Modeling, simulation, and kinetic analysis of a heterogeneous reaction system for the enzymatic conversion of poorly soluble substrate.

We developed a kinetic model that describes a heterogeneous reaction system consisting of a solid substrate suspension for the production of D-amino acid using D-hydantoinase. As a biocatalyst, mass-produced free and whole cell enzymes were used. The heterogeneous reaction system involves dissolution of a solid substrate, enzymatic conversion of the dissolved D-form substrate, spontaneous racemization of an L-form substrate to D-form, and deactivation of the enzyme. In the case of using whole cell enzymes, transfer of the dissolved substrate and product through the cell membrane was considered. The kinetic parameters were determined from experiments, literature data, and by using Marquardt's method of nonlinear regression analysis. The model was simulated using the kinetic parameters and compared with experimental data, and a good agreement was observed between the experimental results and the simulation ones. Factors affecting the kinetics of the heterogeneous reaction system were analyzed on the basis of the kinetic model, and the efficiency of the reaction systems using free and whole cell enzymes was also compared.     

Contribution of Lys276 to the conformational flexibility of the active site of glutamate decarboxylase from Escherichia coli.

Glutamate decarboxylase is a pyridoxal 5'-phosphate-dependent enzyme responsible for the irreversible alpha-decarboxylation of glutamate to yield 4-aminobutyrate. In Escherichia coli, as well as in other pathogenic and nonpathogenic enteric bacteria, this enzyme is a structural component of the glutamate-based acid resistance system responsible for cell survival in extremely acidic conditions (pH < 2.5). The contribution of the active-site lysine residue (Lys276) to the catalytic mechanism of E. coli glutamate decarboxylase has been determined. Mutation of Lys276 into alanine or histidine causes alterations in the conformational properties of the protein, which becomes less flexible and more stable. The purified mutants contain very little (K276A) or no (K276H) cofactor at all. However, apoenzyme preparations can be reconstituted with a full complement of coenzyme, which binds tightly but slowly. The observed spectral changes suggest that the cofactor is present at the active site in its hydrated form. Binding of glutamate, as detected by external aldimine formation, occurs at a very slow rate, 400-fold less than that of the reaction between glutamate and pyridoxal 5'-phosphate in solution. Both Lys276 mutants are unable to decarboxylate the substrate, thus preventing detailed investigation of the role of this residue on the catalytic mechanism. Several lines of evidence show that mutation of Lys276 makes the protein less flexible and its active site less accessible to substrate and cofactor.     

Enzyme reaction kinetics in organic solvents: A theoretical kinetic model and comparison with experimental observations

A theoretical kinetic model has been developed in order to describe the enzyme reaction in organic solvents. In this model the hydration of the enzyme molecule was examined and the equilibrium kinetic constants expressed in terms of thermodynamic activity. Analysis of a proposed kinetic model shows that the enzyme reaction rate in organic solvents is determined by two factors: substrate solvation and enzyme hydration, which are determined by the activity coefficient of the substrate and the water activity of the reaction media, respectively. The activity coefficient of the substrate and the water activity have been calculated using the UNIFAC equation to analyze the effects of organic solvents on the rate of enzyme reaction, and the results were compared with experimental data. Predictions of the proposed model were found to be in good agreement with previous experimental observations.     

Comparison of kinetic and end-point microdensitometry for the direct quantitative histochemical assessment of cytochrome oxidase activity

Synopsis Cytochrome oxidase activity has been assessed by a method of kinetic microdensitometry which involves applying tissue sections to gel films containing phenylamine substrates and measuring the rate of azine dye production by continuously recording the rate of change in extinction. Optimum conditions for the technique were defined, and the results compared with those obtained by conventional end-point microdensitometry in which sections are incubated in histochemical substrate solutions and azine dye production estimated by a single measurement of extinction at the end of the incubation period. When compared with biochemically-determined enzyme activity, kinetic microdensitometry gave a better index of the proportionate activity of cytochrome oxidase in various normal tissues than did end-point microdensitometry. In addition, the degree of inhibition of cytochrome oxidase activity in tissues removed from cyanide-poisoned animals was assessed more reliably by kinetic microdensitometry than by end-point measurements. With end-point microdensitometry, the reaction is non-linear over the comparatively long incubation times required and there is also a spontaneous reactivation of cyanide-inhibited cytochrome oxidase during incubation and thus a progressively increased rate of substrate utilization. In contrast, with kinetic microdensitometry the initial linear reaction rate is measured before significant reactivation occurs. Kinetic microdensitometry can be used for direct dynamic quantitation of enzyme activity in tissues or cells; it may be a valuable technique for quantitative histochemical confirmation or extension of biochemical studies; and it appears to be a reliable direct quantitative histochemical method for investigatingin vivo inhibition of enzyme activity, where spontaneous reactivation of the enzyme-inhibitor complex may occur.     

Studies of the mechanism of glutamine synthetase utilizing pH-dependent behavior in catalysis and binding

The pKa values of enzyme groups of Escherichia coli glutamine synthetase which affect catalysis and/or substrate binding were determined by measuring the pH dependence of Vmax and V/K. Analysis of these data revealed that two enzyme groups are required for catalysis with apparent pKa values of approximately 7.1 and 8.2. The binding of ATP is essentially independent of pH in the range studied while the substrate ammonia must be deprotonated for the catalytic reaction. Using methylamine and hydroxylamine in place of ammonia, the pKa value of the deprotonated amine substrate as expressed in the V/K profiles was shifted to a lower pKa value for hydroxylamine and a higher pKa value for methylamine. These data indicate that the amine substrate must be deprotonated for binding. Hydroxylamine is at least as good a substrate as ammonia judged by the kinetic parameters whereas methylamine is a poor substrate as expressed in both the V and V/K values. Glutamate binding was determined by monitoring fluorescence changes of the enzyme and the data indicate that a protonated residue (pKa = 8.3 +/- 0.2) is required for glutamate binding. Chemical modification by reductive methylation with HCHO indicated that the group involved in glutamate binding most likely is a lysine residue. In addition, the Ki value for the transition state analog, L-3-amino-3-carboxy-propanesulfonamide was measured as a function of pH and the results indicate that an enzyme residue must be protonated (pKa = 8.2 +/- 0.1) to assist in binding. A mechanism for the reaction catalyzed by glutamine synthetase is proposed from the kinetic data acquired herein. A salt bridge is formed between the gamma-phosphate group of ATP and an enzyme group prior to attack by the gamma-carboxyl of glutamate on ATP to form gamma-glutamyl phosphate. The amine substrate subsequently attacks gamma-glutamyl phosphate resulting in formation of the tetrahedral adduct before phosphate release. A base on the enzyme assists in the deprotonation of ammonia during its attack on gamma-glutamyl phosphate or after the protonated carbinol amine is formed. Based on the kinetic data with the three amine substrates, catalysis is not rate-limiting through the pH range 6-9.     

Deviations from michaelis-menten behaviour of plant glutamate dehydrogenase with ammonium as variable substrate

A stopped flow kinetic analysis has been performed with a homogeneous protein fraction of plant glutamate dehydrogenase. The enzyme exerts strong negative cooperativity with ammonium as variable substrate. The limiting initial rate constants for low substrate concentrations, as calculated from the kinetic data, indicate that the catalytic efficiency of the enzyme increases at low ammonium concentrations. From this it becomes evident that the reductive amination reaction is highly adaptive to the ammonium environment.     

Atypical kinetics of immobilized firefly luciferase

The kinetic properties of collagen-bound firefly luciferase have been investigated. Under definite hydrodynamic conditions with low agitation in the reaction medium, the observed behavior is modified compared to the enzyme free in solution: reducing the stirring rate decreases the observed enzymatic activity. But diffusional resistances alone cannot account for these atypical kinetics though mass transfer may certainly play an important role during the transient state of the bioluminescent reaction. After immobilization, the time necessary to reach the steady state increased from 300 ms to 3 min and the two substrates, luciferin and ATP, behave differently with respect to the enzyme: The nature of the saturating substrate first in contact with the bound enzyme is not indifferent suggesting that immobilization can reveal behaviors or mechanisms which are not visualized with the free enzyme.     

Dihydrofolate reductase from Escherichia coli: the kinetic mechanism with NADPH and reduced acetylpyridine adenine dinucleotide phosphate as substrates

Kinetic studies on the reaction catalyzed by dihydrofolate reductase from Escherichia coli have been undertaken with the aim of characterizing further the kinetic mechanism of the reaction. For this purpose, the kinetic properties of substrates were determined by measurement of (a) initial velocities over a wide range of substrate concentrations and (b) the stickiness of substrates in ternary enzyme complexes. Stickiness is defined as the rate at which a substrate reacts to give products relative to the rate at which that substrate dissociates. Stickiness was determined by varying the viscosity of reaction mixtures and the concentration of one substrate in the presence of a saturating concentration of the other substrate. The results indicate that NADPH is sticky in the enzyme-NADPH-dihydrofolate complex, while dihydrofolate is much less sticky in this complex. At higher concentrations, NADPH functions as an activator through the formation of an enzyme-NADPH-tetrahydrofolate from which tetrahydrofolate is released more rapidly than from an enzyme-tetrahydrofolate complex. Higher concentrations of dihydrofolate also cause enzyme activation, and it appears that this effect is due to the ability of dihydrofolate to displace tetrahydrofolate from a binary enzyme complex through the formation of a transitory enzyme-tetrahydrofolate-dihydrofolate complex. As NADPH and dihydrofolate function as activators and as NADPH behaves as a sticky substrate, the kinetic mechanism of the dihydrofolate reductase reaction with the natural substrates is steady-state random. By contrast with NADPH, reduced 3-acetylpyridine adenine dinucleotide phosphate exhibits only slight stickiness and does not function as an activator.(ABSTRACT TRUNCATED AT 250 WORDS)     

The kinetic mechanism of the reactions catalyzed by the glutamate synthase from Azospirillum brasilense

The reactions catalyzed by glutamate synthase from Azospirillum brasilense have been investigated by a combination of absorption spectroscopy, steady-state kinetic measurements and experiments with stereospecifically labelled substrate. The data show that both L-glutamine-dependent and ammonia-dependent reactions of the glutamate synthase from A. brasilense follow an identical two-site uni-uni bi-bi kinetic mechanism, in which the enzyme is alternately reduced by NADPH and oxidized by the iminoglutarate formed on addition of ammonia to the C2 of 2-oxoglutarate. The spectroscopic experiments support the involvement of the enzyme chromophores (flavins and iron-sulfur centers) in both reactions. Finally, using stereospecifically labelled NADPH, we showed that the enzyme from Azospirillum is specific for the transfer of the 4S hydrogen of NADPH. During the catalysis of both L-glutamine-dependent and ammonia-dependent reactions, this hydrogen atom equilibrates with the solvent. The data obtained with glutamate synthase from A. brasilense, a diazotroph, differ significantly from those regarding the ammonia-dependent reaction of other glutamate synthases. The ammonia-dependent activity of glutamate synthase from Azospirillum is not physiologically significant, representing only a segment of the overall physiological L-glutamine-dependent activity and requiring the enzyme flavins and iron-sulfur centers. Finally, the data are not consistent with the hypothesis [Geary, L. E. & Meister, A. (1977) J. Biol. Chem. 252, 3501-3508] that the small subunit of glutamate synthase is endowed with a glutamate-dehydrogenase-like activity.     

Rational polynomial equation as an unbiased approach for the kinetic studies of Drosophila melanogaster acetylcholinesterase reaction mechanism

The hydrolysis of substrates by cholinesterases does not follow the Michaelis–Menten reaction mechanism. The well-known inhibition by excess substrate is often accompanied by an unexpectedly high activity at low substrate concentrations. It appears that these peculiarities are the consequence of an unusual architecture of the active site, which conducts the substrate molecule over many stages before it is cleaved and released. Structural and kinetic data also suggest that two substrate molecules can attach at the same time to the free, as well as to the acetylated, enzyme. We present a procedure which provides an unbiased framework for mathematical modelling of such complex reaction mechanisms. It is based on regression analysis of a rational polynomial using classical initial rate data. The determination of polynomial degree reveals the number of independent parameters that can be evaluated from the available information. Once determined, these parameters can substantially facilitate the construction and evaluation of a kinetic model reflecting the expected molecular events in an enzymic reaction. We also present practical suggestions for testing the postulated kinetic model, using an original thermodynamic approach and an isolated effect in a specifically mutated enzyme.     

KINETICS OF BRAIN GLUTAMATE DECARBOXYLASE. INTERACTIONS WITH GLUTAMATE,PYRIDOXAL 5-PHOSPHATE AND GLUTAMATE-PYRIDOXAL 5-PHOSPHATE SCHIFF BASE1

Abstract— The kinetic behavior of glutamate decarboxylase from mouse brain was analyzed in a wide range of glutamate and pyridoxal 5′-phosphate concentrations, approaching three limit conditions: (I) in the absence of glutamate-pyridoxal phosphate Schiff base; (II) when all glutamate is trapped in the form of Schiff base; (III) when all pyridoxal phosphate is trapped in the form of Schiff base. The experimental results in limit condition (I) are consistent with the existence of two different enzyme activities, one dependent and the other independent of free pyridoxal phosphate. The results obtained in limit conditions (II) and (III) give further support to this postulation. These data show that the free pyridoxal phosphate-dependent activity can be abolished when either all substrate or all cofactor are in the form of Schiff base. The free pyridoxal phosphate-independent activity is also abolished when all substrate is trapped as Schiff base, but it is not affected by the conversion of free pyridoxal phosphate into the Schiff base. A kinetic and mechanistic model for brain glutamate decarboxylase activity, which accounts for these observations as well as for the results of previous dead end-inhibition studies, is postulated. Computer simulations of this model, using the experimentally obtained kinetic constants, reproduced all the observed features of the enzyme behavior. The possible implications of the kinetic model for the regulation of the enzyme activity are discussed.     

Diffusion-dependent rates for the hydrolysis reaction catalyzed by glyoxalase II from rat erythrocytes

Glyoxalase II from rat erythrocytes is a near optimal catalyst for the hydrolysis of S-D-lactoylglutathione in the sense that the magnitude of kcat/Km is limited, in large part, by the rate constant for diffusion-controlled encounter between substrate and active site. The experimental basis for this conclusion is derived from the dependencies of the kinetic properties of the enzyme on solution viscosity (pH 7, Ic = 0.1 M, 25 degrees C). When sucrose is used as a viscogenic agent, kcat/Km for S-D-lactoylglutathione (8.8 x 10(5) M-1 s-1) decreases markedly with increasing solution viscosity. This effect appears not to be due to a sucrose-induced change in the intrinsic kinetic properties of the enzyme, since kcat/Km for the slow substrate S-acetylglutathione (3.7 x 10(4) M-1 s-1) is nearly independent of solution viscosity. Quantitative treatment of the data using Stoke's law indicates that the rate of hydrolysis of S-D-lactoylglutathione will be approximately 50% diffusion limited when [substrate] much less than Km; the encounter complex between enzyme and substrate partitions nearly equally between product formation and dissociation to form free enzyme and substrate. The same conclusion is reached when glycerol is used as a viscogenic agent, once the apparent activation effect of glycerol on the intrinsic activity of the enzyme is taken into account. Finally, the rate of formation of the encounter complex between substrate and active site may be governed to a significant extent by charge-charge interactions.(ABSTRACT TRUNCATED AT 250 WORDS)     

Kinetic parameters for the elimination reaction catalyzed by triosephosphate isomerase and an estimation of the reaction's physiological significance

Kinetic parameters for triosephosphate isomerase catalysis of the elimination reaction of an equilibrium mixture of dihydroxyacetone phosphate (DHAP) and D-glyceraldehyde-3-phosphate (DGAP) to form methylglyoxal and phosphate ion are reported for the enzyme from rabbit muscle. Pseudo-first-order rate constants for the disappearance of substrate (kelim) were determined for reactions at [Enzyme] much greater than [Substrate]. The second-order rate constant kEnz = 10.1 M-1 s-1 was determined from a plot of kelim against enzyme concentration. The kinetic parameters, determined from a steady-state kinetic analysis at [Substrate] much greater than [Enzyme], are kcat = 0.011 s-1, Km = 0.76 mM, and kcat/Km = 14 M-1 s-1. The estimated rate-constant ratio for partitioning of the enzyme-bound intermediate between protonation at carbon 2 and elimination, 1,000,000, is much larger than the ratio of 6.5 determined for the reaction of the enediolate phosphate in a loose complex with quinuclidinonium cation, a small buffer catalyst. There is a 10(5)-10(8)-fold decrease in the rate constant for the elimination reaction of the enediolate phosphate when this species binds to triosephosphate isomerase. The kinetic parameters for the elimination reaction catalyzed by the native triosephosphate isomerase and for the reaction catalyzed by a mutant form of the enzyme, which is missing a segment that forms hydrogen bonds with the phosphate group of substrate [Pompliano, D. L., Peyman, A., & Knowles, J. R. (1990) Biochemistry 29, 3186-3194] are similar.(ABSTRACT TRUNCATED AT 250 WORDS)     

"Clotspeed," a mathematical simulation of the functional properties of prothrombinase

Prothrombinase is a Ca2+-dependent, 1:1, enzymatic complex of Factor Xa and Factor Va that assembles on the surface of negatively charged phospholipid vesicles or platelets. It catalyzes the proteolytic conversion of prothrombin to the blood-clotting enzyme thrombin. Experimentally determined kinetic parameters, plus Kd and n values for the interaction of substrate, cofactor (Factor Va), and serine protease (Factor Xa) for both phospholipid and each other, were used to develop a model that simulates the functional properties of the enzymatic complex. Through the use of a desk-top computer and a program designated "Clotspeed," the distribution of enzymatic components and substrate between the bulk fluid and phospholipid is determined for a given set of initial concentrations of reaction components. Simulated reaction rates are then calculated from the calculated distributions, fractional binding, and local and bulk concentration of reactants. Predicted behavior includes formal Michaelis-Mentenlike properties for the reaction, increasing apparent Km with increased levels of phospholipid, and apparent inhibition by excess substrate, enzyme, and phospholipid. Inhibition by excess enzyme and phospholipid was demonstrated experimentally in quantitative agreement with predicted results. The model is useful in that it rationalizes well the seemingly unusual properties of prothrombinase in straightforward physical terms, provides a means of rationally choosing experimental conditions to both further test and refine the model, and explores the properties not only of prothrombinase but also other blood-clotting or surface-bound enzymatic complexes.     

Characterization of the kinetics of phospholipase C activity toward mixed micelles of sodium deoxycholate and dimyristoylphosphatidylcholine

Phospholipase C catalyzed hydrolysis of dimyristoyl phosphatidylcholine (DMPC) in phospholipid-bile salt mixed micelles was studied with particular attention on the relationship between interfacial enzyme activity and the physicochemical properties of substrate aggregates. Steady state kinetics is observed and it is argued that conditions for steady state exist because the enzyme encounters a steady supply of substrate by hopping between micelles at a rate faster than the chemical reaction rate. An existing kinetic model is reformulated to a more usable form. This presents a new approach to treating the kinetic data and allows extraction of the kinetic parameters of the model from the activity dependence on micellar lipid substrate surface concentration. The kinetic parameters were found to depend on the physicochemical properties of substrate aggregates, but remain constant over a range of lipid and bile salt concentrations. The substrate aggregates were characterized by time-resolved fluorescence quenching (TRFQ). The activity values and the micelle sizes group into two sets: (i) larger micelles for bile salt/lipid 5 with lower activity and longer steady state ( approximately 10 min). At least two sets of parameters, for bile salt/lipid 5, characterize the kinetics. Higher enzyme-micelle dissociation constant and lower catalytic rate are found for the group of smaller micelles. An explanation supporting our finding is that as micelles become smaller the overlap area for enzyme-micelle binding decreases, leading to weaker binding. Consequently the enzyme dissociation constant increases. Extension of the present approach to other phospholipases and substrates to establish its generality and correlation between micelle size and the catalytic rate are areas for future investigations.     

Phosphate-Activated Glutaminase in the Crude Mitochondrial Fraction (P2 Fraction) from Human Brain Cortex

The kinetics and other properties of phosphate-activated glutaminase have for the first time been studied in the crude mitochondrial fraction (P2 fraction) from human brain. The enzyme is for unexplained reasons inactivated postmortem. The enzyme activity decreases by storing the tissue or homogenate at 37 degrees C. The inactivation is not caused by formation of a dialysable inhibiting compound. No large proteolytic degradation has occurred, since the phosphate-activated glutaminase-like immunoreactive band did not disappear during the storage. The molecular weight of the subunit of the enzyme as determined by immunoblots of sodium dodecyl sulfate-treated homogenates from human brain is estimated to be approximately 64 K. The enzyme has been shown to have a pH optimum of 8.6; it is activated by phosphate, inhibited by glutamate, and partially inhibited by ammonia. Double-inverse plots of enzyme activity against phosphate are concave-upward, and more so in the presence of an inhibitor. The inhibition by glutamate appears to be noncompetitive with the substrate glutamine, and competitive with the activator phosphate. These kinetic properties are not significantly different from our earlier observations concerning phosphate-activated glutaminase from pig brain and pig kidney.     

Kinetic modeling of omega-transamination for enzymatic kinetic resolution of alpha-methylbenzylamine

A kinetic model for omega-transaminase from Bacillus thuringiensis JS64 was developed by using the King-Altman method to simulate the kinetic resolution of alpha-methylbenzylamine (alpha-MBA). Starting from a ping-pong bi-bi mechanism, a complete kinetic model including substrate inhibition only in the reverse reaction (i.e., transamination between acetophenone and L-alanine) was developed. The asymmetric synthesis of (S)-alpha-MBA proved to be difficult due to a much lower maximum reverse reaction rate than the maximum forward reaction rate, thermodynamically exergonic forward reaction (i.e., transamination between (S)-alpha-MBA and pyruvate), and the severe product and substrate inhibition of the reverse reaction. Experimental values for kinetic parameters show that the product inhibition constant of (S)-alpha-MBA is the most important parameter on determining the resolution reaction rate, suggesting that the resolution reaction rate will be very low unless (S)-alpha-MBA strongly inhibits the reverse reaction. Using the kinetic model, the kinetic resolution of alpha-MBA in aqueous buffer was simulated, and the simulation results showed a high degree of consistency with experimental data over a range of reaction conditions. Various simulation results suggest that the crucial bottleneck in the kinetic resolution of alpha-MBA lies mainly in the accumulation of acetophenone in reaction media as the reaction proceeds, whereas L-alanine exerts a little inhibitory effect on the reaction. The model predicts that removing acetophenone produced during the reaction can enhance the reaction rate dramatically. Indeed, the biphasic reaction system is capable of extracting acetophenone from the aqueous phase, showing a much higher reaction rate compared to a monophasic reaction system. The kinetic model was also useful in predicting the properties of other, better enzymes as well as the optimal concentrations of amino acceptor and enzyme in the resolution reaction.