scholarly journals Binding energy and catalysis. Fluorinated and deoxygenated glycosides as mechanistic probes of Escherichia coli (lacZ) β-galactosidase

1992 ◽  
Vol 286 (3) ◽  
pp. 721-727 ◽  
Author(s):  
J D McCarter ◽  
M J Adam ◽  
S G Withers
Keyword(s):  
Escherichia Coli ◽  
Transition State ◽  
Kinetic Parameters ◽  
Parent Compound ◽  
Order Rate Constant ◽  
Linear Free Energy Relationship ◽  
Electronic Effects ◽  
Linear Free Energy ◽  
Covalent Intermediate ◽  
Hydrolysis Of

Kinetic parameters for the hydrolysis of a series of deoxy and deoxyfluoro analogues of 2′,4′-dinitrophenyl beta-D-galactopyranoside by Escherichia coli (lacZ) beta-galactosidase have been determined and rates found to be two to nine orders of magnitude lower than that for the parent compound. These large rate reductions result primarily from the loss of transition-state binding interactions due to the replacement of sugar hydroxy groups, and such interactions are estimated to contribute at least 16.7 kJ (4 kcal).mol-1 to binding at the 3, 4 and 6 positions and more than 33.5 kJ (8 kcal).mol-1 at the 2 position. The existence of a linear free-energy relationship between log(kcat./Km) for these compounds and the logarithm of the first-order rate constant for their spontaneous hydrolysis demonstrates that electronic effects are also important and provides direct evidence for oxocarbonium ion character in the enzymic transition state. A covalent intermediate which turns over only extremely slowly (t1/2 = 45 h) accumulates during hydrolysis of the 2-deoxyfluorogalactoside, and kinetic parameters for its formation have been determined. This intermediate is nonetheless catalytically competent, since it re-activates much more rapidly in the presence of the transglycosylation acceptors methanol or glucose, thereby providing support for the notion of a covalent intermediate during hydrolysis of the parent substrates.

scholarly journals Determination of the Mechanism of Nucleophilic Reaction of Fenitrothion Using Substituted Phenoxide Nucleophiles in Aqueous Media

2021 ◽  
pp. 40-46
Author(s):  
E. G. Amadi ◽  
C. I. Egwuatu ◽  
C. U. Okoro ◽  
F. O. Obumselu ◽  
M. U. Onuoha
Keyword(s):  
Transition State ◽  
Rate Constant ◽  
Aqueous Media ◽  
Order Rate Constant ◽  
Second Order ◽  
Bond Rupture ◽  
Linear Free Energy Relationship ◽  
Order Rate ◽  
Linear Free Energy ◽  
Nucleophilic Displacement

The mechanism of the nucleophilic displacement reaction at the phosphorus centre of organophosphates was determined. Phenoxide nucleophiles were reacted with fenitrothion (O,O-dimethyl O-(3-methyl-4-nitrophenyl) phosphorothioate) in water at 25oC and pseudo-first order rate constant measurements taken. Second-order rate constant (kNuc) was determined for the different concentrations of nucleophiles while the second-order rate constant (klg) for the investigation of 2,4-dichlorophenoxide ion with and series of aryl phosphorothioate esters was also determined. Linear free energy relationship was further determined using the Brϕnsted-type plot. The plots are linear over a range of pKaNuc of 7.15-11.10 that straddles the pKa of the leaving 3-methyl-4-nitrophenoxide ion (pKa = 7.20) with statistically acceptable linear correlations (R2 = 0.987) and (R2 = 0.980). The linearity in the traditional Brϕnsted-type plots shows the sensitivity of the nucleophilic displacement to the basicity of the nucleophiles and hence is consistent with a single transition-state mechanism whose barrier to decomposition is low hence concerted. Analysis of the values of βNuc, βLg and βeq (0.734) with the effective charge distribution in the transition state shows that it has no positive character. The Leffler index presents bond formation being slightly ahead of bond rupture.

scholarly journals Kinetics of alkoxysilanes hydrolysis: An empirical approach

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Ahmed A. Issa ◽  
Marwa El-Azazy ◽  
Adriaan S. Luyt
Keyword(s):  
Reaction Mechanisms ◽  
Hydrolysis Reaction ◽  
Hydrolysis Rate ◽  
Coupling Agents ◽  
Linear Free Energy Relationship ◽  
Acidic Media ◽  
Linear Free Energy ◽  
Primary Materials ◽  
Kinetics Of ◽  
Hydrolysis Of

AbstractAlkoxysilanes and organoalkoxysilanes are primary materials in several industries, e.g. coating, anti-corrosion treatment, fabrication of stationary phase for chromatography, and coupling agents. The hydrolytic polycondensation reactions and final product can be controlled by adjusting the hydrolysis reaction, which was investigated under a variety of conditions, such as different alkoxysilanes, solvents, and catalysts by using gas chromatography. The hydrolysis rate of alkoxysilanes shows a dependence on the alkoxysilane structure (especially the organic attachments), solvent properties, and the catalyst dissociation constant and solubility. Some of the alkoxysilanes exhibit intramolecular catalysis. Hydrogen bonding plays an important role in the enhancement of the hydrolysis reaction, as well as the dipole moment of the alkoxysilanes, especially in acetonitrile. There is a relationship between the experimentally calculated polarity by the Taft equation and the reactivity, but it shows different responses depending on the solvent. It was found that negative and positive charges are respectively accumulated in the transition state in alkaline and acidic media. The reaction mechanisms are somewhat different from those previously suggested. Finally, it was found that enthalpy–entropy compensation (EEC) effect and isokinetic relationships (IKR) are exhibited during the hydrolysis of CTES in different solvents and catalysts; therefore, the reaction has a linear free energy relationship (LFER).

scholarly journals Overproduction and Biochemical Characterization of the Chryseobacterium meningosepticum BlaB Metallo-β-Lactamase

2002 ◽  
Vol 46 (6) ◽  
pp. 1921-1927 ◽  
Author(s):  
Sandrine Vessillier ◽  
Jean-Denis Docquier ◽  
Sandrine Rival ◽  
Jean-Marie Frere ◽  
Moreno Galleni ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Ion Exchange ◽  
Kinetic Parameters ◽  
Culture Supernatant ◽  
Biochemical Characterization ◽  
Expression System ◽  
T7 Promoter ◽  
Hydrolysis Of

ABSTRACT The BlaB metallo-β-lactamase of Chryseobacterium meningosepticum CCUG4310 was overproduced in Escherichia coli by means of a T7 promoter-based expression system. The overproducing system, scaled up in a 15-liter fermentor, yielded approximately 10 mg of BlaB protein per liter, mostly released in the culture supernatant. The enzyme was purified by two ion-exchange chromatographic steps with an overall yield of 66%. Analysis of the kinetic parameters revealed efficient activities (k cat/Km ratios of >106 M−1 s−1) toward most penam and carbapenem compounds, with the exception of the 6-α-methoxypenam derivative temocillin and of biapenem, which were poorer substrates. Hydrolysis of cephalosporins was overall less efficient, with a remarkable variability that was largely due to variable affinities of the BlaB enzyme for different compounds. BlaB was also able to hydrolyze serine-β-lactamase inhibitors, including β-iodopenicillanate, sulbactam and, although less efficiently, tazobactam.

Hydrolysis of the thorium(IV) ion in sodium nitrate medium

1982 ◽  
Vol 60 (11) ◽  
pp. 1298-1303 ◽  
Author(s):  
Nikola B. Milić ◽  
Terezija M. Šuranji
Keyword(s):  
Sodium Nitrate ◽  
Metal Ion ◽  
Linear Free Energy Relationship ◽  
Emf Method ◽  
Nitrate Medium ◽  
Linear Free Energy ◽  
Metal Ion Hydrolysis ◽  
Stability Constants Of Complexes ◽  
The Stability ◽  
Hydrolysis Of

The hydrolysis of the thorium(IV) ion in sodium nitrate medium was studied by the emf method at 25 °C. The data show that the extent of the thorium hydrolysis depends both on the concentration of thorium and the sodium nitrate medium. Thus, at a definite pH, the extent of the hydrolysis of thorium increases with increasing its concentration, but decreases with increasing concentration and the hydration energy of the medium ions. The stability constants of complexes Th2(OH)26+, Th3(OH)57+, and Th6(OH)159+ also slightly differ going from one concentration of sodium nitrate to another. The observed effect of the medium is in agreement with the linear free energy relationship proposed for the metal ion hydrolysis.

scholarly journals Catalytic mechanism of α-retaining glucosyl transfer by Corynebacterium callunae starch phosphorylase: the role of histidine-334 examined through kinetic characterization of site-directed mutants

2005 ◽  
Vol 387 (2) ◽  
pp. 437-445 ◽  
Author(s):  
Alexandra SCHWARZ ◽  
Francesco Maria PIERFEDERICI ◽  
Bernd NIDETZKY
Keyword(s):  
Steady State ◽  
Transition State ◽  
Catalytic Mechanism ◽  
Single Step ◽  
Free Enzyme ◽  
Linear Free Energy Relationship ◽  
Wild Type ◽  
Starch Phosphorylase ◽  
Linear Free Energy ◽  
Corynebacterium Callunae

Purified site-directed mutants of Corynebacterium callunae starch phosphorylase in which His-334 was replaced by an alanine, glutamine or asparagine residue were characterized by steady-state kinetic analysis of enzymic glycosyl transfer to and from phosphate and studies of ligand binding to the active site. Compared with wild-type, the catalytic efficiencies for phosphorolysis of starch at 30 °C and pH 7.0 decreased approx. 150- and 50-fold in H334Q (His334→Gln) and H334N mutants, and that of H334A was unchanged. In the direction of α-glucan synthesis, selectivity for the reaction with G1P (α-D-glucose 1-phosphate) compared with the selectivity for reaction with α-D-xylose 1-phosphate decreased from a wild-type value of ∼20000 to 2600 and 100 in H334N and H334Q respectively. Binding of G1P to the free enzyme was weakened between 10-fold (H334N, H334Q) and 50-fold (H334A) in the mutants, whereas binding to the complex of enzyme and α-glucan was not affected. Quenching of fluorescence of the pyridoxal 5′-phosphate cofactor was used to examine interactions of the inhibitor GL (D-gluconic acid 1,5-lactone) with wild-type and mutant enzymes in transient and steady-state experiments. GL binding to the free enzyme and the enzyme–phosphate complex occurred in a single step. The 50-fold higher constant (Kd) for GL dissociation from H334Q bound to phosphate resulted from an increased off-rate for the ligand in the mutant, compared with wild-type. A log-log correlation of catalytic-centre activity for phosphorolysis of starch with a reciprocal Kd value established a linear free-energy relationship (slope=1.19±0.07; r2=0.991) across the series of wild-type and mutant enzymes. It reveals that GL in combination with phosphate has properties of a transition state analogue and that the His-334 side chain has a role in selectively stabilizing the transition state of the reaction.

Substituent Effects on the Chymotrypsin-Catalyzed Hydrolysis of Specific Ester Substrates

1971 ◽  
Vol 49 (2) ◽  
pp. 210-217 ◽  
Author(s):  
R. E. Williams ◽  
M. L. Bender
Keyword(s):  
Free Energy ◽  
Rate Constants ◽  
Substituent Effects ◽  
Phenyl Group ◽  
Linear Free Energy Relationship ◽  
Acylation Reaction ◽  
General Base ◽  
Linear Free Energy ◽  
Base Mechanism ◽  
Hydrolysis Of

The substituent effect on the chymotrypsin-catalyzed hydrolysis of several phenyl esters of specific substrates has been studied. The second-order acylation rate constants (kcat/Km(app)) obey a linear free energy relationship with ρ = +0.63 for phenyl hippurates and ρ = +0.46 for phenyl N-benzyloxycarbonyl-L-tryptophanates when substituents are introduced into the phenyl group of the ester function. These results further support the previously proposed general acid – general base mechanism for the acylation reaction and the formation of a tetrahedral intermediate in the course of the reaction.

Hydrolysis of haloacetonitriles: LINEAR FREE ENERGY RELATIONSHIP, kinetics and products

1999 ◽  
Vol 33 (8) ◽  
pp. 1938-1948 ◽  
Author(s):  
Victor Glezer ◽  
Batsheva Harris ◽  
Nelly Tal ◽  
Berta Iosefzon ◽  
Ovadia Lev
Keyword(s):  
Free Energy ◽  
Linear Free Energy Relationship ◽  
Linear Free Energy ◽  
Hydrolysis Of

Linear free energy relationship and kinetic isotope effects as measures for the transition-state variation — A case of the neophyl system

2005 ◽  
Vol 83 (9) ◽  
pp. 1606-1614 ◽  
Author(s):  
Salai Cheettu Ammal ◽  
Hiroshi Yamataka
Keyword(s):  
Free Energy ◽  
Transition State ◽  
Density Functional ◽  
Isotope Effects ◽  
Kinetic Isotope Effects ◽  
Single Step ◽  
Linear Free Energy Relationship ◽  
Linear Free Energy ◽  
Kinetic Isotope ◽  
State Variation

Ab initio calculations at the MP2/6-31G* level and density functional theory (B3LYP/6-311+G**) calculations have been performed on acid-catalyzed ionizations of substituted neophyl alcohols to investigate whether a variation of the transition-state (TS) structure is reflected in the kinetic isotope effects (KIE) and linear free energy relationship. The effect of substituents on KIEs, TS structures, and activation and reaction energies was calculated. This study revealed that a curved Brønsted-type plot could arise for a single-step process from the variation of TS structure with the substituent, whereas the Hammett plots with a dual-parameter treatment can not detect such TS variation. The variation of KIEs at various positions of neophyl alcohol reflects the variation of TS structures in a manner consistent with the More O'Ferrall – Jencks type reaction diagram analyses.Key words: transition-state variation, substituent effect, kinetic isotope effect, linear free energy relationship.

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