Two-substrate Enzyme Research Articles

Rotating disc electrode characterization of immobilized glucose oxidase

The kinetic properties of glucose oxidase (EC 1.1.3.4) which has been covalently immobilized to a rotating glassy carbon electrode surface have been investigated. Analysis of the rotation rate dependence of the hydrogen peroxide-derived current suggests that oxygen mass transport to the enzyme-electrode surface is rate controlling at low rotation rates. Only as the diffusion layer approaches zero thickness (i.e., infinitely fast rotation rate) does mass transport become unimportant. A diffusion-free glucose K m for air-saturated buffer is found to be 66 m M using this methodology. The importance of mass transport restrictions in two-substrate enzymes such as glucose oxidase is discussed in the context of biosensor design.

A microcomputer program for fitting two-substrate enzyme rate equations

This paper describes a microcomputer program written in BASIC for fitting different two-substrate enzyme kinetic mechanisms. The present program is a complement of one which was recently published and was developed to fit different models of enzyme inhibition [1]. The complete program resulting from this complementation allows the fitting of the most usual rate equations found in steady-state studies of enzyme kinetics. The program based on a non-linear least-squares regression, can be run on any microcomputer having the CP M operating system. The initial rate equation for a steady-state random bisubstrate mechanism was chosen to evaluate the performance of the program, with contrived data of known error structure.

Chronocoulometric techniques in the study of computerized enzyme electrode systems

The electrochemical transient of a two-substrate enzyme electrode was studied theoretically and experimentally. Operation of such electrodes in the chronocoulometric mode leads to increased electrode sensitivity and makes possible the retrieval of useful information on transport and kinetics parameters. Digital simulation was used to solve the kinetics and transport equations and to produce the theoretical chronocoulometric response. A glucose electrode based on glucose oxidase crosslinked to different matrices was tested with air oxygen and p-benzoquinone as the cosubstrate. A computerized electrochemical system was employed for electrode potential control and data acquisition and analysis.

Kinetic analysis of lactate dehydrogenase in cultured chondrocytes by quantitative cytochemistry.

Kinetic analysis of lactate dehydrogenase activity in intact cultured chondrocytes was performed in situ by coupling cell culture and microcytophotometry. Cells were cultured on glass microscope slides divided into eight chambers and studied during the growth cycle in monolayer areas. Lactate dehydrogenase activity was assayed by the reduction of neotetrazolium in the presence of phenazine methosulfate. Quantification of formazan deposits within the cells was performed by scanning and integrating microdensitometry at the isosbestic wavelength of 585 nm. Results indicate the following (a) A kinetic characterization was possible: apparent constants, Km and Ks of this two-substrate enzyme were graphically determined Ks = 1.05 +/- 0.08 and 0.56 +/- 0.05 mM for lactate and NAD respectively and Km = 0.64 +/- 0.03 and 0.37 +/- 0.02 mM for lactate and NAD respectively. (b) Inhibition by lactate concentrations above 10 mM and pyruvate concentration of 1 mM, is in agreement with the well known high anaerobic glycolytic metabolism of chondrocytes. This was confirmed by electrophoresis on cellulose acetate which demonstrated a M3-H isoenzyme form in cultured chick chondrocytes. This study shows that microcytophotometric analysis of lactate dehydrogenase in cultured chondrocytes may be an interesting alternative to mass culture cells followed by classical biochemical studies.

Model of a two-substrate enzyme electrode for glucose.

Dans cette electrode, les enzymes sont immobilisees a l'interieur d'une membrane attachee a un detecteur electrochimique de l'oxygene, cosubstrat de la reaction enzymatique. Le modele mathematique presente concerne les phenomenes de reaction et de diffusion ayant lieu a l'interieur de la membrane

Simplified treatment of two-substrate enzyme kinetics

This paper attempts to show that the extension from single substrate to multi substrate kinetics, particularly two-substrate cases, need not necessarily require the investment of a great deal of additional class time.

II. Transient phase of two-substrate enzyme systems

The time dependence in the transient phase of the two-substrate enzyme systems which evolve according to the random ternary-complex, ordered ternary-complex, ping-pong bi-bi and Theorell-Chance mechanisms has been studied. With this purpose, the equations derived in paper I have been applied. This has allowed to propose a method in order to obtain the rate constant values of these enzyme reactions. In addition, we show that, from experimental knowledge of the induction periods of the ligand species, it is possible to give a method which allows to discriminate between these mechanisms.

Effect of pH on the process of ternary-complex interconversion in the liver-alcohol-dehydrogenase reaction.

1. Kinetic relationships referring to multiple-turnover conditions have been derived for the slowest exponential transient appearing in two-substrate enzyme reactions proceeding by an ordered ternary-complex mechanism. The validity of these and previously derived theoretical relationships for this mechanism has been tested by application to the liver alcohol dehydrogenase reaction. 2. All essential features of the transient-state kinetics of alcohol oxidation by NAD+ in the liver alcohol dehydrogenase system can be qualitatively and quantitatively explained in view of the compulsory-order mechanism in the proposed scheme. There is no kinetic evidence for any half-of-the-sites reactivity of the enzyme. A consistent set of rate constants is reported for the enzymic oxidation of benzyl alcohol at pH 8.75. 3. Transient-state rate parameters for benzyl alcohol/benzaldehyde catalysis by liver alcohol dehydrogenase have been determined at different pH. The interpretation of such rate parameters is critically discussed with reference to their informative value for the purpose of determination of rate constants (k and k') for the process of ternary-complex interconversion in the proposed scheme. It is concluded that the apparent rate constant (k') for hydride transfer from benzyl alcohol to NAD+ is dependent on a proton dissociation step with a pKa of 6.4, whereas the rate constant (k) for hydride transfer from NADH to benzaldehyde exhibits no corresponding dependence on proton association. 4. The asymmetric pH dependence of the forward and reverse rate of ternary-complex interconversion during liver alcohol dehydrogenase catalysis appears to reflect an obligatory step of alcohol/alcoholate ion equilibration occurring at the ternary-complex level. It is suggested that the observed pKa 6.4 dependence of the transient rate of alcohol oxidation can be attributed to a coupled acid-base system involving minimally the enzyme-bound alcohol and the protein residues Ser-48 and His-51.

Optimization of methods for aspartate aminotransferase and alanine aminotransferase.

Conditions for accurate measurement of catalytic activity of aspartate aminotransferase and alanine aminotransferase in human serum have been reinvestigated. The basic variables (kind of buffer, buffer concentration, pH, ion effects, and the influence of pyridoxal-5-phosphate) can now be considered optimized. On this basis, the kinetic parameters of both aminotransferases were determined, i.e., Michaelis and inhibitor constants for substrates and reaction products. With a mathematical approach for two-substrate enzyme reactions the substrate concentrations were calculated from the viewpoints "most economical," "most convenient," and "lowest variability." Also the conditions for the indicator reactions have been newly defined with respect to a kinetic model. All calculated data were rechecked experimentally and it can be shown that both approaches fully agree. Furthermore, we show that the mathematical approach allows more precise recommendations for optimized methods. For technical reasons, the catalytic activity of aspartate aminotransferase in human serum can only be measured as a 0.96 fraction of its theoretical maximum velocity, the catalytic activity of alanine aminotransferase as a 0.91 fraction. The assay conditions for a Reference Method are finally described and recommendations are made for optimized routine methods for determination of the catalytic activity of these transferases in human serum.

“Hit-and-run” mechanism for d-glucuronate reduction catalyzed by d-mannonate:NAD oxidoreductase of Escherichia coli

d-mannonate:NAD oxidoreductase ( d-mannonate:NAD + 5-oxidoreductase, EC 1.1.1.57) from Escherichia coli normally catalyzes the reaction: d- fructuronate + NADH ⇌ d-mannonate + NAD + [4,5]. It can also catalyze the reduction of d-glucuronate according to the equation: d- glucuronate + NADH → l-gulonate + NAD + . Kinetic evidence is given for the second reaction to proceed in two steps: (i) NADH binds reversibly the free enzyme to form a binary complex E · NADH; (ii) d-glucuronate reacts subsequently with the binary complex to give products, without formation of an intermediary ternary complex E · NADH · glucuronate. The rate of the overall reaction obeys the classical Michaelis-Menten law with respect to NADH concentration ( K 1 = 0.03 mM) but is proportional to d-glucuronate concentration in the range 0–100 mM. Results concerning the action of several inhibitors on d-glucuronate reduction were interpreted satisfactorily by developing models based on mechanism involving an enzyme carrying one binding site for the alternative substrate d-mannonate and one binding site for the coenzyme NADH (Table I). In any case the experimental values of the dissociation constants for the complexes enzyme-effector were deduced. NAD and ATP act as strict competitive inhibitors ( K 2 = 0.60 mM and 20 mM) able to bind the free enzyme only. In the case of ATP, a second binding site with a high affinity ( K 3 = 0.02 mM) is unmasked when the first binding site is saturated. Mannonate and altronate behave as strictly non-competitive inhibitors able to bind equally the free enzyme and the binary complex E · NADH. l-Gulonate acts as a mixed non-competitive inhibitor able to bind much easily the free enzyme ( K 4 = 0.41 mM) than the complex E · NADH ( K 2 = 131 mM). In no case was d-glucuronate shown to be able to react with complexes other than E · NADH. Such an unusual mechanism for a two-substrate enzyme looks similar to the sequence found by Chance for horse-radish peroxidase [6].

The Transient‐State Kinetics of Two‐Substrate Enzyme Systems Operating by an Ordered Ternary‐Complex Mechanism

Transient-state kinetic equations, applicable to single-turnover experiments with limiting amounts of substrate and catalytic-suicide experiments with substrates in excess to enzyme, have been derived for two-substrate enzyme reactions occurring by an ordered ternary-complex mechanism. The relative magnitude and dependence on substrate concentrations of rates and amplitudes for the exponential transients corresponding to this mechanism. It is shown that ordered ternary-complex reactions under standard assay conditions may exhibit a biphasic transient rate behaviour, and the precise conditions under which two transients can be detected are described and discussed.

Influence of premixing on transient-phase kinetics for two-substrate enzyme systems

Transient-phase kinetic equations are worked out, using the Laplace transform method, for two-substrate enzyme reactions occurring by the Theorell-Chance, ping pong bi bi and ordered ternary-complex mechanisms (the equations for the random ternary-complex mechanism are too complex to be useful). Two sets of premixing conditions were considered: (i) E not premixed with the first substrate A, i.e., E | A | B, E | A + B or E + B | A; and (ii) E premixed with the first substrate A, i.e., E + A | B. For each mechanism and premixing conditions two cases were treated: (a) enzyme concentration limiting, i.e., e 0 ⪡ a 0, b 0 , and (b) concentration of first substrate A limiting, a 0 ⪡ e 0, b 0 . In all cases (a) gives rise to a transient phase followed by a steady state, the transient phase being represented by two or more exponential terms. In all cases (b) gives no steady state; the concentrations of products X and Y rise to a final value of a 0 in a manner represented by two or more exponential terms. Premixing of type (ii) leads to a more rapid initial rise than that of type (i), in Cases a and b. Some results on horse liver alcohol dehydrogenase are shown to be consistent with the equations derived for a Theorell-Chance mechanism; there is no evidence for the participation of two types of active sites on the enzyme.

Model Studies of the Sequential Random-Order Two-Substrate Enzyme Mechanism.

Model Studies of the Sequential Random-Order Two-Substrate Enzyme Mechanism.

Kinetic mechanism of phosphorylase a. I. Initial velocity studies.

Initial rate studies on rabbit muscle phosphorylase a were carried out in order to assign a kinetic mechanism to this enzyme, which plays an important role in the control of glycogen metabolism. Initial velocities were measured with varied concentrations of both substrates in each reaction direction, both in the presence and the absence of the modifier AMP. Data were analyzed with double-reciprocal plots and secondary replots of slopes and intercepts to yield kinetically derived dissociation constants which may be compared with dissociation constants determined by independent methods. Inhibition studies using UDP-glucose as a substrate analogue are also reported. Inhibition is competitive with glucose 1-phosphate and P1 and noncompetitive with glycogen.A suitable rate equation for this system has been derived, and it should apply to other two-substrate enzyme–modifier systems exhibiting similar kinetics. The results of this detailed kinetic analysis, in conjunction with the isotope exchange studies at equilibrium which are reported in the following paper, suggest the kinetic mechanism of phosphorylase a to be rapid equilibrium random Bi Bi, i.e. random addition of substrates to the enzyme, with ternary complex interconversion as the rate-limiting step in the reaction sequence.

Alternative Allosteric Effects Exerted by End Products upon a Two-Substrate Enzyme in Rhodomicrobium vannielii

Abstract The regulatory enzyme, 3-deoxy-d-arabino-heptulosonate 7-phosphate synthetase of Rhodomicrobium vannielii was studied. Both phenylalanine and tyrosine are competitive inhibitors with respect to the substrate, erythrose 4-phosphate. Tryptophan does not influence enzyme activity per se, but potentiates the effect of phenylalanine or tyrosine. Kinetic experiments led to the conclusion that alternative kinetic pathways to the ternary complex existed, and that the one forming (phosphoenolpyruvate-enzyme) as the initial binary complex was kinetically preferred. Accordingly, the ratio of the two substrates had a marked influence upon reaction velocity. The observed ability of phenylalanine and tyrosine to either activate or inhibit enzyme activity, depending upon the ratio of the two substrates was consistent with the postulated mechanism. An extremely wide range of enzyme activity exists, on the one hand, because of the stimulation of activity by one or two amino acid end products, and on the other hand, because of the potent concerted inhibition in the simultaneous presence of all three amino acids. The physiological consequences of this pattern of control for 3-deoxy-d-arabino-heptulosonate 7-phosphate synthetase are discussed.

RElationships between rapid equilibrium conditions and linearization of the reciprocal rate equation for the sequential random two-substrate enzyme mechanism.

RElationships between rapid equilibrium conditions and linearization of the reciprocal rate equation for the sequential random two-substrate enzyme mechanism.

Kinetic Characteristics of the Sequential Random Order Two-substrate Enzyme Mechanism.

Kinetic Characteristics of the Sequential Random Order Two-substrate Enzyme Mechanism.

Effects of modifiers on the kinetics of two-substrate enzyme reactions.

Steady state rate equations have been derived for ordered bi bi and ping pong bi bi reactions in which there are (a) one or two nonsubstrate modifiers, (b) two different binding sites for a single nonsubstrate modifier, (c) one or two substrates acting as modifiers, and (d) both nonsubstrate modifiers and substrates acting as modifiers. The deviation of these equations from the Michaelis–Menten equation is shown and methods are suggested by which many of these mechanisms can be distinguished experimentally.

A comparative study of some mechanisms describing interaction effects in a dimeric, two-substrate enzyme catalysed reaction

A number of mechanisms are described which, when applied to a dimeric two substrate enzyme, lead to a second degree relationship between the initial velocity of the enzyme catalysed reaction and a variable substrate concentration, the concentration of the second substrate being held constant. The mechanisms fall into three classes. In the first, the subunits of the enzyme are independent of one another, but alternate pathways leading to product formation exist in each subunit. In the second class, a linear mechanism operates within each subunit, but the subunits interact with one another. In the third class, a temporary dissociation of the enzyme into smaller units is an integral part of the substrate binding process. These models are examined in four main ways: (i) the slope of the Hill plots is determined (ii) the form of the relationship between the initial velocity of product formation and substrate concentrations is investigated (iii) a matrix rank analysis of initial rates of substrate binding is described and (iv) the time course of substrate binding is determined. As a result, it appears that characteristic differences between the models exist and may serve to discriminate between them.

Rearrangement of the Ingraham-Makower equation for a two-substrate enzyme reaction for practical application

A rate equation for two-substrate enzyme reaction has been proposed by Ingraham & Makower (1954), and by Dixon & Webb (1958). This paper attempts to rearrange the equation for the purpose of practical application. Both the double-reciprocal relation and that of “ a/v versus a” are represented by hyperbolas indicating substrate effects, either substrate-acceleration or substrate-inhibition. The substrate effects appear as a pair of reverse phenomena, that is, substrate-acceleration by one of the two substrates and substrate-inhibition by the other one, according to mathematical expressions of these. A cyclic sequence of the intermediate complexes is indicated by an interrelation of the partial rate-constants. A graphical method to estimate both the maximum rate of the reaction and a dissociation constant of the EAB ternary complex is achieved by plotting 1/ v-ordinates of the double-reciprocal hyperbolas at the 1/ v-axis against the 1/ b-values, reciprocals of the concentrations of the second substrate B.