Mixed inhibition of the oxidoreductase activity of xanthine oxidase by Pd2+ ion

Sau, Apurba Kumar; Mondal, Madhu Sudan; Mitra, Samaresh

doi:10.1039/c39940001547

Cited by 6 publications

(1 citation statement)

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“…The IC 50 is the concentration of inhibitor necessary to produce a 50% decrease in enzyme activity at an arbitrary concentration of substrate, and thus it requires only n i separate reactions, as opposed to n s × n i . It must be emphasized that IC 50 values are relatively information-poor as they do not reveal the inhibition mode, which is essential knowledge for structural characterization and optimization of the inhibitor-bound form (E, ES, or both), , as well as for discriminating among different enzymatic mechanisms for multisubstrate enzymes . Furthermore, IC 50 values cannot be quantitatively interpreted in terms of binding affinity unless the inhibition mode is known a priori.…”

Section: Discussionmentioning

confidence: 99%

Complete Kinetic Characterization of Enzyme Inhibition in a Single Isothermal Titration Calorimetric Experiment

2018

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Techniques for rapidly measuring both the strength and mode of enzyme inhibitors are crucial to lead generation and optimization in drug development. Isothermal titration calorimetry (ITC) is emerging as a powerful tool for measuring enzyme kinetics with distinct advantages over traditional techniques. ITC measures heat flow, a feature of nearly all chemical reactions, and gives an instantaneous readout of enzyme velocity, eliminating the need for artificial substrates or postreaction processing. In principle, ITC is an ideal method for characterizing enzyme inhibition. However, existing ITC experiments are not well-suited to rapid throughput and few studies to date have employed this approach. We have developed a new ITC experiment, in which substrate and inhibitor are premixed in the injection syringe, that yields complete kinetic characterization of an enzyme inhibitor in an hour or less. This corresponds to savings in time and material of 5-fold or greater compared to previous ITC methods. We validated the approach using the trypsin inhibitor benzamidine as a model system, recapitulating both its competitive inhibition mode and binding constant. Our approach combines the rapid throughput of optimized spectroscopic assays with the universality and precision of ITC-based methods, providing substantially improved inhibitor characterization for biochemistry and drug development applications.

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Section: Discussionmentioning

confidence: 99%

Complete Kinetic Characterization of Enzyme Inhibition in a Single Isothermal Titration Calorimetric Experiment

2018

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Mixed activation and mixed inhibition of the oxidoreductase activity of Rhus vernicifera laccase by different Pd²⁺ ion concentration

Liang

Wang

1999

Chin. J. Chem.

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AMracthnetic experiments were performed to study the effects of P 8 + ion on the oxidation of 5,6-&ro-~2.3-dicyanohydroqtunone catalyzed by Rhus Vern;C$ma laccase under condition of pH 4.5 and 30 * 0.1 T .' h e results showed that the mixed activation could be observed when Pd.+ ion was at low concentrations. The competitive and non-competitive activation constants were 9 x la6 and 2 x 10' moVL, respectively. With the increase of P 8 + ion concentration, the activation was g r a d d y converted into mixed inhibition, and the cornpetitive and non-competitive inhibition constants were 6 x 1@ and 32 x 10' moVL, respectively. KevwordsRhlls vemc$kra laccase, palladium(n), mixed activation, rnixed inhibition.Laccase (EC 1.10.3.2) , which contains a blue (type 1 ) copper and a trinuclear copper cluster comprised of a normal (type 2) and a binuclear (type 3 ) copper center, catalyzes the oxidation of diphenolir substrates by oxygen, producing quinones, phenol coupling products, and water as the sole oxygen reduction p d u c t . Diphenolic substrates participate laccase-enzymatic reaction in terms of the phenolate rnonoanion in the rate-determining step for electron t r a n~f e r .~ As the first-order dissociation of most diphenolic substrates occurs around pH 8.0, and at h s circumstance hydrolysis of most metal ions may take place, so the reports of effects of metal ions on the oxidation of diphenolic substrates catalyzed by laccase were rare. There only were several examples5'' that H&+ ion could be selectively substituted at the type 1 site in laccase, forming the type 1 mercury derivative of laccase, and the derivative remained 5 % catalytic activity of native laccase, but they could not study the effect of HZ + ion on the oxidation catalyzed by laccase directly as the limit mentioned above. In our previous studies we found that the first-order dissociation of 5,6-d1bmmo-2,3-dicyanohy~none ( DDBQH2) occurred at pH 4.0. This finding makes it convenient to study the effects of metal ions on catalytic activity of laccase. In addition, ran: earth metal

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Altered redox affinity of xanthine oxidase active sites by copper(II) ions

Mondal

Mitra

1999

Proc. Indian Acad. Sci. (Chem. Sci.)

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The interaction of Cu 2+ ion with the redox centres of xanthine oxidase (XO) has been investigated using optical difference spectroscopic measurements. Anaerobic enzyme-reduction experiments using controlled and excess substrates (xanthine and NADH) have been performed to investigate the perturbation of XO active sites in the presence of Cu 2÷ ions. The results indicate an overall alteration in the redox affinities (i.e. affinity to accept electrons) of the active sites of XO by Cu 2+ ion.

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Mixed inhibition of the oxidoreductase activity of xanthine oxidase by Pd2+ ion

Cited by 6 publications

References 10 publications

Complete Kinetic Characterization of Enzyme Inhibition in a Single Isothermal Titration Calorimetric Experiment

Complete Kinetic Characterization of Enzyme Inhibition in a Single Isothermal Titration Calorimetric Experiment

Mixed activation and mixed inhibition of the oxidoreductase activity of Rhus vernicifera laccase by different Pd²⁺ ion concentration

Altered redox affinity of xanthine oxidase active sites by copper(II) ions

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Mixed inhibition of the oxidoreductase activity of xanthine oxidase by Pd2+ ion

Cited by 6 publications

References 10 publications

Complete Kinetic Characterization of Enzyme Inhibition in a Single Isothermal Titration Calorimetric Experiment

Complete Kinetic Characterization of Enzyme Inhibition in a Single Isothermal Titration Calorimetric Experiment

Mixed activation and mixed inhibition of the oxidoreductase activity of Rhus vernicifera laccase by different Pd2+ ion concentration

Altered redox affinity of xanthine oxidase active sites by copper(II) ions

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Mixed activation and mixed inhibition of the oxidoreductase activity of Rhus vernicifera laccase by different Pd²⁺ ion concentration