scholarly journals Kinetic studies of the active sites functioning in the quinohemoprotein fructose dehydrogenase

FEBS Letters ◽  
1993 ◽  
Vol 318 (1) ◽  
pp. 23-26 ◽  
Author(s):  
Jovita Marcinkeviciene ◽  
Gillis Johansson
Keyword(s):  
Active Sites ◽  
Kinetic Studies ◽  
Fructose Dehydrogenase

Hydroxylation of C–H bonds at carboxylate-bridged diiron centres

2005 ◽  
Vol 363 (1829) ◽  
pp. 861-877 ◽  
Author(s):  
Stephen J Lippard
Keyword(s):  
Active Sites ◽  
Bond Activation ◽  
Kinetic Studies ◽  
Methyl Radical ◽  
Α Subunit ◽  
Model Complexes ◽  
Soluble Methane Monooxygenase ◽  
Related Enzyme ◽  
High Valent ◽  
Rate Limiting

Nature uses carboxylate-bridged diiron centres at the active sites of enzymes that catalyse the selective hydroxylation of hydrocarbons to alcohols. The resting diiron(III) state of the hydroxylase component of soluble methane monooxygenase enzyme is converted by two-electron transfer from an NADH-requiring reductase into the active diiron(II) form, which subsequently reacts with O 2 to generate a high-valent diiron(IV) oxo species (Q) that converts CH 4 into CH 3 OH. In this step, C–H bond activation is achieved through a transition state having a linear C⋯H⋯O unit involving a bound methyl radical. Kinetic studies of the reaction of Q with substrates CH 3 X, where X=H, D, CH 3 , NO 2 , CN or OH, reveal two classes of reactivity depending upon whether binding to the enzyme or C–H bond activation is rate-limiting. Access of substrates to the carboxylate-bridged diiron active site in the hydroxylase (MMOH) occurs through a series of hydrophobic pockets. In the hydroxylase component of the closely related enzyme toluene/ o -xylene monooxygenase (ToMOH), substrates enter through a wide channel in the α-subunit of the protein that tracks a course identical to that found in the structurally homologous MMOH. Synthetic models for the carboxylate-bridged diiron centres in MMOH and ToMOH have been prepared that reproduce the stoichiometry and key geometric and physical properties of the reduced and oxidized forms of the proteins. Reactions of the diiron(II) model complexes with dioxygen similarly generate reactive intermediates, including high-valent species capable not only of hydroxylating pendant C–H bonds but also of oxidizing phosphine and sulphide groups.

scholarly journals Rate-determining processes and the number of simultaneously active sites of d-glyceraldehyde 3-phosphate dehydrogenase

1971 ◽  
Vol 122 (1) ◽  
pp. 71-77 ◽  
Author(s):  
D. R. Trentham
Keyword(s):  
Oxidative Phosphorylation ◽  
Active Sites ◽  
Dissociation Constants ◽  
Kinetic Studies ◽  
Competitive Inhibitor ◽  
Low Ph ◽  
Conformation Change ◽  
High Ph ◽  
Rate Determining Step ◽  
Transient Experiments

Transient kinetic studies of the reversible oxidative phosphorylation of d-glyceraldehyde 3-phosphate catalysed by d-glyceraldehyde 3-phosphate dehydrogenase show that all four sites of the tetrameric lobster enzyme are simultaneously active, apparently with equal reactivity. The rate-determining step of the oxidative phosphorylation is NADH release at high pH and phosphorolysis of the acyl-enzyme at low pH. For the reverse reaction the rate-determining step is a process associated with NADH binding, probably a conformation change, at high pH and d-glyceraldehyde 3-phosphate release at low pH. NADH has previously been shown to be a competitive inhibitor of the enzyme with respect to d-glyceraldehyde 3-phosphate and vice versa. This is consistent with the mechanism deduced from transient experiments given the additional proviso that 1-arseno-3-phosphoglycerate has a half-life of about 1min or longer at pH7. The dissociation constants of d-glyceraldehyde 3-phosphate and 1,3-diphosphoglycerate to the NAD+-bound enzyme are too large to measure but are nevertheless consistent with the low Km values of these substrates.

scholarly journals Nanostructured faujasite zeolite as metal ion adsorbent: kinetics, equilibrium adsorption and metal recovery studies

2020 ◽  
Author(s):  
Mariana B. Goncalves ◽  
Djanyna V. C. Schmidt ◽  
Fabiana S. dos Santos ◽  
Daniel F. Cipriano ◽  
Gustavo R. Gonçalves ◽  
...  
Keyword(s):  
Ion Exchange ◽  
Metal Ions ◽  
Metal Ion ◽  
Active Sites ◽  
Metal Removal ◽  
High Surface ◽  
High Surface Area ◽  
Kinetic Studies ◽  
Crystallization Time ◽  
X Ray

Abstract The hydrothermal synthesis of nano-faujasite has been successfully performed and the effects of some crystallization parameters were investigated, along with the use of this material as a heavy-metal ion adsorbent. X-ray diffraction patterns have shown that the structure of the nano-faujasite is strongly dependent on both the crystallization time and the alkalinity of the synthesis medium. According to N2 physisorption, X-ray fluorescence, SEM/EDS, and solid state 29Si and 27Al NMR data, the produced nano-faujasite consists of a solid with low molar Si/Al ratio (1.7), with high availability of ion exchange sites and high surface area/small particle size, allowing easy diffusion of metal ions to adsorbent active sites. As a consequence, an excellent performance on removal of Cd2+, Zn2+ and Cu2+ ions was found for this solid. The adsorption capacity followed the order Cd2+ (133 mg·g−1) > Zn2+ (115 mg·g−1) > Cu2+ (99 mg·g−1), which agrees with the order of increasing absolute values of the hydration energy of the metal ions. Kinetic studies and adsorption isotherms showed that the metal ion removal takes place by ion exchange on the monolayer surface of the nano-faujasite. The electrochemical recovery of copper in metallic form exhibited an efficiency of 80.2% after 120 min, which suggests that this process can be adequately implemented for full-scale metal removal.

The Effects of Substrate Concentration on the Mg-Adenosine Triphosphatase Activity of Myosin

1975 ◽  
Vol 53 (12) ◽  
pp. 1282-1287 ◽  
Author(s):  
T. Nihei ◽  
C. A. Filipenko
Keyword(s):  
Steady State ◽  
Substrate Concentration ◽  
Active Sites ◽  
Adenosine Triphosphatase ◽  
Kinetic Studies ◽  
Heavy Meromyosin ◽  
Adenosine Triphosphatase Activity ◽  
Steady State Rate ◽  
State Rate ◽  
Substrate Concentrations

Using myosin, heavy meromyosin, and subfragment-1 the steady state rate of Mg-modified adenosine triphosphatase (Mg-ATPase) was determined over a range of substrate concentrations between 10−8 M and 5 × 10−3 M, at 0.5 M and 0.05 M KCl (pH 7.4 at 20 °C). At the substrate concentrations below 10−5 M, myosin Mg-ATPase was observed to show that two active sites interact, as suggested by the analysis of transient kinetic studies (Walz, F. G., Jr.: J. Theor. Biol. 41, 357–373 (1973)). The increase in the activity at Mg-ATP concentrations higher than 10−4 M corresponds to the binding of Mg-ATP to myosin sites not responsible for the catalytic action. With heavy meromyosin and subfragment-1, the activity was best expressed by the Michaelis equation. With heavy meromyosin, the activation at high ATP concentrations is detectable, though not as pronounced as with myosin, but not with subfragment-1.

scholarly journals Kinetic Studies of the Glycerolysis of Urea to Glycerol Carbonate in the Presence of Amberlyst-15 as Catalyst

2021 ◽  
Vol 16 (1) ◽  
pp. 52-62
Author(s):  
Hary Sulistyo ◽  
Wahyudi Budi Sediawan ◽  
Reviana Inda Dwi Suyatno ◽  
Indah Hartati
Keyword(s):  
Kinetic Model ◽  
Active Sites ◽  
Outer Part ◽  
Kinetic Studies ◽  
Order Reaction ◽  
Second Order ◽  
Glycerol Carbonate ◽  
Glycerol Concentration ◽  
Second Order Reaction ◽  
Amberlyst 15

Amberlyst-15, a strong acidic ion-exchange resin, has showed as a potential and an effective catalyst for the glycerolysis process of urea to glycerol carbonate. In this work, the kinetic model of the urea glycerolysis over Amberlyst-15 catalyst was investigated. The kinetic model was developed by considering simultaneous steps of urea dissolution in glycerol, mass transfer of urea and glycerol from the bulk of the liquid into the outer part of the catalyst, diffusion of urea and glycerol into the inner part of the particle through the catalyst pores, and irreversible second order reaction of urea and glycerol on the active sites. The irreversibility of second order reaction of urea glycerolysis was validated and proven. The proposed kinetic model was simulated and validated with the experimental data. The kinetic studies show that mechanism proposed works well. Furthermore, the activation energy was found to be 145.58 kJ.mol−1 and the collision factor was in 8.00×1010 (m3)2.kg−1.mol−1.s−1. The simulation result shows that the predicted liquid temperatures were close to the experimental temperature data. It also gave glycerol concentration profile inside the catalyst particle as a function of glycerolysis time and position. Copyright © 2021 by Authors, Published by BCREC Group. This is an open access article under the CC BY-SA License (https://creativecommons.org/licenses/by-sa/4.0). 

The oxyanion hole of Pseudomonas fluorescens mannitol 2-dehydrogenase: a novel structural motif for electrostatic stabilization in alcohol dehydrogenase active sites

2009 ◽  
Vol 425 (2) ◽  
pp. 455-463 ◽  
Author(s):  
Mario Klimacek ◽  
Bernd Nidetzky
Keyword(s):  
Pseudomonas Fluorescens ◽  
Serine Proteases ◽  
Active Sites ◽  
Hydride Transfer ◽  
Kinetic Studies ◽  
Chemical Conversion ◽  
Single Site ◽  
Structural Motif ◽  
Oxyanion Hole ◽  
Electrostatic Stabilization

The side chains of Asn191 and Asn300 constitute a characteristic structural motif of the active site of Pseudomonas fluorescens mannitol 2-dehydrogenase that lacks precedent in known alcohol dehydrogenases and resembles the canonical oxyanion binding pocket of serine proteases. We have used steady-state and transient kinetic studies of the effects of varied pH and deuterium isotopic substitutions in substrates and solvent on the enzymatic rates to delineate catalytic consequences resulting from individual and combined replacements of the two asparagine residues by alanine. The rate constants for the overall hydride transfer to and from C-2 of mannitol, which were estimated as ~ 5×102 s−1 and ~ 1.5×103 s−1 in the wild-type enzyme respectively, were selectively slowed, between 540- and 2700-fold, in single-site mannitol 2-dehydrogenase mutants. These effects were additive in the corresponding doubly mutated enzyme, suggesting independent functioning of the two asparagine residues in catalysis. Partial disruption of the oxyanion hole in single-site mutants caused an upshift, by ≥1.2 pH units, in the kinetic pK of the catalytic acid-base Lys295 in the enzyme–NAD+–mannitol complex. The oxyanion hole of mannitol 2-dehydrogenase is suggested to drive a precatalytic conformational equilibrium at the ternary complex level in which the reactive group of the substrate is ‘activated’ for chemical conversion through its precise alignment with the unprotonated side chain of Lys295 (mannitol oxidation) and C=O bond polarization by the carboxamide moieties of Asn191 and Asn300 (fructose reduction). In the subsequent hydride transfer step, the two asparagine residues provide ~ 40 kJ/mol of electrostatic stabilization.

scholarly journals Rat small-intestinal β-galactosidases. Kinetic studies with three separated fractions

1968 ◽  
Vol 110 (1) ◽  
pp. 143-150 ◽  
Author(s):  
Nils-Georg Asp ◽  
Arne Dahlqvist
Keyword(s):  
Active Sites ◽  
Kinetic Studies ◽  
Competitive Inhibitor ◽  
Small Intestinal Mucosa ◽  
Lactase Activity ◽  
Freezing And Thawing ◽  
Ph Optimum ◽  
Measurable Rate ◽  
Small Intestinal ◽  
Hydrolysis Of

1. Three fractions of β-galactosidase activity from the rat small-intestinal mucosa were separated chromatographically. Two of these fractions had an acid pH optimum at 3–4, and the third one had a more neutral pH optimum at 5·7. 2. The two ‘acid’ β-galactosidase fractions had considerably lower Km values for hetero β-galactosides than for lactose. The Vmax. values were similar for all the substrates used (lactose, phenyl β-galactoside, o-nitrophenyl β-galactoside, p-nitrophenyl β-galactoside and 6-bromo-2-naphthyl β-galactoside). No difference could be detected between the two ‘acid’ fractions with respect to their enzymic properties (pH optimum, Km for the different substrates, Ki for lactose as an inhibitor of the hydrolysis of hetero β-galactosides, Ki for phenyl β-galactoside as an inhibitor of the hydrolysis of lactose, and relative Vmax. for the hydrolysis of different substrates). These two fractions probably represent different forms of the same enzyme. 3. The ‘neutral’ fraction had similar Km values for all the substrates hydrolysed, but with lactose as substrate the Vmax. was much higher than with the hetero β-galactosides. This fraction did not split phenyl β-galactoside or 6-bromo-2-naphthyl β-galactoside at a measurable rate. 4. Lactose was a competitive inhibitor of the hetero β-galactosidase activities of all the three fractions, and Ki for lactose as an inhibitor in each case was the same as Km for the lactase activity. Phenyl β-galactoside was a competitive inhibitor of the lactase activity of all the three fractions. These facts strongly indicate that in all the three fractions lactose is hydrolysed by the same active sites as the hetero β-galactosides. 5. Human serum albumin stabilized the separated enzymes against inactivation by freezing and thawing.

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