. From this standard curve, the amount of sIgA from saliva of either normal or immunocompromised patients can be calculated from the Ag-Ab complex peak area.     

A new technique for mapping epitope specificities of monoclonal antibodies using quasi-elastic light scattering spectroscopy

Quasi-elastic light scattering (QLS) was used to determine relative epitope specificities of a group of anti-bovine serum albumin (BSA) monoclonal antibodies (MAb). QLS is a non-invasive technique which can determine the mean size and size distributions of macromolecular scatterers by analysis of the fluctuations in the intensity of laser light scattering. When two MAbs are mixed together with antigen, QLS detects the complex formation which results from the Ag-Ab reaction, and can easily distinguish between the large complexes formed by interaction of non-competitive MAbs and the smaller complexes formed by competitive MAbs. In this report, the competitive or non-competitive behavior of six anti-BSA MAbs were assessed by radioimmunoassay (RIA) and QLS analysis. The results obtained by QLS analysis confirmed the RIA findings indicating that the six MAbs examined can be categorized into three distinct, non-interacting groups. QLS analysis represents a simple, and extremely rapid technique for epitope mapping studies.     

Effects of thermodynamic nonideality in kinetic studies

Experimental evidence is presented for concentration dependence of the pseudo-firstorder rate constant describing the rate of inversion of sucrose by 2 m HCl; and also of the increase in maximal velocity for the catalytic reduction of pyruvate by lactate dehydrogenase that results from addition of the inert macromolecular solutes bovine serum albumin, ovalbumin, and Dextran T70. These somewhat unusual and seemingly diverse observations are examined in terms of a theory formulated on the basis of two equilibrium reactions, the first describing complex formation between two reactants, and the second isomerization of that complex to an activated state prior to product formation. This formulation permits consideration of activity coefficient ratios relevant to the equilibria and the expression of these ratios as power series in total solution composition. Quantitative assessment of the experimental results is made possible in these terms by estimating the magnitudes of the constant coefficients of the virial expansions as excluded volumes. It is concluded that the result observed in the sucrose inversion study finds rational explanation in thermodynamic nonideality factors governing the overall equilibrium between the reactants and the activated complex of sucrose and hydronium ion. For the enzyme-catalyzed reaction the same general equation applies but particular attention is given to the simplified form that is relevant to high substrate concentrations, where, in the absence of inert compounds, the conventional maximal velocity is approached. In this region an increase in velocity observed upon addition of an inert macromolecular component may be considered explicitly in terms of excluded volume effects related to a shape change in the isomerization between enzyme-substrate complex and its activated state.     

Sodium + potassium-activated ATPase of mammalian brain. Regulation of phosphatase activity.

1. The K+-nitrophenylphosphatase activity associated with mammalian brain (Na+ + K+)-ATPase displays K+ activation curves that have intermediary plateaus and maxima in the presence of less than saturating concentrations of Na+. Zero Na+ and saturating Na+ produce sigmoid K+-activation curves with low and high K+ affinities respectively. 2. ATP inhibits K+-activated nitrophenylphosphatase through both competitive and non-competitive mechanisms. ATP is synergistic with Na+ in the mechanism which converts the enzyme from low to high K+ affinity. 3. The Na+ and K+ interactions can be accounted for by equations which describe a model with separate regulatory sites for Na+ and K+ and with K+- requiring catalytic site which is only accessible in one of the two principal conformational stages of the enzyme. 4. The effects of ATP can be accounted for by the same model through interactions at a single nucleotide binding site. Inhibition which is competitive with K+ and non-competitive with substrate arises from stabilization of the inactive enzyme conformation. Inhibition which is non-competitive with K+ and competitive with substrate results from interactions with the active enzyme conformation. The synergism between Na+ and ATP appears to arise as a consequence of the formation of phosphoryl enzyme. 5. A model for (Na+ + K+)-ATPase is discussed which involves in-phase coupling of subunit interactions as suggested by these studies.     

High pressure enzyme kinetics of dextransucrase.

The pressure dependence of enzymatic dextran formation has been observed up to 1000 at for several substrate concentrations. First order denaturation effects could be separated from the thermodynamic effects, which lead to a volume of 30.4 to 44.0 ccm per mole for the formation and -13.6ccm per mole for the activation of the enzyme-substrate complex. Denaturation depends on the substrate concentration. This leads to the conslusion that only the free enzyme is denatured, wheras the ES complex is stable.     

Reflections on the kinetics of substrate binding

The principle questions which are still being asked about enzyme-substrate complex formation are examined. Data for the maximum rates of collision complex formation are reviewed. The limitations of past experimental procedures and the potentialities of new approaches are discussed together with methods that allow the distinction between collision complexes and subsequent events.     

Free Energy Diagram for the Heterogeneous Enzymatic Hydrolysis of Glycosidic Bonds in Cellulose

Kinetic and thermodynamic data have been analyzed according to transition state theory and a simplified reaction scheme for the enzymatic hydrolysis of insoluble cellulose. For the cellobiohydrolase Cel7A from Hypocrea jecorina (Trichoderma reesei), we were able to measure or collect relevant values for all stable and activated complexes defined by the reaction scheme and hence propose a free energy diagram for the full heterogeneous process. For other Cel7A enzymes, including variants with and without carbohydrate binding module (CBM), we obtained activation parameters for the association and dissociation of the enzyme-substrate complex. The results showed that the kinetics of enzyme-substrate association (i.e. formation of the Michaelis complex) was almost entirely entropy-controlled and that the activation entropy corresponded approximately to the loss of translational and rotational degrees of freedom of the dissolved enzyme. This implied that the transition state occurred early in the path where the enzyme has lost these degrees of freedom but not yet established extensive contact interactions in the binding tunnel. For dissociation, a similar analysis suggested that the transition state was late in the path where most enzyme-substrate contacts were broken. Activation enthalpies revealed that the rate of dissociation was far more temperature-sensitive than the rates of both association and the inner catalytic cycle. Comparisons of one- and two-domain variants showed that the CBM had no influence on the transition state for association but increased the free energy barrier for dissociation. Hence, the CBM appeared to promote the stability of the complex by delaying dissociation rather than accelerating association.     

Characterization of a cellodextrin glucohydrolase with soluble oligomeric substrates: experimental results and modeling of concentration-time-course data

A previously isolated cellodextrin glucohydrolase (beta-glucosidase) from Trichoderma reesei QM 9414 is characterized using beta-1,4-glucose oligomers with defined degrees of polymerization as soluble substrates. The enzyme splits off glucose units from the nonreducing chain ends of cellooligomers. Besides this hydrolytic activity there is also evidence for transfer activity depending on the concentration and degree of polymerization of substrates. Concentration-time-course data have been gathered for the degradation of cellobiose, cellotriose, cellotetraose, cellopentaose, and cellohexaose covering a wide range of enzyme and substrate concentrations. A Michaelis-Menten type kinetic model has been developed, which is able to satisfactorily describe the complex system of parallel and series reactions during the conversion of oligomers to glucose. The only kind of inhibition considered is competitive inhibition by the final product glucose. The model takes into account the formation of multiple enzyme-substrate complexes and is limited to those conditions, in which no transglucosylation products are observed. Cellodextrins with higher degrees of polymerization are found to be better substrates for this enzyme than is the dimer cellobiose.     

The role of the tryptophan-62 residue in the structure and function of lysozyme]

The thermostability and thermodinamics of formation of the enzyme-substrate complex of two oxidation products of chicken egg lysozyme with the tryptophane-62 residue modified to N'-formylkinurenine (with 2.5% activity) and kinurenine (with 27.5% activity) have been studied. In thermostability and pH effect on the substrate binding the lysozyme oxidation products do not differ from native lysozyme. The data obtained and thermodynamical characteristics of the enzyme-substrate complex formation suggest that the chemical nature of the 62 residue does not significantly affect the conformational properties of lysozyme, however, having a strongly pronounced effect on the binding of substrate and hence the total enzyme activity.     

Molecular modeling of formate dehydrogenase: the formation of the Michaelis complex

The formation of the reactive enzyme-substrate complex of formate dehydrogenase has been investigated by molecular dynamics techniques accounting for different conformational states of the enzyme. Simulations revealed that the transport of substrate to the active site through the substrate channel proceeds in the open conformation of enzyme due to the crucial role of the Arg284 residue acting as a vehicle. However, formate binding in the active site of the open conformation leads to the formation of a nonproductive enzyme-substrate complex. The productive Michaelis complex is formed only in the closed enzyme conformation after the substrate and coenzyme have bound, when required rigidity of the binding site and reactive formate orientation due to interactions with Arg284, Asn146, Ile122, and His332 residues is attained. Then, the high occupancy (up to 75%) of the reactive substrate-coenzyme conformation is reached, which was demonstrated by hybrid quantum mechanics/molecular mechanics simulations using various semiempirical Hamiltonians.     

Inhibition of the Trichoderma reesei cellulases by cellobiose is strongly dependent on the nature of the substrate

The inhibition effect of cellobiose on the initial stage of hydrolysis when cellobiohydrolase Cel 7A and endoglucanases Cel 7B, Cel 5A, and Cel 12A from Trichoderma reesei were acting on bacterial cellulose and amorphous cellulose that were [(3)H]- labeled at the reducing end was quantified. The apparent competitive inhibition constant (K(i)) for Cel 7A on [(3)H]-bacterial cellulose was found to be 1.6 +/- 0.5 mM, 100-fold higher than that for Cel 7A acting on low-molecular-weight model substrates. The hydrolysis of [(3)H]-amorphous cellulose by endoglucanases was even less affected by cellobiose inhibition with apparent K(i) values of 11 +/- 3 mM and 34 +/- 6 mM for Cel 7B and Cel 5A, respectively. Contrary to the case for the other enzymes studied, the release of radioactive label by Cel 12A was stimulated by cellobiose, possibly due to a more pronounced transglycosylating activity. Theoretical analysis of the inhibition of Cel 7A by cellobiose predicted an inhibition analogous to that of mixed type with two limiting cases, competitive inhibition if the prevalent enzyme-substrate complex without inhibitor is productive and conventional mixed type when the prevalent enzyme-substrate complex is nonproductive.     

On true and apparent Michaelis constants in enzymology. III. Is it linear dependence between apparent michaelis constant and limiting rate and is it possible to determine the substrate constant value using this dependence?

The Slater-Bonner method which is used for graphic determination of substrate constant (Ks) by linear dependence of apparent Michaelis constant (Km(app)) on the limiting rate (V(app)) of enzyme-catalysed reactions with activator participation has been critically analysed. It has been shown that although it is possible to record the mechanisms of such reactions as a scheme similar to Michaelis-Menten model which allow to find correlation Km(app) and V(app) as equation Km(app) = Ks + V(app)/k1[E]0 ([E]0 is a total enzyme concentration, k1 is a rate constant of enzyme-substrate complex formation from free enzyme and substrate) in order to calculate Ks and individual rate constants (k1, k(-1)), but this approach for investigation of all reactions with activator participation ought not to be used. The above equation is not obeyed in general, it may be true for some mechanisms only or under certain ratios of kinetic parameters of enzyme-catalysed reactions.     

Properties of a complex of Fe(III)-soybean lipoxygenase-1 and 4-nitrocatechol

Fe(III)-soybean lipoxygenase-1 yields with 4-nitrocatechol a green coloured 1 : 1 complex, which shows at pH 7.0 absorption maxima at 385 nm and 650 nm. The formation of this complex is reversible. The circular dichroism spectrum of the complex of Fe(III)-lipoxygenase-1 and 4-nitrocatechol has a positive band at around 380 nm and a negative band at around 450 nm and is significantly different from that of the Fe(III)-enzyme as such. 4-Nitrocatechol can be displaced from the green complex by 13-L-hydroperoxy-cis-9, trans-11-octadecadienoic acid, resulting in the formation of the blue complex between the Fe(III)-enzyme and 13-L-hydroperoxy-cis-9,trans-11-octadecadienoic acid both under aerobic and anaerobic conditions. Also linoleic acid competes with 4-nitrocatechol for the binding site on the Fe(III)-enzyme, as can be demonstrated under anaerobic conditions, ultimately leading to reduction of the Fe(III)-enzyme. The oxygenation of linoleic acid by Fe(III)-lipoxygenase-1 is inhibited by 4-nitrocatechol. From steady-state kinetics a non-competitive inhibition pattern is obtained. Probably it has to be considered as pseudo non-competitive because of the slow establishment of the complex equilibrium. An inhibition constant (K4NC) of 16.3 microM is found. On prolonged incubation of Fe(III)-lipoxygenase-1 and 4-nitrocatechol the green complex converts into a brown species. This conversion is found to be coupled with a change in the nature of the inhibition from reversible to irreversible. A complex between native lipoxygenase-1 and 4-nitrocatechol is found to be unlikely.     

Mechanism of Carboxypeptidase Y Catalyzed Hydrolysis and Aminolysis Reactions

The reaction mechanism of carboxypeptidase Y catalyzed reactions is investigated. Presteady state and steady state kinetic measurements are performed on the hydrolysis and aminolysis of an ester and an amide substrate. It is found that deacylation is the rate determining step in hydrolysis of the ester, pivalic acid 4-nitrophenol and acylation in that of the amide, succinyl-L-alanyl-L-alalyl-L-propyl-L-phenylalanine 4-nitroanilide.

The kinetic effects observed in the presence of a nucleophile, L-valine amide, where aminolysis occurs in parallel to the hydrolysis reaction are analysed in details. The results are described satisfactorily by a reaction scheme which involves the binding of the added nucleophile, (i) to the free enzyme, resulting in a simple competitive effect, and (ii) to the acyl-enzyme with the formation of a complex between the enzyme and the aminolysis product, the dissociation of which is rate determining. That scheme can account for both increases and decreases of kinetic parameter values as a function of the nucleophile concentration. There is no indication of binding of the nucleophile to the enzyme-substrate complex before acylation takes place.     

A graphical method for determining inhibition parameters for partial and complete inhibitors.

A new simple graphical method is described for the determination of inhibition type and kinetic parameters of an enzyme reaction without any replot. The method consists of plotting experimental data as v/(vo--v) versus the reciprocal of the inhibitor concentration at different substrate concentrations, where v and vo represent the velocity in the presence and in the absence of the inhibitor respectively with a given concentration of the substrate. Partial inhibition gives straight lines that converge on the abscissa at a point away from the origin, whereas complete inhibition gives lines that go through the origin. The inhibition constants of enzymes and the reaction rate constant of the enzyme-substrate-inhibitor complex can be calculated from the abscissa and ordinate intercepts of the plot. The relationship between the slope of the plot and the substrate concentration shows characteristic features depending on the inhibition type: for partial competitive inhibition, the straight line converging on the abscissa at--Ks, the dissociation constant of the enzyme-substrate complex; for non-competitive inhibition, a constant slope independent of the substrate concentration; for uncompetitive inhibition, a hyperbola decreasing with the increase in the substrate concentration; for mixed-type inhibition, a hyperbola increasing with the increase in the substrate concentration. The properties of the replot are useful in confirmation of the inhibition mechanism.