Kinetics of the reaction catalyzed by inositol dehydrogenase from Bacillus subtilis and inhibition by fluorinated substrate analogs

2006 ◽  
Vol 84 (4) ◽  
pp. 522-527 ◽  
Author(s):  
Richard Daniellou ◽  
Hongyan Zheng ◽  
David RJ Palmer
Keyword(s):  
Bacillus Subtilis ◽  
Competitive Inhibitor ◽  
Enzyme Mechanism ◽  
Substrate Analogs ◽  
Kinetic Isotope ◽  
Steady State Kinetic ◽  
Dehydrogenase Enzyme ◽  
Key Enzyme ◽  
Inositol Dehydrogenase ◽  
Kinetics Of

Inositol dehydrogenase (EC 1.1.1.18) from Bacillus subtilis catalyzes the oxidation of myo-inositol to scyllo-inosose by transfer of the equatorial hydride of the substrate to NAD+. This is a key enzyme in the metabolism of myo-inositol, a primary carbon source for soil bacteria. In light of our recent discovery that the enzyme has a broad substrate spectrum while maintaining high stereoselectivity, we seek a more thorough understanding of the enzyme and its active site. We have examined the kinetics of the recombinant enzyme, and synthesized fluorinated substrate analogues as competitive inhibitors. We have evaluated all rate constants in the ordered, sequential Bi Bi mechanism. No steady-state kinetic isotope effect is observed using myo-[2-2H]-inositol, indicating that the chemical step of the reaction is not rate-limiting. We have synthesized the substrate analogs 2-deoxy-2-fluoro-myo-inositol, its equatorial analog 1-deoxy-1-fluoro-scyllo-inositol, the gem-difluorinated analog 1-deoxy-1,1-difluoro-scyllo-inositol, and the sugar analog α-D-glucosyl fluoride. Of these, 1-deoxy-1-fluoro-scyllo-inositol showed no inhibition, while all others tested had Ki values comparable to the Km values of the analogous substrates myo-inositol and α-D-glucose.Key words: inositol dehydrogenase, enzyme mechanism, kinetics, competitive inhibitor, substrate analogue.

scholarly journals Kinetics of nitrogenase of Klebsiella pneumoniae. Heterotropic interactions between magnesium-adenosine 5′-diphosphate and magnesium-adenosine 5′-triphosphate

1977 ◽  
Vol 165 (2) ◽  
pp. 255-262 ◽  
Author(s):  
R N F Thorneley ◽  
A Cornish-Bowden
Keyword(s):  
Electron Transfer ◽  
Steady State ◽  
Protein A ◽  
Competitive Inhibitor ◽  
Electron Transfer Reaction ◽  
H2 Evolution ◽  
Fe Protein ◽  
Steady State Kinetics ◽  
Steady State Kinetic ◽  
Kinetics Of

The effects of MgADP and MgATP on the kinetics of a pre-steady-state electron-transfer reaction and on the steady-state kinetics of H2 evulution for nitrogenase proteins of K. pneumoniae were studied. MgADP was a competitive inhibitor of MgATP in the MgATP-induced electron transfer from the Fe-protein to the Mo-Fe-protein. A dissociation constant K′i = 20 micron was determined for MgADP. The release of MgADP or a coupled conformation change in the Fe-protein of K.pneumoniae occurred with a rate comparable with that of electron transfer, k approximately 2 × 10(2)S-1. Neither homotropic nor heterotropic interactions involving MgATP and MgADP were observed for this reaction. Steady-state kinetic data for H2 evolution exhibited heterotropic effects between MgADP and MgATP. The data have been fitted to symmetry and sequential-type models involving conformation changes in two identical subunits. The data suggest that the enzyme can bind up to molecules of either MgATP or MgADP, but is unable to bind both nucleotides simultaneously. The control of H2 evolution by the MgATP/MgADP ratio is not at the level of electron transfer between the Fe- and Mo-Fe-proteins.

scholarly journals The mechanism of action of β-galactosidase. Effect of aglycone nature and α-deuterium substitution on the hydrolysis of aryl galactosides

1973 ◽  
Vol 133 (1) ◽  
pp. 89-98 ◽  
Author(s):  
Michael L. Sinnott ◽  
Ian J. L. Souchard
Keyword(s):  
Steady State ◽  
Isotope Effects ◽  
Ion Pair ◽  
Enzyme Mechanism ◽  
Kinetic Isotope ◽  
Deuterium Substitution ◽  
Steady State Kinetic ◽  
Hydrolysis Of ◽  
State Kinetic ◽  
Step Mechanism

1. Steady-state kinetic parameters for the β-galactosidase-catalysed hydrolysis of 13 aryl β-d-galactopyranosides show no simple dependence on aglycone acidity. 2. α-Deuterium kinetic isotope effects (kH/kD) for seven of these substrates, measured under steady-state conditions with [S]»Km, vary from 1.00 for poor substrates to 1.25 for hydrolysis of the galactosyl-enzyme. 3. Methanolysis of the galactosyl-enzyme in 1.5m-methanol increases KH/kD for degalactosylation, but leaves that for hydrolysis of ‘slow’ substrates unchanged. 4. These data are incompatible with a simple two-step mechanism. A scheme consisting of a conformation change, liberation of a galactopyranosyl cation in an intimate ion-pair, non-productive but preferential collapse of the ion-pair to a covalent species and reaction of the galactosyl enzyme through the ion-paired form is proposed. 5. This scheme is used to rationalize previously puzzling data about the enzyme mechanism.

Kinetic Aspects of the Interaction of Blood Clotting Enzymes

1968 ◽  
Vol 19 (03/04) ◽  
pp. 364-367 ◽  
Author(s):  
H. C Hemker ◽  
P. W Hemker
Keyword(s):  
Enzyme Kinetics ◽  
Blood Clotting ◽  
Competitive Inhibition ◽  
Competitive Inhibitor ◽  
Kinetics Of

SummaryThe enzyme kinetics of competitive inhibition under conditions prevailing in clotting tests are developed and a method is given to measure relative amounts of a competitive inhibitor by means of the t — D plot.

Cyclization and acid-catalyzed hydrolysis of O-benzoylbenzamidoximes

1986 ◽  
Vol 51 (12) ◽  
pp. 2786-2797
Author(s):  
František Grambal ◽  
Jan Lasovský
Keyword(s):  
Reaction Rate ◽  
Kinetic Isotope ◽  
Acid Base Equilibrium ◽  
Mineral Acids ◽  
Kinetics Of Formation ◽  
Acid Catalyzed ◽  
Ph Scale ◽  
Kinetics Of ◽  
Hydrolysis Of ◽  
Derivatives Of

Kinetics of formation of 1,2,4-oxadiazoles from 24 substitution derivatives of O-benzoylbenzamidoxime have been studied in sulphuric acid and aqueous ethanol media. It has been found that this medium requires introduction of the Hammett H0 function instead of the pH scale beginning as low as from 0.1% solutions of mineral acids. Effects of the acid concentration, ionic strength, and temperature on the reaction rate and on the kinetic isotope effect have been followed. From these dependences and from polar effects of substituents it was concluded that along with the cyclization to 1,2,4-oxadiazoles there proceeds hydrolysis to benzamidoxime and benzoic acid. The reaction is thermodynamically controlled by the acid-base equilibrium of the O-benzylated benzamidoximes.

scholarly journals Recognition of hydrogen isotopomers by an open-cage fullerene

2013 ◽  
Vol 371 (1998) ◽  
pp. 20110629 ◽  
Author(s):  
Yasujiro Murata ◽  
Shih-Ching Chuang ◽  
Fumiyuki Tanabe ◽  
Michihisa Murata ◽  
Koichi Komatsu
Keyword(s):  
Equilibrium Constants ◽  
Equilibrium Mixture ◽  
Size Difference ◽  
Binding Affinities ◽  
Kinetic Isotope ◽  
Molecular Sizes ◽  
Lower Temperature ◽  
Kinetics Of ◽  
Pressure Hydrogen

We present our study on the recognition of hydrogen isotopes by an open-cage fullerene through determination of binding affinity of isotopes H 2 /HD/D 2 with the open-cage fullerene and comparison of their relative molecular sizes through kinetic-isotope-release experiments. We took advantage of isotope H 2 /D 2 exchange that generated an equilibrium mixture of H 2 /HD/D 2 in a stainless steel autoclave to conduct high-pressure hydrogen insertion into an open-cage fullerene. The equilibrium constants of three isotopes with the open-cage fullerene were determined at various pressures and temperatures. Our results show a higher equilibrium constant for HD into open-cage fullerene than the other two isotopomers, which is consistent with its dipolar nature. D 2 molecule generally binds stronger than H 2 because of its heavier mass; however, the affinity for H 2 becomes larger than D 2 at lower temperature, when size effect becomes dominant. We further investigated the kinetics of H 2 /HD/D 2 release from open-cage fullerene, proving their relative escaping rates. D 2 was found to be the smallest and H 2 the largest molecule. This notion has not only supported the observed inversion of relative binding affinities between H 2 and D 2 , but also demonstrated that comparison of size difference of single molecules through non-convalent kinetic-isotope effect was applicable.

scholarly journals Kinetic Analysis of tRNA-Directed Transcription Antitermination of the Bacillus subtilis glyQS Gene In Vitro

2004 ◽  
Vol 186 (16) ◽  
pp. 5392-5399 ◽  
Author(s):  
Frank J. Grundy ◽  
Tina M. Henkin
Keyword(s):  
Bacillus Subtilis ◽  
Assay System ◽  
Nascent Transcript ◽  
Vitro Assay ◽  
T Box ◽  
Transcription Antitermination ◽  
Transcriptional Pausing ◽  
Transcription Complexes ◽  
Kinetics Of

ABSTRACT Binding of uncharged tRNA to the nascent transcript promotes readthrough of a leader region transcription termination signal in genes regulated by the T box transcription antitermination mechanism. Each gene in the T box family responds independently to its cognate tRNA, with specificity determined by base pairing of the tRNA to the leader at the anticodon and acceptor ends of the tRNA. tRNA binding stabilizes an antiterminator element in the transcript that sequesters sequences that participate in formation of the terminator helix. tRNAGly-dependent antitermination of the Bacillus subtilis glyQS leader was previously demonstrated in a purified in vitro assay system. This assay system was used to investigate the kinetics of transcription through the glyQS leader and the effect of tRNA and transcription elongation factors NusA and NusG on transcriptional pausing and antitermination. Several pause sites, including a major site in the loop of stem III of the leader, were identified, and the effect of modulation of pausing on antitermination efficiency was analyzed. We found that addition of tRNAGly can promote antitermination as long as the tRNA is added before the majority of the transcription complexes reach the termination site, and variations in pausing affect the requirements for timing of tRNA addition.

scholarly journals Interaction of difluoro-oxaloacetate with aspartate transaminase

1977 ◽  
Vol 161 (2) ◽  
pp. 383-387 ◽  
Author(s):  
P A Briley ◽  
R Eisenthal ◽  
R Harrison ◽  
G D Smith
Keyword(s):  
Steady State ◽  
Competitive Inhibitor ◽  
Aspartate Transaminase ◽  
Steady State Kinetic ◽  
High Concentrations ◽  
State Kinetic ◽  
Uncompetitive Inhibitor

Diffluoro-oxaloacetate behaves as a competitive inhibitor of 2-oxoglutarate and as an uncompetitive inhibitor with respect to aspartate in steady-state kinetic experiments with cytoplasmic aspartate transaminase. In the presence of high concentrations of aspartate transaminase, difluoro-oxaloacetate is slowly transaminated to difluoro-aspartate, suggesting its use as a kinetic probe to study the reactions of the aminic form of the enzyme.

α-Retaining glucosyl transfer catalysed by trehalose phosphorylase from Schizophyllum commune: mechanistic evidence obtained from steady-state kinetic studies with substrate analogues and inhibitors

2001 ◽  
Vol 360 (3) ◽  
pp. 727-736 ◽  
Author(s):  
Bernd NIDETZKY ◽  
Christian EIS
Keyword(s):  
Steady State ◽  
Transition State ◽  
Schizophyllum Commune ◽  
Catalytic Efficiency ◽  
Kinetic Studies ◽  
Competitive Inhibitor ◽  
Chemical Mechanism ◽  
Steady State Kinetic ◽  
State Kinetic ◽  
Trehalose Phosphorylase

Fungal trehalose phosphorylase is classified as a family 4 glucosyltransferase that catalyses the reversible phosphorolysis of α,α-trehalose with net retention of anomeric configuration. Glucosyl transfer to and from phosphate takes place by the partly rate-limiting interconversion of ternary enzyme–substrate complexes formed from binary enzyme–phosphate and enzyme–α-d-glucopyranosyl phosphate adducts respectively. To advance a model of the chemical mechanism of trehalose phosphorylase, we performed a steady-state kinetic study with the purified enzyme from the basidiomycete fungus Schizophyllum commune by using alternative substrates, inhibitors and combinations thereof in pairs as specific probes of substrate-binding recognition and transition-state structure. Orthovanadate is a competitive inhibitor against phosphate and α-d-glucopyranosyl phosphate, and binds 3×104-fold tighter (Ki≈ 1μM) than phosphate. Structural alterations of d-glucose at C-2 and O-5 are tolerated by the enzyme at subsite +1. They lead to parallel effects of approximately the same magnitude (slope = 1.14; r2 = 0.98) on the reciprocal catalytic efficiency for reverse glucosyl transfer [log (Km/kcat)] and the apparent affinity of orthovanadate determined in the presence of the respective glucosyl acceptor (log Ki). An adduct of orthovanadate and the nucleophile/leaving group bound at subsite +1 is therefore the true inhibitor and displays partial transition state analogy. Isofagomine binds to subsite −1 in the enzyme–phosphate complex with a dissociation constant of 56μM and inhibits trehalose phosphorylase at least 20-fold better than 1-deoxynojirimycin. The specificity of the reversible azasugars inhibitors would be explained if a positive charge developed on C-1 rather than O-5 in the proposed glucosyl cation-like transition state of the reaction. The results are discussed in the context of α-retaining glucosyltransferase mechanisms that occur with and without a β-glucosyl enzyme intermediate.

scholarly journals Mitochondrial adenosine triphosphatase from human placenta--inhibition by free magnesium ions of ITP hydrolysis.

1994 ◽  
Vol 41 (1) ◽  
pp. 39-44 ◽  
Author(s):  
Z Aleksandrowicz
Keyword(s):  
Competitive Inhibition ◽  
Competitive Inhibitor ◽  
Hill Coefficient ◽  
Negative Cooperativity ◽  
Magnesium Ions ◽  
Beef Liver ◽  
Bicarbonate Ions ◽  
The Hill ◽  
Kinetics Of ◽  
Free Magnesium

The effects of Mg2+ and bicarbonate on the kinetics of ITP hydrolysis by soluble ATPase (F1) from human placental mitochondria were studied. Increasing amounts of Mg2+ at fixed ITP concentration, caused a marked activation of F1 followed by inhibition at higher Mg2+ concentration. The appropriate substrate for the mitochondrial F1 seems to be the MgITP complex as almost no ITP was hydrolysed in the absence of magnesium. Mg2+ behaved as a competitive inhibitor towards the MgITP complex. In this respect the human placental enzyme differ from that from other sources such as yeast, beef liver or rat liver. The linearity of the plot presenting competitive inhibition by free Mg2+ of MgITP hydrolysis (in the presence of activating bicarbonate anion) suggests that both Mg2+ and MgITP bind to the same catalytic site (Km(MgITP) = 0.46 mM, Ki(Mg) = 4 mM). When bicarbonate was absent in the ITPase assay, placental F1 exhibited apparent negative cooperativity in the presence of 5 mM Mg2+, just as it did with MgATP as a substrate under similar conditions. Bicarbonate ions eliminated the negative cooperativity with respect to ITP (as the Hill coefficient of 0.46 was brought to approx. 1), and thus limited inhibition by free Mg2+. The results presented suggest that the concentration of free magnesium ions may be an important regulatory factor of the human placental F1 activity.

Sign in / Sign up

Export Citation Format

Share Document