scholarly journals Determination of the mechanism and kinetic constants for hog kidney γ-glutamyltransferase

1976 ◽  
Vol 157 (3) ◽  
pp. 609-617 ◽  
Author(s):  
J W London ◽  
L M Shaw ◽  
D Fetterolf ◽  
D Garfinkel
Keyword(s):  
Initial Velocity ◽  
Competitive Inhibition ◽  
Linear Regression Analysis ◽  
Kinetic Constants ◽  
Reaction Products ◽  
Gamma Glutamyl ◽  
Ping Pong ◽  
Donor Acceptor ◽  
Michaelis Constants ◽  
Hog Kidney

The initial-velocity kinetics of hog kidney gamma-glutamyltransferase were studied. Glutamate gamma-(4-nitroanilide) and its 3-carboxy derivative, glutamate gamma-(3-carboxy-4-nitroanilide), served as gamma-glutamyl donors, and glycylglycine as an acceptor. Reaction products were identified by paper chromatography and amino acid analysis. Inhibited Ping Pong mechanisms and a comprehensive initial- velocity expression were developed which account for the observed simultaneous gamma-glutamyl transfer and autotransfer, competitive inhibition by glycylglycine, and non-competitive inhibition by the carboxy donor. The validity of the proposed Ping Pong mechanisms are supported by enzyme-velocity data obtained with constant ratios of acceptor to donor concentrations. Kinetic constants were determined by a non-linear regression analysis. With glutamate gamma-(4-nitroanilide) as the donor, Michaelis constants for the donor, acceptor and donor-acting-as-acceptor are 1.87, 24.9, and 2.08 mM respectively. With glutamate gamma-(3-carboxy-4-nitroanilide) as the donor, these Michaelis constants are 1.63, 16.6, and 12.3 mM. Glyclyglycine competitive inhibition constants with the parent donor and its carboxy derivative are 275 and 205 mM respectively; the non-competitive inhibition constant of the carboxy donor is 34 mM.

The Kinetics of Inhibition of the Action of Thrombin by the Fibrinopeptides Formed during the Clotting of Fibrinogen

1966 ◽  
Vol 16 (01/02) ◽  
pp. 277-295 ◽  
Author(s):  
A Silver ◽  
M Murray
Keyword(s):  
Amino Acids ◽  
Competitive Inhibition ◽  
Amorphous Material ◽  
Peptide Bond ◽  
Initial Reaction ◽  
Reaction Products ◽  
Coagulation Mechanism ◽  
Kinetics Of ◽  
Kinetics Of Inhibition ◽  
Burk Plot

SummaryVarious investigators have separated the coagulation products formed when fibrinogen is clotted with thrombin and identified fibrinopeptides A and B. Two other peaks are observed in the chromatogram of the products of coagulation, but these have mostly been dismissed by other workers. They have been identified by us as amino acids, smaller peptides and amorphous material (37). We have re-chromatographed these peaks and identified several amino acids. In a closed system of fibrinogen and thrombin, the only reaction products should be fibrin and peptide A and peptide B. This reasoning has come about because thrombin has been reported to be specific for the glycyl-arginyl peptide bond. It is suggested that thrombin also breaks other peptide linkages and the Peptide A and Peptide B are attacked by thrombin to yield proteolytic products. Thrombin is therefore probably not specific for the glycyl-arginyl bond but will react on other linkages as well.If the aforementioned is correct then the fibrinopeptides A and B would cause an inhibition with the coagulation mechanism itself. We have shown that an inhibition does occur. We suggest that there is an autoinhibition to the clotting mechanism that might be a control mechanism in the human body.The experiment was designed for coagulation to occur under controlled conditions of temperature and time. Purified reactants were used. We assembled an apparatus to record visually the speed of the initial reaction, the rate of the reaction, and the density of the final clot formed after a specific time.The figures we derived made available to us data whereby we could calculate and plot the information to show the mechanism and suggest that such an inhibition does exist and also further suggest that it might be competitive.In order to prove true competitive inhibition it is necessary to fulfill the criteria of the Lineweaver-Burk plot. This has been done. We have also satisfied other criteria of Dixon (29) and Bergman (31) that suggest true competitive inhibition.

scholarly journals Aspartate aminotransferase. The effects of ionic concentration on kinetic constants of both isoenzymes

1968 ◽  
Vol 106 (3) ◽  
pp. 581-586 ◽  
Author(s):  
T. R. C. Boyde
Keyword(s):  
Aspartate Aminotransferase ◽  
Specific Effect ◽  
Ionic Concentration ◽  
Phosphate Concentration ◽  
Kinetic Constants ◽  
Sharp Minimum ◽  
Ionic Concentrations ◽  
Michaelis Constants ◽  
Added Salt

1. The Michaelis constants for both isoenzymes for both substrates depend strongly on ionic concentration, being approximately proportional to phosphate concentration over considerable ranges. This is probably an effect of anions only. 2. In the absence of added salt, Km (2-oxoglutarate) (anionic isoenzyme) is so small as to be indeterminate. 3. Km (l-aspartate) (anionic isoenzyme) passes through a sharp minimum at about 3·3mm-phosphate. It is not clear whether this is a specific effect of phosphate. 4. Both substrates are inhibitory at sufficiently low ionic concentrations. 5. A modified graphical procedure is described for the derivation of the kinetic constants.

scholarly journals Kinetic mechanism of the dimeric ATP sulfurylase from plants

2013 ◽  
Vol 33 (4) ◽  
Author(s):  
Geoffrey E. Ravilious ◽  
Jonathan Herrmann ◽  
Soon Goo Lee ◽  
Corey S. Westfall ◽  
Joseph M. Jez
Keyword(s):  
Initial Velocity ◽  
Competitive Inhibition ◽  
Tight Binding ◽  
Atp Synthesis ◽  
Kinetic Mechanism ◽  
Titration Calorimetry ◽  
Atp Sulfurylase ◽  
Dead End ◽  
Sequential Mechanism ◽  
Enzyme Functions

In plants, sulfur must be obtained from the environment and assimilated into usable forms for metabolism. ATP sulfurylase catalyses the thermodynamically unfavourable formation of a mixed phosphosulfate anhydride in APS (adenosine 5′-phosphosulfate) from ATP and sulfate as the first committed step of sulfur assimilation in plants. In contrast to the multi-functional, allosterically regulated ATP sulfurylases from bacteria, fungi and mammals, the plant enzyme functions as a mono-functional, non-allosteric homodimer. Owing to these differences, here we examine the kinetic mechanism of soybean ATP sulfurylase [GmATPS1 (Glycine max (soybean) ATP sulfurylase isoform 1)]. For the forward reaction (APS synthesis), initial velocity methods indicate a single-displacement mechanism. Dead-end inhibition studies with chlorate showed competitive inhibition versus sulfate and non-competitive inhibition versus APS. Initial velocity studies of the reverse reaction (ATP synthesis) demonstrate a sequential mechanism with global fitting analysis suggesting an ordered binding of substrates. ITC (isothermal titration calorimetry) showed tight binding of APS to GmATPS1. In contrast, binding of PPi (pyrophosphate) to GmATPS1 was not detected, although titration of the E•APS complex with PPi in the absence of magnesium displayed ternary complex formation. These results suggest a kinetic mechanism in which ATP and APS are the first substrates bound in the forward and reverse reactions, respectively.

Gamma-glutamyltransferase: Substrate inhibition, kinetic mechanism, and assay conditions.

1976 ◽  
Vol 22 (4) ◽  
pp. 417-421 ◽  
Author(s):  
J H Stromme ◽  
L Theodorsen
Keyword(s):  
Clinical Chemistry ◽  
Substrate Inhibition ◽  
Kinetic Mechanism ◽  
Reaction Conditions ◽  
Gamma Glutamyltransferase ◽  
Gamma Glutamyl ◽  
Dead End ◽  
Ping Pong ◽  
Competitive Substrate ◽  
Assay Conditions

Abstract Gamma-glutamyltransferase activity in serum is shown to be competitively inhibited by the two substrates gamma-glutamyl-4-nitroanilide and glycylglycine. Awareness of this is of importance when one is choosing final reaction conditions for the assay of the enzyme. Gamma-glutamyltransferase probably acts by a "ping-pong bi-bi" kinetic mechanism, which fits with the double competitive substrate inhibition demonstrated. The product, 4-nitro-aniline, appears to be an uncompetitive dead-end inhibitor of both substrates. Various amino acids, particularly glycine and L-alanine, inhibit the enzyme. Their inhibition patterns are uncompetitive with glycylglycine and competitive with gamma-glutamyl-4-nitroanilide. On the basis of the present and other studies, the Scandinavian Society for Clinical Chemistry and Clinical Physiology is going to recommend for routine use a gamma-glutamyltransferase method in which the final concentrations of gamma-glutamyl-4-nitroanilide and glycylglycine are 4 and 75 mmol/liter, respectively.

scholarly journals Estimating the Product Inhibition Constant from Enzyme Kinetic Equations Using the Direct Linear Plot Method in One-Stage Treatment

Catalysts ◽  
2020 ◽  
Vol 10 (8) ◽  
pp. 853
Author(s):  
Pedro L. Valencia ◽  
Bastián Sepúlveda ◽  
Diego Gajardo ◽  
Carolina Astudillo-Castro
Keyword(s):  
Least Squares Method ◽  
Competitive Inhibition ◽  
Kinetic Equations ◽  
Initial Reaction Rate ◽  
Product Inhibition ◽  
Kinetic Constants ◽  
Initial Reaction ◽  
Mathematical Treatment ◽  
One Stage ◽  
Linear Plot

A direct linear plot was applied to estimate kinetic constants using the product’s competitive inhibition equation. The challenge consisted of estimating three kinetic constants, Vmax, Km, and Kp, using two independent variables, substrates, and product concentrations, in just one stage of mathematical treatment. The method consisted of combining three initial reaction rate data and avoiding the use of the same three product concentrations (otherwise, this would result in a mathematical indetermination). The direct linear plot method was highly superior to the least-squares method in terms of accuracy and robustness, even under the addition of error. The direct linear plot method is a reliable and robust method that can be applied to estimate Kp in inhibition studies in pharmaceutical and biotechnological areas.

scholarly journals Steady-state kinetics of malonyl-CoA synthetase from Bradyrhizobium japonicum and evidence for malonyl-AMP formation in the reaction

1994 ◽  
Vol 297 (2) ◽  
pp. 327-333 ◽  
Author(s):  
Y S Kim ◽  
S W Kang
Keyword(s):  
Steady State ◽  
Bradyrhizobium Japonicum ◽  
Initial Velocity ◽  
Catalytic Mechanism ◽  
Kinetic Mechanism ◽  
Product Inhibition ◽  
Steady State Kinetics ◽  
Malonyl Coa ◽  
Michaelis Constants ◽  
Inhibition Studies

Malonyl-CoA synthetase catalyses the formation of malonyl-CoA directly from malonate and CoA with hydrolysis of ATP into AMP and PP1. The catalytic mechanism of malonyl-CoA synthetase from Bradyrhizobium japonicum was investigated by steady-state kinetics. Initial-velocity studies and the product-inhibition studies with AMP and PPi strongly suggested ordered Bi Uni Uni Bi Ping Pong Ter Ter system as the most probable steady-state kinetic mechanism of malonyl-CoA synthetase. Michaelis constants were 61 microM, 260 microM and 42 microM for ATP, malonate and CoA respectively, and the value for Vmax, was 11.2 microM/min. The t.l.c. analysis of the 32P-labelled products in a reaction mixture containing [gamma-32P]ATP in the absence of CoA showed that PPi was produced after the sequential addition of ATP and malonate. Formation of malonyl-AMP, suggested as an intermediate in the kinetically deduced mechanism, was confirmed by the analysis of 31P-n.m.r. spectra of an AMP product isolated from the 18O-transfer experiment using [18O]malonate. The 31P-n.m.r. signal of the AMP product appeared at 0.024 p.p.m. apart from that of [16O4]AMP, indicating that one atom of 18O transferred from [18O]malonate to AMP through the formation of malonyl-AMP. Formation of malonyl-AMP was also confirmed through the t.l.c. analysis of reaction mixture containing [alpha-32P]ATP. These results strongly support the ordered Bi Uni Uni Bi Pin Pong Ter Ter mechanism deduced from initial-velocity and product-inhibition studies.

gamma-Glutamyltransferase in human serum: an analysis of kinetic models.

1981 ◽  
Vol 27 (2) ◽  
pp. 303-307 ◽  
Author(s):  
H E Solberg ◽  
L Theodorsen ◽  
J H Strømme
Keyword(s):  
Kinetic Models ◽  
Competitive Inhibition ◽  
Acceptor Site ◽  
Velocity Measurements ◽  
Gamma Glutamyltransferase ◽  
Donor Substrate ◽  
Ping Pong ◽  
Decreasing Ph ◽  
Best Fit ◽  
Assay Conditions

Abstract The purpose of the study was to elucidate details of the kinetic model for gamma-glutamyltransferase when assayed with gamma-glutamyl-3-carboxy-4-nitroanilide and glycylglycine as substrates. Data from several sets of initial velocity measurements were fitted by nonlinear regression to a set of different kinetic models. gamma-Glutamyltransferase acts by a "ping-pong bi-bi" mechanism. A model encompassing transfer and autotransfer, competitive inhibition by the acceptor substrate, and no inhibition by the donor substrate gives the best fit to the experimental data. Effects of spectrophotometric nonlinearity may simulate noncompetitive inhibition by the donor substrate. The nonlinearity is dependent on the absorption of the incubation mixture and therefore is related to the concentration of the donor substrate and the wavelength (405-412 nm) used to monitor the reaction. With decreasing pH the autotransfer fraction decreases and the binding of the donor substrate to the acceptor site increases, simulating an increased competitive inhibition by the donor substrate. These results are of importance when elaborating optimum assay conditions for gamma-glutamyltransferase in serum.

scholarly journals Kinetic properties of spermine synthase from bovine brain

1983 ◽  
Vol 215 (3) ◽  
pp. 669-676 ◽  
Author(s):  
R L Pajula
Keyword(s):  
Initial Velocity ◽  
Substrate Inhibition ◽  
Product Inhibition ◽  
Bovine Brain ◽  
Kinetic Properties ◽  
Methylthioadenosine Phosphorylase ◽  
Spermine Synthase ◽  
Michaelis Constants ◽  
Inhibition Studies ◽  
Competitive Product

A kinetic analysis including initial-velocity and product-inhibition studies were performed with spermine synthase purified from bovine brain. The enzyme activity was assayed in the presence of 5′-methylthioadenosine phosphorylase as an auxiliary enzyme to prevent the accumulation of the inhibitory product, 5′-methylthioadenosine, and thus to obtain linearity of the reaction with time. Initial-velocity studies gave intersecting or converging linear double-reciprocal plots. No substrate inhibition by decarboxylated S-adenosylmethionine was observed at concentrations up to 0.4 mM. Apparent Michaelis constants were 60 microM for spermidine and 0.1 microM for decarboxylated S-adenosylmethionine. Spermine was a competitive product inhibitor with respect to decarboxylated S-adenosylmethionine, but a mixed one with respect to the other substrate, spermidine. 5′-Methylthioadenosine showed a mixed inhibition with both substrates, predominantly competitive with respect to decarboxylated S-adenosylmethionine and predominantly uncompetitive with respect to spermidine. The observed kinetic and inhibition patterns are consistent with a compulsory-order mechanism, where both substrates add to the enzyme before products can be released.

scholarly journals Properties and mechanism of action of pyruvate, phosphate dikinase from leaves

1969 ◽  
Vol 114 (1) ◽  
pp. 117-125 ◽  
Author(s):  
T. J. Andrews ◽  
M. D. Hatch
Keyword(s):  
Mechanism Of Action ◽  
Sugar Cane ◽  
Initial Velocity ◽  
Isotope Exchange ◽  
Reverse Direction ◽  
Pyruvate Phosphate Dikinase ◽  
Reaction Proceeds ◽  
Michaelis Constants ◽  
Phosphate Groups ◽  
Mechanism Of The Reaction

1. Sugar-cane leaf pyruvate,Pi dikinase was prepared free of enzymes that would interfere with studies on the stoicheiometry and mechanism of the reaction it catalyses. The reaction was unequivocally shown to involve the conversion of equimolar amounts of pyruvate, ATP and Pi into phosphoenolpyruvate, AMP and PPi. 2. The purified enzyme was stable at pH8·3 only if stored at about 20° in the presence of Mg2+ and a thiol-reducing reagent, care being taken to prevent the oxidation of the thiol. 3. The apparent Michaelis constants for phosphoenolpyruvate and PPi were 0·11mm and 0·04mm respectively and that for AMP was less than 4μm. 4. At pH8·3 the initial velocity of the reaction was about 6 times as fast in the direction towards phosphoenolpyruvate synthesis as in the reverse direction. 5. With the exception of ATP, all the products of the reaction in both directions were inhibitory. 6. The phosphate groups of PPi were derived from Pi and from the terminal phosphate of ATP. 7. Isotope-exchange studies indicated that the reaction proceeds in the following steps: Enzyme+ATP+Pi ⇌ Enzyme–P+AMP+PPi Enzyme–P+pyruvate ⇌ Enzyme+phosphoenolpyruvate

Kinetic studies of mutant human erythrocyte adenine phosphoribosyltransferases

1968 ◽  
Vol 46 (7) ◽  
pp. 703-706 ◽  
Author(s):  
J. Frank Henderson ◽  
Helen R. Miller ◽  
William N. Kelley ◽  
Frederick M. Rosenbloom ◽  
J. Edwin Seegmiller
Keyword(s):  
Reaction Mechanism ◽  
Human Erythrocyte ◽  
Enzyme Activities ◽  
Kinetic Studies ◽  
Kinetic Constants ◽  
Heat Inactivation ◽  
Adenine Phosphoribosyltransferase ◽  
Michaelis Constants ◽  
Low Degrees ◽  
Reduced Activity

The kinetic constants and reaction mechanism of human erythrocyte adenine phosphoribosyltransferase from individuals whose enzyme activities have high, intermediate, or low degrees of stability to heat inactivation, and in a human mutant with reduced activity of this enzyme, have been measured. The Michaelis constants for one or both substrates are different from normal in seven mutants.

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