scholarly journals Substrate analogues as probes of the catalytic mechanism of l-mandelate dehydrogenase from Rhodotorula graminis

1994 ◽  
Vol 297 (3) ◽  
pp. 647-652 ◽  
Author(s):  
O Smékal ◽  
G A Reid ◽  
S K Chapman
Keyword(s):  
Transition State ◽  
Catalytic Mechanism ◽  
Activation Parameters ◽  
Linear Free Energy Relationship ◽  
Free Energies ◽  
Linear Free Energy ◽  
Kinetic Isotope ◽  
Substrate Analogues ◽  
Reaction Constant ◽  
Delta G

A detailed kinetic analysis of the oxidation of mono-substituted mandelates catalysed by L-(+)-mandelate dehydrogenase (L-MDH) from Rhodotorula graminis has been carried out to elucidate the role of the substrate in the catalytic mechanism. Values of Km and kcat. (25 degrees C, pH 7.5) were determined for mandelate and eight substrate analogues. Values of the activation parameters, delta H++ and delta S++ (determined over the range 5-37 degrees C), for mandelate and all substrate analogues were compensatory resulting in similar low values for free energies of activation delta G++ (approx. 60 kJ.mol-1 at 298.15 K) in all cases. A kinetic-isotope-effect value of 1.1 +/- 0.1 was observed using D,L-[2-2H]mandelate as substrate and was invariant over the temperature range studied. The logarithm of kcat. values for the enzymic oxidation of mandelate and all substrate analogues (except 4-hydroxymandelate) showed good correlation with Taft's dual substituent constant omega (where omega = omega I + 0.64 omega +R) and gave a positive reaction constant value, rho, of 0.36 +/- 0.07. This linear free-energy relationship was verified by analysing the data using isokinetic methods. These findings support the hypothesis that the enzyme-catalysed reaction proceeds via the same transition state for each substrate and indicates that this transition state is relatively nonpolar but has an electron-rich centre at the alpha-carbon position.

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.

Kinetics of Pb(IV) Oxidation of Substituted Methyl Mandelates — A L.F. E. R. Study

1986 ◽  
Vol 41 (4) ◽  
pp. 467-472
Author(s):  
Sudheer K. Banerjee ◽  
R. Shanker ◽  
Om P. Sachdeva
Keyword(s):  
Bond Rupture ◽  
Linear Free Energy Relationship ◽  
First Order ◽  
Keto Esters ◽  
Linear Free Energy ◽  
Kinetic Isotope ◽  
Rate Limiting Step ◽  
Catalysed Reaction ◽  
Reaction Constant ◽  
Rate Limiting

Lead tetra acetate (Pb(IV) or LTA) oxidation of the esters X -C6H4-CH(OH)-COOCH3(X = H, m-NO2, p-NO2, m-Cl, p-Cl, p-Br. p-CH3 and p-C2H5) gives corresponding keto esters. The reaction is first order in [Pb(IV)] and second order in [Ester], and is catalysed by pyridine and 2,6-lutidine. The pyridine catalysed reaction is first order each in [Pb(IV)]. [Ester] and [Pyridine]. A kinetic isotope effect is observed in oxidation of methyl m andelate for both uncatalysed reaction (kH/kD = 4.2) and pyridine catalysed reaction (kH/kD = 1.8). Activation param eters for both uncatalysed and pyridine catalysed reactions are evaluated. The results are in accord with Linear Free Energy Relationship (L.F.E.R.) and the reaction constant (ϱ = +0.75) obtained supports proton transfer in the ɑ-C-H bond rupture in the rate limiting step. The role pf pyridine in catalysis, as a ligand or as a base is discussed

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.

Kinetics and mechanism of bromination of styrenes

1967 ◽  
Vol 45 (2) ◽  
pp. 167-173 ◽  
Author(s):  
Keith Yates ◽  
W. V. Wright
Keyword(s):  
Activation Parameters ◽  
Second Order ◽  
Linear Free Energy Relationship ◽  
Reaction Products ◽  
Order Process ◽  
Third Order ◽  
Linear Free Energy ◽  
Reaction Constant ◽  
Relationship Of ◽  
Kinetics Of

The kinetics of bromination of six substituted styrènes (3-fluoro-, 3-chloro-, 3-bromo-, 3,4-dichloro-, 3-nitro-, and 4-nitro-) in anhydrous acetic acid have been investigated at several temperatures. At 25.3 °C the reactions follow the rate expression [Formula: see text]The rate constants for the second order process show a good linear free energy relationship of the log k versus σ type with ρ = − 2.24. (The value obtained at 35.3 °C is − 1.93.) No simple rate-substituent dependence is obtained for the more complex third order process. Activation parameters have been obtained for the second order brominations, the ΔS≠ values being large and negative. Bromination of styrene in the presence of a large excess of acetate or nitrate gives only two products in each case, the α,β-dibromide and the α –acetoxy β-bromide or α -nitrato- β -bromide respectively.The magnitude of the reaction constant ρ, the values of ΔS≠, and the reaction products all support a mechanism involving a highly unsymmetrical bromonium ion intermediate.

scholarly journals Rationalisation of the activities of phenolic (vitamin E-type) antioxidants

2003 ◽  
Vol 17 (4) ◽  
pp. 753-762
Author(s):  
Christopher J. Rhodes ◽  
Thuy T. Tran ◽  
Philip Denton ◽  
Harry Morris
Keyword(s):  
Free Energy ◽  
Vitamin E ◽  
Gibbs Free Energy ◽  
State Theory ◽  
Linear Free Energy Relationship ◽  
Free Energies ◽  
Gibbs Free Energy Change ◽  
Linear Free Energy ◽  
Free Energy Of Activation ◽  
Enthalpy And Entropy

Using Transition-State Theory, experimental rate constants, determined over a range of temperatures, for reactions of vitamin E type antioxidants are analysed in terms of their enthalpies and entropies of activation. It is further shown that computational methods may be employed to calculate enthalpies and entropies, and hence Gibbs Free Energies, for the overall reactions. Within the Linear Free Energy Relationship (LFER) assumption, that the Gibbs Free Energy of activation is proportional to the overall Gibbs Free Energy change for the reaction, it is possible to rationalise, and even to predict, the relative contributions of enthalpy and entropy for reactions of interest, involving potential antioxidants.

scholarly journals Human DNMT1 transition state structure

2016 ◽  
Vol 113 (11) ◽  
pp. 2916-2921 ◽  
Author(s):  
Quan Du ◽  
Zhen Wang ◽  
Vern L. Schramm
Keyword(s):  
Transition State ◽  
Dna Methyltransferase ◽  
Catalytic Mechanism ◽  
Isotope Effects ◽  
Nucleophilic Attack ◽  
Kinetic Isotope ◽  
Methyl Transfer ◽  
Major Transition ◽  
Methylation Patterns ◽  
Rate Limiting

Human DNA methyltransferase 1 (DNMT1) maintains the epigenetic state of DNA by replicating CpG methylation signatures from parent to daughter strands, producing heritable methylation patterns through cell divisions. The proposed catalytic mechanism of DNMT1 involves nucleophilic attack of Cys1226 to cytosine (Cyt) C6, methyl transfer from S-adenosyl-l-methionine (SAM) to Cyt C5, and proton abstraction from C5 to form methylated CpG in DNA. Here, we report the subangstrom geometric and electrostatic structure of the major transition state (TS) of the reaction catalyzed by human DNMT1. Experimental kinetic isotope effects were used to guide quantum mechanical calculations to solve the TS structure. Methyl transfer occurs after Cys1226 attack to Cyt C6, and the methyl transfer step is chemically rate-limiting for DNMT1. Electrostatic potential maps were compared for the TS and ground states, providing the electronic basis for interactions between the protein and reactants at the TS. Understanding the TS of DNMT1 demonstrates the possibility of using similar analysis to gain subangstrom geometric insight into the complex reactions of epigenetic modifications.

Some quantitative relationships for ionization reactions at high pressures

1975 ◽  
Vol 28 (5) ◽  
pp. 945 ◽  
Author(s):  
BS El'yanov ◽  
SD Hamann
Keyword(s):  
High Pressures ◽  
Good Description ◽  
Ionization Constants ◽  
Linear Free Energy Relationship ◽  
Free Energies ◽  
Ion Hydration ◽  
Linear Free Energy ◽  
Linear Relationships ◽  
Wide Range ◽  
Ionization Equilibria

A simple formula is proposed to describe the pressure dependence of the variable Φ in El?yanov and Gonikberg's linear free energy relationship for ionization reactions in solution at high pressure. ��� The expression, given in equations (10) and (12), provides a good description of the influence of pressure on ionization equilibria in aqueous solutions. It permits El'yanov's general linear relationships between Φ and ionization free energies, enthalpies and entropies, pH and Hammett's p parameter, to be expressed in terms of the pressure in convenient analytical forms. ��� The formula is shown to be consistent with the simple electrostatic theory of ion hydration, allowing for the effect of pressure on the dielectric constant of water. Combined with the theory, it provides a general means of predicting ionization constants over a wide range of pressures and temperatures simply from knowledge of the changes in molar volume, enthalpy and entropy which accompany the reactions at atmospheric pressure.

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.

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