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Ping-pong Mechanism (ping-pong + mechanism)

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Chemistry100%

Selected Abstracts

Mosquito NADPH-cytochrome P450 oxidoreductase: kinetics and role of phenylalanine amino acid substitutions at leu86 and leu219 in CYP6AA3-mediated deltamethrin metabolism

ARCHIVES OF INSECT BIOCHEMISTRY AND PHYSIOLOGY (ELECTRONIC), Issue 4 2010
Songklod Sarapusit
Abstract The NADPH-cytochrome P450 oxidoreductase (CYPOR) enzyme is a membrane-bound protein and contains both FAD and FMN cofactors. The enzyme transfers two electrons, one at a time, from NADPH to cytochrome P450 enzymes to function in the enzymatic reactions. We previously expressed in Escherichia coli the membrane-bound CYPOR (flAnCYPOR) from Anopheles minimus mosquito. We demonstrated the ability of flAnCYPOR to support the An. minimus CYP6AA3 enzyme activity in deltamethrin degradation in vitro. The present study revealed that the flAnCYPOR purified enzyme, analyzed by a fluorometric method, readily lost its flavin cofactors. When supplemented with exogenous flavin cofactors, the activity of flAnCYPOR-mediated cytochrome c reduction was increased. Mutant enzymes containing phenylalanine substitutions at leucine residues 86 and 219 were constructed and found to increase retention of FMN cofactor in the flAnCYPOR enzymes. Kinetic study by measuring cytochrome c,reducing activity indicated that the wild-type and mutant flAnCYPORs followed a non-classical two-site Ping-Pong mechanism, similar to rat CYPOR. The single mutant (L86F or L219F) and double mutant (L86F/L219F) flAnCYPOR enzymes, upon reconstitution with the An. minimus cytochrome P450 CYP6AA3 and a NADPH-regenerating system, increased CYP6AA3-mediated deltamethrin degradation compared to the wild-type flAnCYPOR enzyme. The increased enzyme activity could illustrate a more efficient electron transfer of AnCYPOR to CYP6AA3 cytochrome P450 enzyme. Addition of extra flavin cofactors could increase CYP6AA3-mediated activity supported by wild-type and mutant flAnCYPOR enzymes. Thus, both leucine to phenylalanine substitutions are essential for flAnCYPOR enzyme in supporting CYP6AA3-mediated metabolism. © 2010 Wiley Periodicals, Inc. [source]

Characterization of carbonic anhydrase from Neisseria gonorrhoeae

FEBS JOURNAL, Issue 6 2001
Björn Elleby
We have investigated the steady state and equilibrium kinetic properties of carbonic anhydrase from Neisseria gonorrhoeae (NGCA). Qualitatively, the enzyme shows the same kinetic behaviour as the well studied human carbonic anhydrase II (HCA II). This is reflected in the similar pH dependencies of the kinetic parameters for CO2 hydration and the similar behaviour of the kinetics of 18O exchange between CO2 and water at chemical equilibrium. The pH profile of the turnover number, kcat, can be described as a titration curve with an exceptionally high maximal value of 1.7 × 106 s,1 at alkaline pH and a pKa of 7.2. At pH 9, kcat is buffer dependent in a saturable manner, suggesting a ping-pong mechanism with buffer as the second substrate. The ratio kcat/Km is dependent on two ionizations with pKa values of 6.4 and 8.2. However, an 18O-exchange assay identified only one ionizable group in the pH profile of kcat/Km with an apparent pKa of 6.5. The results of a kinetic analysis of a His66,Ala variant of the bacterial enzyme suggest that His66 in NGCA has the same function as a proton shuttle as His64 in HCA II. The kinetic defect in the mutant can partially be overcome by certain buffers, such as imidazole and 1,2-dimethylimidazole. The bacterial enzyme shows similar Ki values for the inhibitors NCO,, SCN, and N3, as HCA II, while CN, and the sulfonamide ethoxzolamide are considerably weaker inhibitors of the bacterial enzyme than of HCA II. The absorption spectra of the adducts of Co(II)-substituted NGCA with acetazolamide, NCO,, SCN,, CN, and N3, resemble the corresponding spectra obtained with human Co(II)-isozymes I and II. Measurements of guanidine hydrochloride (GdnHCl)-induced denaturation reveal a sensitivity of the CO2 hydration activity to the reducing agent tris(2-carboxyethyl)phosphine (TCEP). However, the A292/A260 ratio was not affected by the presence of TCEP, and a structural transition at 2.8,2.9 m GdnHCl was observed. [source]

The high-resolution structure of pig heart succinyl-CoA:3-oxoacid coenzyme A transferase

ACTA CRYSTALLOGRAPHICA SECTION D, Issue 7 2010
Shu-Fen Coker
The enzyme succinyl-CoA:3-oxoacid coenzyme A transferase (SCOT) participates in the metabolism of ketone bodies in extrahepatic tissues. It catalyses the transfer of coenzyme A (CoA) from succinyl-CoA to acetoacetate with a classical ping-pong mechanism. There is biochemical evidence that the enzyme undergoes conformational changes during the reaction, but no domain movements have been reported in the available crystal structures. Here, a structure of pig heart SCOT refined at 1.5,Å resolution is presented, showing that one of the four enzyme subunits in the crystallographic asymmetric unit has a molecule of glycerol bound in the active site; the glycerol molecule is hydrogen bonded to the conserved catalytic glutamate residue and is likely to occupy the cosubstrate-binding site. The binding of glycerol is associated with a substantial relative movement (a 13° rotation) of two previously undefined domains that close around the substrate-binding site. The binding orientation of one of the cosubstrates, acetoacetate, is suggested based on the glycerol binding and the possibility that this dynamic domain movement is of functional importance is discussed. [source]

Crystallization and preliminary X-ray studies of azoreductases from Bacillus sp.

ACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 5 2010

Azoreductases from Bacillus sp. B29 are NADH-dependent flavoenzymes which contain a flavin mononucleotide (FMN) as a prosthetic group and exist as homodimers composed of 23,kDa subunits. These enzymes catalyze the reductive degradation of various azo compounds by a ping-pong mechanism. In order to determine the structure,function relationship of the azo-dye reduction mechanism, an X-ray crystallographic study of azoreductases was performed. Selenomethionine-labelled AzrA (SeMet-AzrA) and AzrC were crystallized by the hanging-drop vapour-diffusion method. A crystal of SeMet-AzrA diffracted to 2.0,Å resolution and was determined to belong to space group P212121, with unit-cell parameters a = 56.9, b = 69.0, c = 105.4,Å. The native crystals of AzrC belonged to space group C2, with unit-cell parameters a = 192.0, b = 56.6, c = 105.5,Å, , = 115.7°, and diffracted to 2.21,Å resolution. [source]