Generalization of the Monod-Wyman-Changeux model for the case of multisubstrate reactions]

A general equation is derived for the rate of multisubstrate reaction catalyzed by oligomeric enzyme E(R, T) liable to concerted transitions Ro in equilibrium To or Ro in equilibrium 2To. It is shown that with some assumptions about the enzymes the rate equations can be constructed from the rates of corresponding reactions catalyzed by a single active site. These single active site rate equations are known for the majority of catalysis mechanisms, otherwise they can be easily deduced. As an example the rate equation is derived for the reaction S1 + S2 + S3 in equilibrium S4 + S5 catalyzed by an oligomeric enzyme according to the ordered ter-bi mechanism.     

Principles of antibody catalysis

Antibodies have now been shown to catalyze a variety of chemical transformations, including hydrolytic, concerted, and bimolecular reactions. The inherent chirality of the antibody binding pocket has been exploited to exert precise stereochemical control over their catalyzed reactions. The mechanisms by which antibodies catalyze reactions are not expected to differ in any general way from those of natural enzymes. Antibodies use their binding energy to stabilize species of higher free energy which appear along the reaction coordinate or effect general acid/base catalysis. The advent of catalytic antibodies promises new catalysts that extend the range of catalysis by proteins to chemical transformations that were not required during the evolution of enzymes.     

3-Hydroxy-3-methylglutaryl-coenzyme A reductase of Haloferax volcanii: role of histidine 398 and attenuation of activity by introduction of negative charge at position 404.

Mutant 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductases of the halophilic archaeon Haloferax volcanii were constructed to test the proposed mechanism that phosphorylation downregulates the activity of higher eukarya HMG-CoA reductases via charge-charge interaction with the active site histidine. To first verify the sequence-based inference that His 398 is the catalytic histidine of the H. volcanii enzyme, enzyme H398Q was constructed, purified, and assayed for catalysis of three reactions: [1] reductive deacylation of HMG-CoA, [2] reduction of mevaldehyde, and [3] oxidative acylation of mevaldehyde. Enzyme H398Q had low activity for catalysis of reaction [1] or [3], but readily catalyzed mevaldehyde reduction. By analogy to hamster HMG-CoA reductase, we conclude that His 398 is the active site histidine. Mutant forms of the 403-residue H. volcanii enzyme were constructed to model phosphorylation and infer whether attenuated activity involved interaction with His 398. Chimeric H. volcanii-hamster enzymes constructed in an effort to create an active, phosphorylatable chimeric enzyme were inactive or not phosphorylated. We therefore added Asp at position 404 to mimic the introduction of negative charge that would accompany phosphorylation. Enzyme 404D/H398Q was inactive for reaction [1] or [3], but catalyzed reaction [2] at 35% the wild-type rate. These observations are consistent with the model that attenuation of catalytic activity results from an ionic interaction between the imidazolium cation of His 398 and the carboxylate anion of Asp 404.     

Modification of the Microenvironment of Enzymes in Organic Solvents. Substitution of Water by Polar Solvents

Enzyme catalysis in water-immiscible organic solvents is strongly influenced by the amount of water present in the reaction mixture. Effects of substitution of part of the water by other polar solvents were studied. In an alcoholysis reaction catalyzed by chymotrypsin deposited on celite, it was possible to exchange half of the water by formamide, ethylene glycol or dimethyl sulfoxide with often increased initial reaction rate. Furthermore, these substitutions caused the suppression of the competing hydrolysis reaction. However, formamide caused enzyme inactivation, and ethylene glycol participated as a reactant in the alcoholysis to some extent, hence dimethyl sulfoxide was considered the best water substitute among the solvents tested. These effects were noted for chymotrypsin catalyzed alcoholysis in several water immiscible solvents and also for interesterification reactions catalyzed by Candida cylindracea lipase on celite. In the latter case a change in the stereoselectivity was observed. At a low water content a high stereoselectivity was observed; when the amount of polar solvent was increased, either by doubling the water content or adding an equal amount of DMSO, the stereoselectivity decreased.     

Hysteresis, multiplicity of stationary states and auto-oscillations in the reversible flow-through reaction]

A mathematical model has been investigated of a reversible flow-through reaction S1 reversible S2 catalyzed by an olygomeric enzyme E(R,T) the protomers of which undergo concerted conformational transitions R reversible T. The isosteric activation of olygomer E by product S2 binding preferably to protomer active sites in conformation R is shown to be a possible cause of hysteresis in the quasi-stationary input characteristic of the reaction, v (s2). The latter determines the rate law of the reaction, provided the concentration of S2 is a quasi-stationary one. The hysteresis of the characteristic v (s1) gives rise to multiple steady states and self-oscillations in the reaction.     

Mechanisms of coupling between DNA recognition specificity and catalysis in EcoRI endonuclease

Proteins that bind to specific sites on DNA often do so in order to carry out catalysis or specific protein-protein interaction while bound to the recognition site. Functional specificity is enhanced if this second function is coupled to correct DNA site recognition. To analyze the structural and energetic basis of coupling between recognition and catalysis in EcoRI endonuclease, we have studied stereospecific phosphorothioate (PS) or methylphosphonate (PMe) substitutions at the scissile phosphate GpAATTC or at the adjacent phosphate GApATTC in combination with molecular-dynamics simulations of the catalytic center with bound Mg2+. The results show the roles in catalysis of individual phosphoryl oxygens and of DNA distortion and suggest that a "crosstalk ring" in the complex couples recognition to catalysis and couples the two catalytic sites to each other.     

Kinetic and chemical mechanisms of shikimate dehydrogenase from Mycobacterium tuberculosis

Mycobacterium tuberculosis shikimate dehydrogenase (MtbSD) catalyzes the fourth reaction in the shikimate pathway, the NADPH-dependent reduction of 3-dehydroshikimate. To gather information on the kinetic mechanism, initial velocity patterns, product inhibition, and primary deuterium kinetic isotope effect studies were performed and the results suggested a steady-state ordered bi-bi kinetic mechanism. The magnitudes of both primary and solvent kinetic isotope effects indicated that the hydride transferred from NADPH and protons transferred from the solvent in the catalytic cycle are not significantly rate limiting in the overall reaction. Proton inventory analysis indicates that one proton gives rise to solvent isotope effects. Multiple isotope effect studies indicate that both hydride and proton transfers are concerted. The pH profiles revealed that acid/base chemistry takes place in catalysis and substrate binding. The MtbSD 3D model was obtained in silico by homology modeling. Kinetic and chemical mechanisms for MtbSD are proposed on the basis of experimental data.     

Investigation on the mechanism of crystallization of soluble protein in the presence of nonionic surfactant

The mechanism of crystallization of soluble, globular protein (lysozyme) in the presence of nonionic surfactant C8E4 (tetraoxyethylene glycol monooctyl ether) was examined using both static and dynamic light scattering. The interprotein interaction was found to be attractive in solution conditions that yielded crystals and repulsive in the noncrystallizing solution conditions. The validity of the second virial coefficient as a criterion for predicting protein crystallization could be established even in the presence of nonionic surfactants. Our experiments indicate that the origin of the change in interactions can be attributed to the adsorption of nonionic surfactant monomers on soluble proteins, which is generally assumed to be the case with only membrane proteins. This adsorption screens the hydrophobic attractive force and enhances the hydration and electrostatic repulsive forces between protein molecules. Thus at low surfactant concentration, the effective protein-protein interaction remains repulsive. Large surfactant concentrations promote protein crystallization, possibly due to the attractive depletion force caused by the intervening free surfactant micelles.     

The defective proton-ATPase of uncD mutants of Escherichia coli. Two mutations which affect the catalytic mechanism

The catalytic characteristics of F1-ATPases from uncD412 and uncD484 mutant strains of Escherichia coli were studied in order to understand how these beta-subunit mutations cause defective catalysis. Both mutant enzymes showed reduced affinity for ATP at the first catalytic site. While uncD412 F1 was similar to normal in other aspects of single site catalysis, uncD484 F1 showed a Keq of bound reactants greatly biased toward bound substrate ATP and an abnormally fast rate of Pi release. Impairment of productive catalytic cooperativity was the major cause of the reduced steady state ("multisite") catalytic rate in both mutant enzymes. Addition of excess ATP to saturate second and/or third catalytic sites did promote ATP hydrolysis and product release at the first catalytic site of uncD412 F1, but the multisite turnover rate was significantly slower than normal. In contrast, with uncD484 F1, addition of excess ATP induced rapid release of ATP from the first catalytic site and so productive catalytic cooperativity was almost completely absent. The results show that both mutations affect properties of the catalytic site and catalytic site cooperativity and further that the relatively more severe uncD484 mutation affects a residue which acts as a determinant of the fate of bound substrate ATP during promotion of catalysis. Taken together with previous studies of uncA mutant F1-ATPases (Wise, J. G., Latchney, L. R., Ferguson, A. M., and Senior, A. E. (1984) Biochemistry 23, 1426-1432) the results indicate that catalytic site cooperativity in F1-ATPases involves concerted beta-alpha-beta intersubunit communication between catalytic sites on the beta-subunits.     

Reaction mechanism of the membrane-bound ATPase of submitochondrial particles from beef heart

Submitochondrial particles from beef heart, washed with dilute solutions of KCl so as to activate the latent, membrane-bound ATPase, F1, may be used to study single site catalysis by the enzyme. [gamma-32P]ATP, incubated with a molar excess of catalytic sites, a condition which favors binding of substrate in only a single catalytic site on the enzyme, is hydrolyzed via a four-step reaction mechanism. The mechanism includes binding in a high affinity catalytic site, Ka = 10(12)M-1, a hydrolytic step for which the equilibrium constant is near unity, and two product release steps in which Pi dissociates from catalytic sites about 10 times more rapidly than ADP. Catalysis by the membrane-bound ATPase also is characterized by a 10(6)-fold acceleration in the rate of net hydrolysis of [gamma-32P]ATP, bound in the high affinity catalytic site, that occurs when substrate is made available to additional catalytic sites on the enzyme. These aspects of the reaction mechanism of the ATPase of submitochondrial particles closely parallel the reaction mechanism determined for solubilized, homogeneous F1 (Grubmeyer, C., Cross, R. L., and Penefsky, H. S. (1982) J. Biol. Chem. 257, 12092-12100). The finding that removal of the enzyme from the membrane does not significantly alter the properties of single site catalysis lends support to models of ATP synthesis in oxidative phosphorylation, catalyzed by membrane-bound F1, that have been based on the study of the soluble enzyme.     

Use of optical biosensors for the study of mechanistically concerted surface adsorption processes

The advent of commercial optical biosensors, such as the BIAcore from Pharmacia and IAsys from Affinity Sensors, has made available to the biochemist a powerful means to examine and characterize the interaction of biological macromolecules with a binding surface. By analysis of the kinetic and equilibrium aspects of the observed experimental adsorption isotherms, rate and affinity constants can be determined. This Review focuses on pertinent aspects of the technology and its use for the performance and quantitative characterization of some various types of mechanistically concerted adsorption behavior.     

Mechanisms of enzyme control as probed by equilibrium exchange rates: patterns of modifier effects with a two substrate, two product system

Measurements of reaction rates at equilibrium by isotopic exchange techniques can give considerable information about the mode of action of modifiers of enzymic reaction rates. To illustrate the various patterns that may be obtained, differing effects of modifiers on the exchange of A with P and of B with Q in the simple enzymic reaction of A + BP + Q are given. For this, reasonable values of rate constants are assumed, and calculations made for random and compulsory binding order systems. Cases where modifiers bind at the catalytic sites of substrate or at other binding sites, and where substrate association, substrate dissociation, covalent interconversion, or total catalytic capacity are modified are considered. Some quite distinctive patterns emerge, among the most interesting being those in which a modifier may block net catalysis yet allow one equilibrium exchange to occur essentially unhindered.     

The effect of electrochemical regeneration upon the enzymatic catalysis of a thermodynamically unfavorable reaction

The association between enzymatic and electrochemical reactions, enzymatic electrocatalysis, had proven to be a very powerful tooth in both analytical and synthetic fields. However, most of the combinations studied have involved enzymatic catalysis of irreversible or quasi-irreversible reaction. In the present work, we have investigated the possibility of applying enzymatic electrocatalysis to a case where the electrochemical reaction drives a thermodynamically unfavorable reversible reaction. Such thermodynamically unfavorable reactions include most of the oxidations catalyzed by dehydrogenases. Yeast alcohol dehydrogenase (E.C. 1.1.1.1) was chosen as a model enzyme because the oxidation of ethanol is thermodynamically very unfavorable and because its kinetics are well known. The electrochemical reaction was the oxidation of NADH which is particularly attractive as a method of cofactor regeneration. Both the electrochemical and enzymatic reactions occur in the same batch reactor in such a way that electrical energy is the only external driving force. Two cases were experimentally and theoretically developed with the enzyme either in solution or immobilized onto the electrode's surface. In both cases, the electrochemical reaction could drive the enzymatic reaction by NADH consumption in solution or directly in the enzyme's microenvironment. However even for a high efficiency of NADH consumption, the rate of enzymatic catalysis was limited by product (acetaldedehyde) inhibition. Extending this observation to the subject of organic synthesis catalyzed by dehydrogenases, we concluded that thermodynamically unfavorable reaction and can only be used in a process if efficient NAD regeneration and product elimination are simultaneously carried out within the reactor.     

Effects of excluded surface area and adsorbate clustering on surface adsorption of proteins I. Equilibrium models

Statistical-thermodynamic models for the equilibrium adsorption of proteins onto homogeneous, locally planar surfaces are presented. An extension of earlier work [R.C. Chatelier, A.P. Minton, Biophys. J. 71 (1996) 2367], the models presented here allow for the formation of a broadly heterogeneous population of adsorbate clusters in addition to excluded volume interactions between all adsorbate species. Calculations are carried out for three simple models for the structure of adsorbate, illustrating similarities and differences in the equilibrium properties of maximally compact clusters, minimally compact clusters and isomerizing clusters. Depending upon the strength of attractive interactions between adsorbate molecules, the resulting equilibrium isotherms may exhibit negative cooperativity, positive cooperativity, essentially no apparent cooperativity, or a mixture of positive cooperativity at low surface density and negative cooperativity at high surface density of adsorbate. The condition of apparent lack of cooperativity, which might naively be interpreted as evidence of a lack of interaction between adsorbate molecules, actually conceals a balance between attractive and repulsive interactions and extensive clustering of adsorbate.     

Effect of water activity on enzyme hydration and enzyme reaction rate in organic solvents

The effects of water on enzyme (protein) hydration and catalytic efficiency of enzyme molecules in organic solvents have been analyzed in terms of the thermodynamic activity of water, which has been estimated by the NRTL or UNIFAC equations. When the amount of water bound to the enzyme was plotted as a function of water activity, the water adsorption isotherms obtained from the water-solvent liquid mixtures were similar to the reported water-vapor adsorption isotherms of proteins. The water adsorption of proteins from the organic media was not significantly dependent on the properties of the solvents or the nature of the proteins. It is also shown that there is a linear relationship between the logarithm of the enzyme reaction rate and water activity. However, the dependence of the enzyme reaction rate on water activity was found to be different depending on the properties of the solvent. The relationship between water activity and other solvent parameters such as solvent hydrophobicity and the solubility of water in the solvent is also discussed.     

Escherichia coli ATP synthase alpha subunit Arg-376: the catalytic site arginine does not participate in the hydrolysis/synthesis reaction but is required for promotion to the steady state

The three catalytic sites of the F(O)F(1) ATP synthase interact through a cooperative mechanism that is required for the promotion of catalysis. Replacement of the conserved alpha subunit Arg-376 in the Escherichia coli F(1) catalytic site with Ala or Lys resulted in turnover rates of ATP hydrolysis that were 2 x 10(3)-fold lower than that of the wild type. Mutant enzymes catalyzed hydrolysis at a single site with kinetics similar to that of the wild type; however, addition of excess ATP did not chase bound ATP, ADP, or Pi from the catalytic site, indicating that binding of ATP to the second and third sites failed to promote release of products from the first site. Direct monitoring of nucleotide binding in the alphaR376A and alphaR376K mutant F(1) by a tryptophan in place of betaTyr-331 (Weber et al. (1993) J. Biol. Chem. 268, 20126-20133) showed that the catalytic sites of the mutant enzymes, like the wild type, have different affinities and therefore, are structurally asymmetric. These results indicate that alphaArg-376, which is close to the beta- or gamma-phosphate group of bound ADP or ATP, respectively, does not make a significant contribution to the catalytic reaction, but coordination of the arginine to nucleotide filling the low-affinity sites is essential for promotion of rotational catalysis to steady-state turnover.     

Poly(L-histidyl-L-alanyl-α-L-glutamic acid). II. Catalysis of p-nitrophenyl acetate hydrolysis

The hydrolysis of p-nitrophenyl acetate is catalyzed by imidazole, free in solution or as the side chain in poly(His-Ala-Glu). This is based on the observations that the reaction is first order in ester and first order in nonprotonated imidazole. Catalysis of p-nitrophenyl acetate hydrolysis is dependent on solvent conditions. The effect of low concentrations of ethanol, dioxane, and trifluoroethanol were investigated. As the concentration of organic solvent is increased, the second-order rate constant for imidazole catalysis decreases. The decrease, however, is greater for imidazole than for poly(His-Ala-Glu). In 2% trifluoroethanol/water solution, free imidazole has twice the catalytic activity of polymeric imidazole, while in 40% trifluoroethanol/water they have equal activity. Since under the latter solvent conditions poly(His-Ala-Glu) is partially α-helical, the relative improvement in polymeric–imidazole catalysis may be attributed to imidazole hydrogen-bonded to a carboxylate ion. With this assumption the carboxylate–imidazole hydrogen-bonded system has been calculated to have three times the base catalytic activity of imidazole.     

Catalytic site occupancy during ATP hydrolysis by MF1-ATPase. Evidence for alternating high affinity sites during steady-state turnover

The mechanism of ATP hydrolysis by the solubilized mitochondrial ATPase (MF1) has been studied under conditions where catalytic turnover occurs at one site, uni-site catalysis (obtained when enzyme is in excess of substrate), or at two sites, bi-site catalysis (obtained when substrate is in excess of enzyme). Pulse-chase experiments support the conclusion that the sites which participate in bi-site catalysis are the same as those which participate in uni-site catalysis. Upon addition of ATP in molar excess to MF1, label that was bound under uni-site conditions dissociates at a rate equal to the rate of bi-site catalysis. Similarly, when medium ATP is removed, label that was bound under bi-site conditions dissociates at a rate equal to the rate of uni-site catalysis. Evidence that a high affinity catalytic site equivalent to the one observed under uni-site conditions participates as an intermediate in bi-site catalysis includes the demonstration of full occupancy of a catalytically competent site during steady-state turnover at nanomolar concentrations of ATP. Improved measurements of the interaction of ADP at a high affinity catalytic site have lead to the revision of several of the rate constants that define uni-site catalysis. The rate constant for unpromoted dissociation of ADP is equal to that for Pi (4 X 10(-3) s-1). The rate of binding ADP at a high affinity chaseable site (Kd = 1 nM) is equal to the rate of binding ATP (4 X 10(6) M-1 s-1). The rate of catalysis obtained when substrate binding at one site promotes product release from an adjacent site (bi-site catalysis) is up to 100,000-fold faster than unpromoted product release (uni-site catalysis).     

Mimic models of peroxidase--kinetic studies of the catalytic oxidation of hydroquinone by H2O2

The reactions of hydroquinone with hydrogen peroxide catalyzed by transition metal ions Cu2+, Fe2+, Fe3+, Co2+ and Mn2+ were investigated in aqueous solution at 25 degrees C. Two copper (II) complexes (bis(dimethylglyoxime) copper(II) and 5,7,7,12,14,14-hexamethyl-1,4,8,11-tetraazacyclotetradeca-4,11-dienatocopper(II)iodide) were prepared. Their catalytic activities on this oxidation were kinetically investigated in aqueous solution and in cetyltrimethylammonium bromide (CTAB) micellar solution at 25 degrees C. The kinetic equations for micellar catalysis and metallomicellar catalysis were established, respectively. CTAB micelle enhances the reaction rate due to its concentrated and electrostatic effects on substrates and/or intermediate. Metallomicelle exhibits remarkable catalytic activity on this oxidation reaction, which is attributed to the active center and the microenvironment effects. Metallomicelle enhances the rate of reaction by activating hydroquinone anion. The presence of co-ligand of imidazole (or pyridine) remarkably increases the catalytic activity of metal complex in micelle system in contrast to it lowers the activity of the complex in aqueous solution. Metallomicelles could be treated as the mimic models of peroxidase.     

A Possible Role for the Asymmetric C-Terminal Domain Dimer of Rous Sarcoma Virus Integrase in Viral DNA Binding

Integration of the retrovirus linear DNA genome into the host chromosome is an essential step in the viral replication cycle, and is catalyzed by the viral integrase (IN). Evidence suggests that IN functions as a dimer that cleaves a dinucleotide from the 3′ DNA blunt ends while a dimer of dimers (tetramer) promotes concerted integration of the two processed ends into opposite strands of a target DNA. However, it remains unclear why a dimer rather than a monomer of IN is required for the insertion of each recessed DNA end. To help address this question, we have analyzed crystal structures of the Rous sarcoma virus (RSV) IN mutants complete with all three structural domains as well as its two-domain fragment in a new crystal form at an improved resolution. Combined with earlier structural studies, our results suggest that the RSV IN dimer consists of highly flexible N-terminal domains and a rigid entity formed by the catalytic and C-terminal domains stabilized by the well-conserved catalytic domain dimerization interaction. Biochemical and mutational analyses confirm earlier observations that the catalytic and the C-terminal domains of an RSV IN dimer efficiently integrates one viral DNA end into target DNA. We also show that the asymmetric dimeric interaction between the two C-terminal domains is important for viral DNA binding and subsequent catalysis, including concerted integration. We propose that the asymmetric C-terminal domain dimer serves as a viral DNA binding surface for RSV IN.