scholarly journals Isocitrate lyase from Phycomyces blakesleeanus. The role of Mg2+ ions, kinetics and evidence for two classes of modifiable thiol groups

1990 ◽  
Vol 272 (2) ◽  
pp. 359-367 ◽  
Author(s):  
J Rúa ◽  
D de Arriaga ◽  
F Busto ◽  
J Soler
Keyword(s):  
Enzyme Inhibitor ◽  
Isocitrate Lyase ◽  
Equilibrium Constants ◽  
Spectrophotometric Titration ◽  
Kinetic Mechanism ◽  
Thiol Groups ◽  
Phycomyces Blakesleeanus ◽  
Denatured State ◽  
Nitrobenzoic Acid ◽  
Dead End

Isocitrate lyase was purified from Phycomyces blakesleeanus N.R.R.L. 1555(-). The native enzyme has an Mr of 240,000. The enzyme appeared to be a tetramer with apparently identical subunits of Mr 62,000. The enzyme requires Mg2+ for activity, and the data suggest that the Mg2(+)-isocitrate complex is the true substrate and that Mg2+ ions act as a non-essential activator. The kinetic mechanism of the enzyme was investigated by using product and dead-end inhibitors of the cleavage and condensation reactions. The data indicated an ordered Uni Bi mechanism and the kinetic constants of the model were calculated. The spectrophotometric titration of thiol groups in Phycomyces isocitrate lyase with 5.5′-dithiobis-(2-nitrobenzoic acid) gave two free thiol groups per subunit of enzyme in the native state and three in the denatured state. The isocitrate lyase was completely inactivated by iodoacetate, with non-linear kinetics. The inactivation data suggest that the enzyme has two classes of modifiable thiol groups. The results are also in accord with the formation of a non-covalent enzyme-inhibitor complex before irreversible modification of the enzyme. Both the equilibrium constants for formation of the complex and the first-order rate constants for the irreversible modification step were determined. The partial protective effect of isocitrate and Mg2+ against iodoacetate inactivation was investigated in a preliminary form.

scholarly journals Kinetic studies of rat liver hexokinase D (glucokinase) in non-co-operative conditions show an ordered mechanism with MgADP as the last product to be released

2003 ◽  
Vol 371 (1) ◽  
pp. 29-38 ◽  
Author(s):  
Octavio MONASTERIO ◽  
María Luz CÁRDENAS
Keyword(s):  
Rat Liver ◽  
Kinetic Studies ◽  
Kinetic Mechanism ◽  
Product Inhibition ◽  
Nitrobenzoic Acid ◽  
Binary Complexes ◽  
Dead End ◽  
Straight Line ◽  
Inactivation Constant ◽  
Reciprocal Value

The kinetic mechanism of rat liver hexokinase D ('glucokinase') was studied under non-co-operative conditions with 2-deoxyglucose as substrate, chosen to avoid uncertainties derived from the co-operativity observed with the physiological substrate, glucose. The enzyme shows hyperbolic kinetics with respect to both 2-deoxyglucose and MgATP2-, and the reaction follows a ternary-complex mechanism with Km = 19.2±2.3mM for 2-deoxyglucose and 0.56±0.05mM for MgATP2-. Product inhibition by MgADP- was mixed with respect to MgATP2- and was largely competitive with respect to 2-deoxyglucose, suggesting an ordered mechanism with 2-deoxyglucose as first substrate and MgADP- as last product. Dead-end inhibition by N-acetylglucosamine, AMP and the inert complex CrATP [the complex of ATP with chromium in the 3+ oxidation state, i.e. Cr(III)—ATP], studied with respect to both substrates, also supports an ordered mechanism with 2-deoxyglucose as first substrate. AMP appears to bind both to the free enzyme and to the E·dGlc complex. Experiments involving protection against inactivation by 5,5′-dithiobis-(2-nitrobenzoic acid) support the existence of the E·MgADP- and E·AMP complexes suggested by the kinetic studies. MgADP-, AMP, 2-deoxyglucose, glucose and mannose were strong protectors, supporting the existence of binary complexes with the enzyme. Glucose 6-phosphate failed to protect, even at concentrations as high as 100mM, and MgATP2- protected only slightly (12%). The inactivation results support the postulated ordered mechanism with 2-deoxyglucose as first substrate and MgADP- as last product. In addition, the straight-line dependence observed when the reciprocal value of the inactivation constant was plotted against the sugar-ligand concentration supports the view that there is just one sugar-binding site in hexokinase D.

A Novel Synthetic Approach to Asymmetric Salen, Dihydrosalen, and Tetrahydrosalen Ligands: Structures and O2-Activating Properties of their Nickel(II) and Cobalt(II) Complexes

1994 ◽  
Vol 49 (8) ◽  
pp. 1089-1100 ◽  
Author(s):  
Arnd Böttcher ◽  
Horst Elias ◽  
Brigitte Eisenmann ◽  
Elke Hilms ◽  
Andreas Huber ◽  
...  
Keyword(s):  
Equilibrium Constants ◽  
Spectrophotometric Titration ◽  
Acetone Solution ◽  
Synthetic Approach ◽  
Coordination Core ◽  
Type Complex ◽  
Square Planar ◽  
Synthetic Procedure ◽  
Derivatives Of ◽  
Methyl Phenyl

A synthetic procedure is described for the preparation of the tetradentate N2O2 ligands H2[H4]L1=6-(2-hydroxyphenyl)-2,5-diaza-3,3-dimethyl-1-(2-hydroxy-3-t-butyl-5-methyl- phenyl)heptane, H2[H2]L1 = 6-(2-hydroxyphenyl)-2,5-diaza-3,3-dimethyl-1-(2-hydroxy-3-t-butyl-5-methylphenyl)-1-heptene, and H2L1 = 6-(2-hydroxyphenyl)-2,5-diaza-3,3-dimethyl-1-(2- hydroxy-3-t-butyl-5-methylphenyl)-1,5-heptadiene, which are asymmetric derivatives of the ligands tetrahydrosalen, dihydrosalen, and salen. Complexes Ni[H4]L1, Ni[H2]L1, NiL1, CoL1, Ni[H4]L2 (([H4]L2)2- = anion of H2[H4]L2 = N,N'-bis(2-hydroxy-3-t-butyl-5-methylbenzyl)- trans-(S,S)-1,2-diaminocyclohexane), and NiL2 were prepared, characterized (VIS and IR ab­sorption, magnetic moment) and subjected to spectrophotometric titration with pyridine, to determine the equilibrium constants for adduct formation. Single crystal X-ray structure analy­ses were carried out for Ni[H2]L1 (monoclinic, P21/n; a = 8.926(4), b = 29.324(7), c = 8.411(4) Å; β = 95.3(1)°; Z = 4), CoL1 (monoclinic, C2/c; a = 25.389(2), b = 18.139(2), c = 10.179(1) Å; β = 112.227(6); Z = 8), and Ni[H4]L2·acetone (tetragonal, P41212; a = 13.928(3), c = 33.698(5) Å; Z = 8). In all of the three complexes, the N2O2-metal coordination core is square-planar. The skeleton of the tetradentate ligand is more or less twisted. The planar cobalt(II) complex CoL1 is a low spin d7 system with μexp = 2.02 BM at 298 K, whereas the planar complexes NiL1, Ni[H2]L1 and Ni[H4]L2 are diamagnetic (μexp = 0.28-0.64 BM). The blue solvate Ni[H4]L1- 3EtOH · H2O is paramagnetic (μexp = 3.04 BM), which points to octahedral coordination. In aerated acetone solution, the tetrahydrosalen-type complex Ni[H4]L1 activates dioxygen and one C-N bond is oxidatively dehydrogenated. The VIS spectrum of the dihydrosalen-type complex formed is virtually identical with that of the aldimine complex Ni[H2]L1. In the pres­ence of dioxygen, Co[H4]L1 and Co[H2]L1 are readily oxidized to CoL1 in acetone solution.

scholarly journals Purification and regulatory properties of isocitrate lyase from Escherichia coli ML308

1988 ◽  
Vol 250 (1) ◽  
pp. 25-31 ◽  
Author(s):  
C MacKintosh ◽  
H G Nimmo
Keyword(s):  
Escherichia Coli ◽  
Isocitrate Lyase ◽  
Random Order ◽  
Kinetic Mechanism ◽  
Competitive Inhibitor ◽  
Intact Cells ◽  
Competing Anions ◽  
Effects Of Ph ◽  
Order Of Magnitude

Isocitrate lyase was purified to homogeneity from Escherichia coli ML308. Its subunit Mr and native Mr were 44,670 +/- 460 and 17,000-180,000 respectively. The kinetic mechanism of the enzyme was investigated by using product and dead-end inhibitors of the cleavage and condensation reactions. The data indicated a random-order equilibrium mechanism, with formation of a ternary enzyme-isocitrate-succinate complex. In an attempt to predict the properties of isocitrate lyase in intact cells, the effects of pH, inorganic anions and potential regulatory metabolites on the enzyme were studied. The Km of the enzyme for isocitrate was 63 microM at physiological pH and in the absence of competing anions. Chloride, phosphate and sulphate ions inhibited competitively with respect to isocitrate. Phosphoenolpyruvate inhibited non-competitively with respect to isocitrate, but the Ki value suggested that this effect was unlikely to be significant in intact cells. 3-Phosphoglycerate was a competitive inhibitor. At the concentration reported to occur in intact cells, this metabolite would have a significant effect on the activity of isocitrate lyase. The available data suggest that the Km of isocitrate lyase for isocitrate is similar to the concentration of isocitrate in E. coli cells growing on acetate, about one order of magnitude higher than the Km determined in vitro in the absence of competing anions.

scholarly journals Isomerization of an enzyme-coenzyme complex in yeast alcohol dehydrogenase-catalysed reactions

2003 ◽  
Vol 68 (2) ◽  
pp. 77-84 ◽  
Author(s):  
Vladimir Leskovac ◽  
Svetlana Trivic ◽  
Draginja Pericin
Keyword(s):  
Alcohol Dehydrogenase ◽  
Rate Constants ◽  
Equilibrium Constants ◽  
Kinetic Mechanism ◽  
Yeast Alcohol Dehydrogenase ◽  
Catalyzed Reactions ◽  
The Individual ◽  
Individual Rate ◽  
Catalyzed Oxidation

In this work, all the rate constants in the kinetic mechanism of the yeast alcohol dehydrogenase-catalyzed oxidation of ethanol by NAD+, at pH 7.0, 25 ?C, have been estimated. The determination of the individual rate constants was achieved by fitting the reaction progress curves to the experimental data, using the procedures of the FITSIM and KINSIM software package of Carl Frieden. This work is the first report in the literature showing the internal equilibrium constants for the isomerization of the enzyme-NAD+ complex in yeast alcohol dehydrogenase-catalyzed reactions.

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.

Fungal trehalose phosphorylase: kinetic mechanism, pH-dependence of the reaction and some structural properties of the enzyme from Schizophyllum commune

2001 ◽  
Vol 356 (3) ◽  
pp. 757-767 ◽  
Author(s):  
Christian EIS ◽  
Mark WATKINS ◽  
Thomas PROHASKA ◽  
Bernd NIDETZKY
Keyword(s):  
Inductively Coupled Plasma ◽  
Metal Ion ◽  
Ph Dependence ◽  
Schizophyllum Commune ◽  
Structural Similarity ◽  
Kinetic Mechanism ◽  
Inductively Coupled ◽  
Peptide Mass ◽  
Dead End ◽  
Trehalose Phosphorylase

Initial-velocity measurements for the phospholysis and synthesis of α,α-trehalose catalysed by trehalose phosphorylase from Schizophyllum commune and product and dead-end inhibitor studies show that this enzyme has an ordered Bi Bi kinetic mechanism, in which phosphate binds before α,α-trehalose, and α-d-glucose is released before α-d-glucose 1-phosphate. The free-energy profile for the enzymic reaction at physiological reactant concentrations displays its largest barriers for steps involved in reverse glucosyl transfer to d-glucose, and reveals the direction of phospholysis to be favoured thermodynamically. The pH dependence of kinetic parameters for all substrates and the dissociation constant of d-glucal, a competitive dead-end inhibitor against d-glucose (Ki = 0.3mM at pH6.6 and 30°C), were determined. Maximum velocities and catalytic efficiencies for the forward and reverse reactions decrease at high and low pH, giving apparent pK values of 7.2–7.8 and 5.5–6.0 for two groups whose correct protonation state is required for catalysis. The pH dependences of kcat/K are interpreted in terms of monoanionic phosphate and α-d-glucose 1-phosphate being the substrates, and of the pK value seen at high pH corresponding to the phosphate group in solution or bound to the enzyme. The Ki value for the inhibitor decreases outside the optimum pH range for catalysis, indicating that binding of d-glucal is tighter with incorrectly ionized forms of the complex between the enzyme and α-d-glucose 1-phosphate. Each molecule of trehalose phosphorylase contains one Mg2+ that is non-dissociable in the presence of metal chelators. Measurements of the 26Mg2+/24Mg2+ ratio in the solvent and on the enzyme by using inductively coupled plasma MS show that exchange of metal ion between protein and solution does not occur at measurable rates. Tryptic peptide mass mapping reveals close structural similarity between trehalose phosphorylases from basidiomycete fungi.

scholarly journals Active-site modification of native and mutant forms of inosine 5′-monophosphate dehydrogenase from Escherichia coli K12

1980 ◽  
Vol 191 (2) ◽  
pp. 533-541 ◽  
Author(s):  
Harry J. Gilbert ◽  
William T. Drabble
Keyword(s):  
Escherichia Coli ◽  
Binding Site ◽  
Enzyme Inactivation ◽  
Enzyme Hydrolysis ◽  
Thiol Groups ◽  
Imp Dehydrogenase ◽  
Nitrobenzoic Acid ◽  
Saturation Kinetics ◽  
Mutant Forms ◽  
Hydrolysis Of

IMP dehydrogenase of Escherichia coli was irreversibly inactivated by Cl-IMP (6-chloro-9-β-d-ribofuranosylpurine 5′-phosphate, 6-chloropurine ribotide). The inactivation reaction showed saturation kinetics. 6-Chloropurine riboside did not inactivate the enzyme. Inactivation by Cl-IMP was retarded by ligands that bind at the IMP-binding site. Their effectiveness was IMP>XMP>GMP»AMP. NAD+ did not protect the enzyme from modification. Inactivation of IMP dehydrogenase was accompanied by a change in λmax. of Cl-IMP from 263 to 290nm, indicating formation of a 6-alkylmercaptopurine nucleotide. The spectrum of 6-chloropurine riboside was not changed by IMP dehydrogenase. With excess Cl-IMP the increase in A290 with time was first-order. Thus it appears that Cl-IMP reacts with only one species of thiol at the IMP-binding site of the enzyme: 2–3mol of Cl-IMP were bound per mol of IMP dehydrogenase tetramer. Of ten mutant enzymes from guaB strains, six reacted with Cl-IMP at a rate similar to that for the native enzyme. The interaction was retarded by IMP. None of the mutant enzymes reacted with 6-chloropurine riboside. 5,5′-Dithiobis-(2-nitrobenzoic acid), iodoacetate, iodoacetamide and methyl methanethiosulphonate also inactivated IMP dehydrogenase. Reduced glutathione re-activated the methanethiolated enzyme, and 2-mercaptoethanol re-activated the enzyme modified by Cl-IMP. IMP did not affect the rate of re-activation of methanethiolated enzyme. Protective modification indicates that Cl-IMP, methyl methanethiosulphonate and iodoacetamide react with the same thiol groups in the enzyme. This is also suggested by the low incorporation of iodo[14C]acetamide into Cl-IMP-modified enzyme. Hydrolysis of enzyme inactivated by iodo[14C]acetamide revealed radioactivity only in S-carboxymethylcysteine. The use of Cl-IMP as a probe for the IMP-binding site of enzymes from guaB mutants is discussed, together with the possible function of the essential thiol groups.

scholarly journals The binding and catalytic activities of forms of ligandin after modification of its thiol groups

1979 ◽  
Vol 177 (2) ◽  
pp. 433-439 ◽  
Author(s):  
T Carne ◽  
E Tipping ◽  
B Ketterer
Keyword(s):  
Thiol Group ◽  
Performic Acid ◽  
Acid Oxidation ◽  
Thiol Groups ◽  
Glutathione S Transferase ◽  
Catalytic Activities ◽  
Nitrobenzoic Acid ◽  
Number Of Binding Sites ◽  
The Common ◽  
Hydrophobic Ligand

Ligandin (glutathione S-transferase B, EC 2.5.1.18)was treated with p-mercuribenzoate, N-(4-dimethylamino-3,5-dinitrophenyl)-maleimide, 5,5,-dithiobis-(2-nitrobenzoic acid), N-ethylmaleimide, iodoacetamide or iodoacetate. Although performic acid oxidation revealed the presence of four cysteines, p-mercuribenzoate and N-(4-dimethylamino-3,5-dinitrophenyl)maleimide, the most effective of the reagents studied, reacted with only three residues. N-Ethylmaleimide and 5,5′-dithiobis-(2-nitrobenzoic acid) each reacted with two cysteines: iodoacetamide reacted with only one cysteine and iodoacetate was essentially unreactive. Modification of three thiol groups decreased both the enzymic and binding activities of ligandin although the number of binding sites was unaffected. Modification of only one or two of the thiol groups had little effect on the ligandin activities. It therefore appears that there is a thiol group in the common hydrophobic-ligand- and substrate-binding site of ligandin. Ligandin was separated into two fractions on CM-cellulose. Both fractions gave the same results with p-mercuribenzoate and iodoacetamide.

Kinetic mechanism of a recombinant Arabidopsis glyoxylate reductase: studies of initial velocity, dead-end inhibition and product inhibition

2007 ◽  
Vol 85 (9) ◽  
pp. 896-902 ◽  
Author(s):  
Gordon J. Hoover ◽  
Gerald A. Prentice ◽  
A. Rod Merrill ◽  
Barry J. Shelp
Keyword(s):  
Initial Velocity ◽  
Kinetic Mechanism ◽  
Competitive Inhibitor ◽  
Product Inhibition ◽  
Succinic Semialdehyde ◽  
In Planta ◽  
Dead End ◽  
Arabidopsis Protein ◽  
Enzyme Functions ◽  
Glyoxylate Reductase

Kinetic analysis of substrate specificity revealed that a recombinant Arabidopsis protein catalyzes the conversion of glyoxylate to glycolate (Km,glyoxylate = 4.5 μmol·L–1) and succinic semialdehyde (SSA) to γ-hydroxybutyrate (Km, SSA = 0.87 mmol·L–1) via an essentially irreversible, NADPH-based mechanism. In this report, the enzyme was further characterized via initial-velocity, dead-end inhibition and product inhibition studies. The kinetic mechanism was ordered Bi Bi, involving the complexation of NADPH to the enzyme before glyoxylate or SSA, and the release of NADP+ before glycolate or γ-hydroxybutyrate, respectively. It can be concluded that the enzyme functions as a NADPH-dependent glyoxylate reductase (EC 1.1.1.79) or possibly an aldehyde reductase (EC 1.1.1.2), and the kinetic mechanism involved is consistent with that found in members of both the aldo-keto reductase and 3-hydroxyisobutyrate dehydrogenase-related superfamilies of enzymes. Since NADP+ was an effective competitive inhibitor with respect to NADPH (Ki = 1–3 µmol·L–1), it is proposed that the ratio of NADPH/NADP+ regulates enzymatic activity in planta.

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