scholarly journals Multiple inhibition of glutathione S-transferase A from rat liver by glutathione derivatives: kinetic analysis supporting a steady-state random sequential mechanism

1979 ◽  
Vol 177 (3) ◽  
pp. 861-868 ◽  
Author(s):  
I Jakobson ◽  
M Warholm ◽  
B Mannervik
Keyword(s):  
Steady State ◽  
Goodness Of Fit ◽  
Linear Regression Analysis ◽  
Rate Equations ◽  
Glutathione S Transferase ◽  
Confidence Levels ◽  
Steady State Kinetics ◽  
Sequential Mechanism ◽  
Replicate Measurements ◽  
Glutathione Derivatives

Glutathione derivatives inhibit glutathione S-transferase A [cf. Biochem. J. (1975) 147, 513–522]. The steady-state kinetics of this inhibition have been investigated in detail by using S-octyglutathione, glutathione disulphide and S-(2-chloro-4-nitrophenyl)glutathione: the last compound is a product of the enzyme-catalused reaction. Interpreted in terms of generalized denotations of inhibition patterns, the compounds were found to be competitive with the substrate glutathione. Double-inhibition experiments involving simultaneous use of two inhibitors indicated exclusive binding of the inhibitors to the enzyme. The discrimination between alternative rate equations has been based on the results of weighted non-linear regression analysis. The experimental error was determined by replicate measurements and was found to increase with velocity. The established error structure was used as a basis for weighting in the regression and to construct confidence levels for the judgement of goodness-of-fit of rate equations fitted to experimental data. The results obtained support a steady-state random model for the mechanism of action of glutathione S-transferase A and exclude a number of simple kinetic models.

scholarly journals A steady-state-kinetic model for formaldehyde dehydrogenase from human liver. A mechanism involving NAD+ and the hemimercaptal adduct of glutathione and formaldehyde as substrates and free glutathione as an allosteric activator of the enzyme

1979 ◽  
Vol 177 (3) ◽  
pp. 869-878 ◽  
Author(s):  
L Uotila ◽  
B Mannervik
Keyword(s):  
Steady State ◽  
Human Liver ◽  
Linear Regression Analysis ◽  
Equilibrium Mixture ◽  
Reaction Scheme ◽  
Formaldehyde Dehydrogenase ◽  
Sequential Reaction ◽  
Steady State Kinetics ◽  
Steady State Kinetic ◽  
Necessary And Sufficient

The steady-state kinetics of formaldehyde dehydrogenase from human liver have been explored. Non-linearities were obtained in v-versus-v[S] plots. It was necessary and sufficient to consider two reactants of the equilibrium mixture of formaldehyde, glutathione and their hemimercaptal adduct for a complete description of the kinetics. A random sequential reaction scheme is proposed in which adduct and beta-NAD+ are the substrates. In addition, glutathione can bind to an allosteric regulatory site and only the glutathione-containing enzyme is considered productive. Various alternative reaction models were examined but no simple alterative was superior to the model chosen. The discrimination was largely based on results of non-linear regression analysis. Several S-substituted glutathione derivatives were tested as activators or inhibitors of the enzyme, but all were without effect. Thio-NAD+, nicotinamide–hypoxanthine dinucleotide and 3-acetylpyridine-adenine dinucleotide could substitute for beta-NAD+ as the nucleotide substrate. alpha-NAD+ and ADP-ribose were competitive inhibitors with respect to beta-NAD+ and non-competitive with glutathione and the adduct. When used simultaneously, the inhibitors were linear competitive versus each other, indicating a single nucleotide-binding site or, if more than one, non-co-operative binding sites.

Steady-state kinetics

2000 ◽  
Author(s):  
Athel Cornish-Bowden
Keyword(s):  
Steady State ◽  
Enzyme Kinetics ◽  
Single Molecule ◽  
Transient State ◽  
Curve Analysis ◽  
Rate Equations ◽  
Progress Curve ◽  
Steady State Kinetics ◽  
Time Regime ◽  
Time Courses

All of chemical kinetics is based on rate equations, but this is especially true of steady-state enzyme kinetics: in other applications a rate equation can be regarded as a differential equation that has to be integrated to give the function of real interest, whereas in steady-state enzyme kinetics it is used as it stands. Although the early enzymologists tried to follow the usual chemical practice of deriving equations that describe the state of reaction as a function of time there were too many complications, such as loss of enzyme activity, effects of accumulating product etc., for this to be a fruitful approach. Rapid progress only became possible when Michaelis and Menten (1) realized that most of the complications could be removed by extrapolating back to zero time and regarding the measured initial rate as the primary observation. Since then, of course, accumulating knowledge has made it possible to study time courses directly, and this has led to two additional subdisciplines of enzyme kinetics, transient-state kinetics, which deals with the time regime before a steady state is established, and progress-curve analysis, which deals with the slow approach to equilibrium during the steady-state phase. The former of these has achieved great importance but is regarded as more specialized. It is dealt with in later chapters of this book. Progress-curve analysis has never recovered the importance that it had at the beginning of the twentieth century. Nearly all steps that form parts of the mechanisms of enzyme-catalysed reactions involve reactions of a single molecule, in which case they typically follow first-order kinetics: . . . v = ka . . . . . . 1 . . . or they involve two molecules (usually but not necessarily different from one another) and typically follow second-order kinetics: . . . v = kab . . . . . . 2 . . . In both cases v represents the rate of reaction, and a and b are the concentrations of the molecules involved, and k is a rate constant. Because we shall be regarding the rate as a quantity in its own right it is not usual in steady-state kinetics to represent it as a derivative such as -da/dt.

scholarly journals The effect of ethanol on the steady-state kinetics of glutathione S -transferase a from rat liver

FEBS Letters ◽  
1979 ◽  
Vol 102 (1) ◽  
pp. 165-168 ◽  
Author(s):  
Inga Jakobsson ◽  
Margareta Warholm ◽  
Bengt Mannervik
Keyword(s):  
Steady State ◽  
Rat Liver ◽  
Glutathione S Transferase ◽  
Steady State Kinetics ◽  
Kinetics Of

scholarly journals The Rv1712 Locus from Mycobacterium tuberculosis H37Rv Codes for a Functional CMP Kinase That Preferentially Phosphorylates dCMP

2009 ◽  
Vol 191 (8) ◽  
pp. 2884-2887 ◽  
Author(s):  
Caroline Thum ◽  
Cristopher Z. Schneider ◽  
Mario S. Palma ◽  
Diógenes S. Santos ◽  
Luiz A. Basso
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Steady State ◽  
Tuberculosis H37rv ◽  
Mycobacterium Tuberculosis H37rv ◽  
Steady State Kinetics ◽  
Sequential Mechanism

ABSTRACT The Mycobacterium tuberculosis cmk gene, predicted to encode a CMP kinase (CMK), was cloned and expressed, and its product was purified to homogeneity. Steady-state kinetics confirmed that M. tuberculosis CMK is a monomer that preferentially phosphorylates CMP and dCMP by a sequential mechanism. A plausible role for CMK is discussed.

scholarly journals A steady-state kinetic model of butyrylcholinesterase from horse plasma

1974 ◽  
Vol 141 (3) ◽  
pp. 825-834 ◽  
Author(s):  
Klas-Bertil Augustinsson ◽  
Tamas Bartfai ◽  
Bengt Mannervik
Keyword(s):  
Steady State ◽  
Molecular Model ◽  
Rate Equations ◽  
Phenyl Acetate ◽  
Thiol Ester ◽  
Regression Methods ◽  
Steady State Kinetics ◽  
Steady State Kinetic ◽  
Hydrolysis Of ◽  
Single Enzyme

The steady-state kinetics of the butyrylcholinesterase-catalysed hydrolysis of butyrylthiocholine and thiophenyl acetate were shown to deviate from Michaelis–Menten kinetics. The ‘best’ empirical rate law was selected by fitting different rate equations to the experimental data by non-linear regression methods. The results were analysed in view of two alternative interpretations: (1) the reaction is catalysed by a mixture of enzymes, or (2) the activity is due to a single enzyme displaying deviations from Michaelis–Menten kinetics. It was concluded that the second alternative applies, and this conclusion was further supported by experiments involving simultaneous hydrolysis of alternative thiol ester substrates (butyrylthiocholine/thiophenyl acetate) as well as alternative thiol ester and oxygen ester substrates (butyrylthiocholine/phenyl acetate; thiophenyl acetate/butyrylcholine; acetylthiocholine/phenyl acetate). On the basis of the conclusion that a single enzyme is responsible for the activity, a molecular model is proposed. This model involves an acylated enzyme, and implies binding to the enzyme of one acyl group and one ester molecule, but not two ester molecules at the same time. Thus butyrylcholinesterase, which is structurally a tetramer, behaves functionally as a co-operative dimer, an interpretation in accordance with available data from active-site titrations.

Functional and structural roles of the glutathione-binding residues in maize (Zea mays) glutathione S-transferase I

2001 ◽  
Vol 358 (1) ◽  
pp. 101-110 ◽  
Author(s):  
Nikolaos E. LABROU ◽  
Luciane V. MELLO ◽  
Yannis D. CLONIS
Keyword(s):  
Steady State ◽  
Catalytic Mechanism ◽  
Limited Proteolysis ◽  
Glutathione S Transferase ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Steady State Kinetics ◽  
Helical Segment ◽  
Α Helix ◽  
Rate Limiting

The isoenzyme glutathione S-transferase (GST) I from maize (Zea mays) was cloned and expressed in Escherichia coli, and its catalytic mechanism was investigated by site-directed mutagenesis and dynamic studies. The results showed that the enzyme promotes proton dissociation from the GSH thiol and creates a thiolate anion with high nucleophilic reactivity by lowering the pKa of the thiol from 8.7 to 6.2. Steady-state kinetics fit well to a rapid equilibrium, random sequential Bi Bi mechanism, with intrasubunit modulation between the GSH binding site (G-site) and the electrophile binding site (H-site). The rate-limiting step of the reaction is viscosity-dependent, and thermodynamic data suggest that product release is rate-limiting. Five residues of GST I (Ser11, His40, Lys41, Gln53 and Ser67), which are located in the G-site, were individually replaced with alanine and their structural and functional roles in the 1-chloro-2,4-dinitrobenzene (CDNB) conjugation reaction were investigated. On the basis of steady-state kinetics, difference spectroscopy and limited proteolysis studies it is concluded that these residues: (1) contribute to the affinity of the G-site for GSH, as they are involved in side-chain interaction with GSH; (2) influence GSH thiol ionization, and thus its reactivity; (3) participate in kcat regulation by affecting the rate-limiting step of the reaction; and (4) in the cases of His40, Lys41 and Gln53 play an important role in the structural integrity of, and probably in the flexibility of, the highly mobile short 310-helical segment of α-helix 2 (residues 35–46), as shown by limited proteolysis experiments. These structural perturbations are probably transmitted to the H-site through changes in Phe35 conformation. This accounts for the modulation of Kcdnbm by His40, Lys41 and Gln53, and also for the intrasubunit communication between the G- and H-sites. Computer simulations using CONCOORD were applied to maize GST I monomer and dimer structures, each with bound lactoylglutathione, and the results were analysed by the essential dynamics technique. Differences in dynamics were found between the monomer and the dimer simulations showing the importance of using the whole structure in dynamic analysis. The results obtained confirm that the short 310-helical segment of α-helix 2 (residues 35–46) undergoes the most significant structural rearrangements. These rearrangements are discussed in terms of enzyme catalytic mechanism.

scholarly journals Steady-state kinetic analysis of aldehyde dehydrogenase from human erythrocytes

1992 ◽  
Vol 287 (1) ◽  
pp. 145-150 ◽  
Author(s):  
G T M Henehan ◽  
K F Tipton
Keyword(s):  
Steady State ◽  
Aldehyde Dehydrogenase ◽  
Initial Rate ◽  
Substrate Inhibition ◽  
Human Erythrocytes ◽  
Steady State Kinetics ◽  
Steady State Kinetic ◽  
Sequential Mechanism ◽  
Kinetics Of

The steady-state kinetics of purified cytoplasmic aldehyde dehydrogenase (EC 1.2.1.3) from human erythrocytes have been studied at 37 degrees C. Previous studies of the enzyme from several mammalian sources, which used a lower assay temperature, have been difficult to interpret because of the substrate activation by acetaldehyde which led to complex kinetic behaviour. At 37 degrees C the initial-rate data do not depart significantly from Michaelis-Menten kinetics. Studies of the variation of initial rates as a function of the concentrations of both substrates and studies of the inhibition by NADH were consistent with a sequential mechanism being followed. High-substrate inhibition by acetaldehyde was competitive with respect to NAD+. The enzyme was not inhibited by the product acetate and thus the results of these studies, although consistent with an ordered mechanism in which NAD+ was the first substrate to bind, were inconclusive. That such a mechanism was followed was confirmed by determination of the initial-rate behaviour in the presence of acetaldehyde and glycolaldehyde as alternative substrates. When the reciprocal of the initial rate of NADH formation was plotted against the acetaldehyde concentration at a series of fixed ratios between that substrate and glycolaldehyde, a linear ‘mixed inhibition’ pattern was obtained, confirming the mechanism to be ordered with NAD+ being the leading substrate and with kinetically significant ternary complex-formation.

Determination of the steady-state kinetics in a spin-1 Ising model using path probability method

2020 ◽  
Vol 34 (30) ◽  
pp. 2050338 ◽  
Author(s):  
Songül Özüm ◽  
Rıza Erdem
Keyword(s):  
Steady State ◽  
Ising Model ◽  
Linear Equations ◽  
Kinetic Equations ◽  
Phenomenological Approach ◽  
Rate Equations ◽  
Probability Method ◽  
Steady State Kinetics ◽  
Path Probability Method

As a continuation of our previously published work, we propose a theoretical framework for the determination of steady-state kinetics in a spin-1 Ising model by the path probability method. The framework is based on the principles of non-equilibrium statistical physics and is quite different from the phenomenological approach. We construct a set of linear kinetic equations for the order parameters using the non-linear dynamic (or rate) equations in the presence of external magnetic field. From the steady-state solutions of the linear equations, an expression for the complex (or dynamic) magnetic susceptibility [Formula: see text] is derived. The temperature dependence of the magnetic dispersion relation [Formula: see text] and magnetic absorption factor [Formula: see text] has been studied in the ferromagnetic (FM) and paramagnetic (PM) phases as well as near the critical regime.

scholarly journals Wheat-germ aspartate transcarbamoylase. Steady-state kinetics and stereochemistry of the binding site for l-aspartate

1979 ◽  
Vol 183 (2) ◽  
pp. 247-254 ◽  
Author(s):  
J E Grayson ◽  
R J Yon ◽  
P J Butterworth
Keyword(s):  
Steady State ◽  
Binding Site ◽  
Dissociation Constant ◽  
Product Inhibition ◽  
Aspartate Transcarbamoylase ◽  
Carbamoyl Phosphate ◽  
Triticum Vulgare ◽  
Steady State Kinetics ◽  
Dead End ◽  
Sequential Mechanism

1. The steady-state kinetics of the bisubstrate reaction catalysed by aspartate transcarbamoylase purified from wheat (Triticum vulgare)-germ have been studied at 25 degrees C, pH 8.5 AND I 0.10-0.12. Initial-velocity and product-inhibition results are consistent with an ordered sequential mechanism in which carbamoyl phosphate is the first substrate to bind, followed by L-aspartate, and carbamoyl aspartate is the first product to leave, followed by Pi. The order of substrate addition is supported by dead-end inhibition studies using pyrophosphate and maleate as inhibitory analogues of the substrates. Product inhibition permitted a minimum value for the dissociation constant of L-aspartate from the ternary complex to be estimated. This minimum is of the same order as the dissociation constant (Ki) of succinate. 2. A range of dicarboxy analogues of L-aspartate were tested as possible inhibitors of the enzyme. These studies suggested that L-aspartate is bound with its carboxy groups in the eclipsed configuration, and that the stereochemical constraints around the binding site are very similar to those reported for the catalytic subunit of the enzyme from Escherichia coli [Davies, Vanaman & Stark (1970) J. Biol. Chem. 245, 1175-1179].

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