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.

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.

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 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.

A QM/MM Investigation of the Catalytic Mechanism of Acetylene Hydratase: Insights into Engineering a More Effective Enzyme

2020 ◽  
Author(s):  
wenyuan zhang ◽  
Eric A.C. Bushnell
Keyword(s):  
Active Site ◽  
Cluster Model ◽  
Reaction Rate ◽  
Catalytic Mechanism ◽  
Single Step ◽  
Wild Type ◽  
Step Process ◽  
Rate Determining Step ◽  
Binding Phase ◽  
Insight Into

In the present investigation, a QM/MM approach was used to better understand the effect of the second environmental shell of the active site on the catalytic conversion of acetylene to acetaldehyde by acetylene hydratase (AH). In addition, the effect of substituting W-coordinating sulfur atoms with selenium atoms was done to provide insight into the influence of the W-coordinating atoms on the catalytic reaction. From the results, it found that the presence of the second shell environment had a significant effect on the reaction. Specifically, in the absence of the MM second shell environment(i.e., QM-cluster model), the reaction rate-determining step is defined by the first proton transfer step. In contrast, for the QM/MM model, the rate-determining step is defined by the water attacking step. Moreover, with the presence of the MM second shell environment, a key intermediate found in the DFT-cluster investigation does not exist in the QM/MM investigation. Rather, what was a two-step process in the DFT-cluster study was calculated to occur in a single step for the QM/MM study. Regarding the sulfur to selenium substitutions, it was found that Gibbs energy for the acetylene binding phase was significantly affected. Notably, the trans-position selenium made the binding of acetylene 65.6 kJ mol-1 less endergonic. Moreover, the overall reaction became 38.2 kJ mol-1 less endergonic compared to the wild type (WT) AH model. Thus, the substitution of key W-coordinating sulfur atoms with selenium atoms may offer a means to enhance the catalytic mechanism of AH considerably.

scholarly journals Properties of the histidine residues of indole-chymotrypsin. Implications for the activation process and catalytic mechanism.

1975 ◽  
Vol 147 (3) ◽  
pp. 411-416 ◽  
Author(s):  
W H Cruickshank ◽  
H Kaplan
Keyword(s):  
Charge Transfer ◽  
Aspartic Acid ◽  
Catalytic Mechanism ◽  
Enzymic Activity ◽  
Activation Process ◽  
Linear Free Energy Relationship ◽  
Relay System ◽  
X Ray ◽  
Histidine Residues ◽  
Linear Free Energy

The use of a linear free-energy relationship shows that both histidine residues of alpha-chymotrypsin and chymotrypsinogen are super-reactive toward 1-fluoro-2,4-dinitrobenzene. The binding of indole to the specificity site of alpha-chymotrypsin causes both histidine residues to become less reactive. On the basis of these results and those from X-ray-crystallographic studies, the following conclusions are made. (1) The super-reactivity of the catalytic-site histidine-57 is due to charge transfer from aspartic acid-102 by means of hydrogen bonding. (2) The aspartic acid-102-histidine-57-serine-195 ‘charge-relay’ system is not complete in the zymogen or native enzyme and only on binding of a suitable substrate or ligand to the specificity site of the enzyme is the charge transfer to serine-195 completed. (3) The lack of substantial enzymic activity in the zymogen is due to the absence of a completed specificity site, and therefore it cannot bind suitable substrates or ligands to induce completion of the charge-relay system.

scholarly journals Mutationally altered rate constants in the mechanism of alkaline phosphatase

1974 ◽  
Vol 141 (3) ◽  
pp. 845-852 ◽  
Author(s):  
Stephen E. Halford ◽  
Milton J. Schlesinger
Keyword(s):  
Alkaline Phosphatase ◽  
Steady State ◽  
Catalytic Mechanism ◽  
Product Formation ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Ph Values ◽  
Modified Enzyme ◽  
The Difference ◽  
The Individual

The hydrolysis of phosphate esters by a mutationally altered alkaline phosphatase from Escherichia coli was studied by both steady-state and transient-kinetic methods. The difference between the catalytic-centre activities of the mutationally altered and the wild-type alkaline phosphatases was found to vary with pH and at optimal pH values the modified enzyme had the higher activity. Stopped-flow experiments at acidic pH values showed that transient product formation by the mutationally altered enzyme was faster than that with the wild-type enzyme whereas the rate of the steady state was slower. In the alkaline pH region, the transient was observed in the reaction of only the modified enzyme and not the wild type. These observations permit a fuller characterization of the individual steps in the catalytic mechanism of alkaline phosphatase than is possible by study of only the wild-type enzyme.

scholarly journals Catalytic-rate improvement of a thermostable malate dehydrogenase by a subtle alteration in cofactor binding

1995 ◽  
Vol 305 (2) ◽  
pp. 539-548 ◽  
Author(s):  
R M Alldread ◽  
D M Halsall ◽  
A R Clarke ◽  
T K Sundaram ◽  
T Atkinson ◽  
...  
Keyword(s):  
Steady State ◽  
Amino Acid ◽  
Glutamic Acid ◽  
Catalytic Mechanism ◽  
Fold Increase ◽  
Mutant Enzyme ◽  
Wild Type ◽  
Thermophilic Enzyme ◽  
Thermus Aquaticus ◽  
Equivalent Position

The nucleotide-binding fold of many NAD(+)-dependent dehydrogenases contains a conserved acidic amino acid residue which hydrogen-bonds with the 2′- and 3′-hydroxy groups of the adenine-ribose of the cofactor. This residue is highly conserved as aspartate in malate dehydrogenases, except in the thermophilic enzyme from Thermus aquaticus B (TaqMDH), which has glutamic acid-41 in the equivalent position. The catalytic mechanism was dissected to investigate the functional significance of this difference in TaqMDH with respect to a mutant enzyme where glutamic acid-41 was replaced by aspartic acid. The mutant enzyme was found to retain a high degree of protein structural stability to both thermal and chemical denaturation. When compared with the wild-type enzyme the mutant had a higher Km and Kd for both reduced and oxidized cofactors (NADH and NAD+) and a 2-3-fold increase in steady-state kcat in both assay directions. The rate-determining step for the reduction of oxaloacetate by wild-type TaqMDH was shown to be the rate of NAD+ release, which was about 2.5-fold higher for the mutant enzyme. This correlates well with the 1.8-fold higher steady-state kcat of the mutant enzyme and represents an improvement in the steady-state kcat of a thermophilic enzyme at moderate temperature by a conservative amino acid substitution which increases the rate of product release.

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