Reduced Forms of the Enzyme

Reduced Forms of the Enzyme. The hydrolysis of the product Schiff-base to release product and generate the aminoquinol form of the cofactor may involve water which has been retained in the active site. Dooley and co-workers have provided direct evidence for copper reduction during the interaction of amine oxidases with substrate under anaerobic conditions (Dooley et al., 1991). By varying the temperature at which EPR spectra were recorded, it was shown for amine oxidases from several sources that there is a temperature dependent equilibrium between Cu V aminoquinol TPQ and Cu / TPQ semiquinone. The Cu / TPQ semi-quinone form was found to be stabilised in the presence of cyanide. The 

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S, substrate R, product P, reduced form of the cosubstrate (mediator) Q, oxidized form of the cosubstrate (mediator) Ei, reduced form of the enzyme E2, oxidized form of the e... 

Unlike the whole-cell system, enzymatic reductions require the addition of a hydride donating cofactor to regenerate the reduced form of the enzyme. Depending on the chosen ADH, the cofactor is usually NADH or NADPH, both of which are prohibitively expensive for use in stoichiometric quantities at scale. Given the criticality of cofactor cost, numerous methods of in situ cofactor regeneration, both chemical and biocatalytic, have been investigated. However, only biocatalytic regeneration has so far proven to be sufficiently selective to provide the cofactor total turnover numbers of at least 10 required in production. 

Figure 2.27 Double catalytic cycle that gives rise to the current in the presence of p-D-glucose. COx(FAD) oxidizes the enzyme substrate p-D-glucose to yield the reduced form of the enzyme GOx(FADH2) that is then reoxidized by the osmium polymer mediator PAH-Os(ll I). This process yields PAH-Os(ll). Electrons should diffuse to the electrode surface by electron...

Oxyanions also affect the coordination chemistry of the metal center (84). Molybdate and tungstate are tightly bound noncompetitive inhibitors (Ki s of ca. 4 (iM) (85). These anions bind to the reduced form of the enzyme, changing the rhombic EPR spectrum of the native enzyme to axial (Figure 1) and affecting the NMR shifts observed (84,85). Comparisons of the ENDOR spectra of reduced uterofenin and its molybdate complex show that molybdate binding causes the loss of iH features which are also lost when the reduced enzyme is placed in deuterated solvent (86). These observations suggest that molybdate displaces a bound water upon complexation. 

Here GO(ox) and GO(red) are the oxidized (native) and reduced forms of the enzyme, respectively. The effect of dioxygen is neglected and the transfer of the first electron from reduced FADH2 at ferricenium ion is considered as the rate-limiting step for simplicity. Equation (30) describes the steady-state rate of the enzymatic ferricenium fading. 

When O2 is the electron acceptor, the reduction can occur in either two-electron steps with FADH2 as reductant and H2O2 as the product or in a one-electron manner with 02 as the product. In the latter case, the reduced form of the flavin could be either FADH2 or FAD Recent studies on the reaction of O2 with reduced xanthine oxidase has shown that reoxidation of the six-electron reduced enzyme by O2 proceeds initially with two sequential two-electron steps to form two moles of H2O2 and the two-electron reduced form of the enzyme. Oxidation of the two-electron reduced form by O2 then proceeds via two-sequential one-electron steps to form two moles of O2 and oxidized enzyme. The differential rate of O2 release is suggestive of one mole arising from the one-electron... 

Metyrapone, another compound widely used as an inhibitor of cytochromes P-450, binds to the reduced form of the enzyme and acts as a noncompetitive inhibitor. 

RGURE 22-47 Allopurinol, an inhibitor of xanthine oxidase. Hypoxanthine is the normal substrate of xanthine oxidase. Only a slight alteration in the structure of hypoxanthine (shaded pink) yields the medically effective enzyme inhibitor allopurinol. At the active site, allopurinol is converted to oxypurinol, a strong competitive inhibitor that remains tightly bound to the reduced form of the enzyme. 

The redox mediator 2,6-dichlorophenol indophenol, can mediate electron transfer from and to the redox enzyme, cytochrome c. The mediator was switched between the oxidized and reduced forms by the application of a potential using optically transparent electrodes in a thin-layer cell. From the absorbances of the oxidized and reduced form of the enzyme, the ratio of their concentrations at various potentials was obtained. Calculate the formal potential E° of the enzyme from the data given in Table E.l. Confirm that the enzyme redox process involves one electron transfer. (Contractor)... 

Indirect Interaction. A substrate is oxidized with an oxidized form of a given enzyme (or coenzyme) to give a corresponding oxidized substrate and a reduced form of the enzyme (or coenzyme). This step is followed directly or indirectly (through the electron-transport system) by the reaction between molecular oxygen and the reduced form of the enzyme to regenerate the active oxidized form of the enzyme. In this way, the oxidation proceeds catalytically. 

A wide variety of oxidations mediated by monooxygenase enzymes are similarly thought to involve oxygen transfer from a high-valent oxoiron intermediate to the substrate (although the mechanistic details are still controversial) [8-11]. However, in this case a stoichiometric cofactor is necessary to regenerate the reduced form of the enzyme resulting in the overall stoichiometry shown in Fig. 4.8. 

Eukaryotes contain two forms of this enzyme, a manganese-containing version located in mitochondria and a copper-zinc-dependent cytosolic form. These enzymes perform the dismutation reaction by a similar mechanism (Figure 18.22). The oxidized form of the enzyme is reduced by superoxide to form oxygen. The reduced form of the enzyme, formed in this reaction, then reacts with a second superoxide ion to form peroxide, which takes up two protons along the reaction path to yield hydrogen peroxide. 

Figure 18.18 Superoxide dismutase mechanism. The oxidized form of superoxide dismutase (Mo ) reacts with one superoxide ion to form O7 and generate the reduced form of the enzyme (Mred)- fhe reduced form then reacts with a second superoxide and two protons to form hydrogen peroxide and regenerate ifie oxidized form of the enzyme.

The x-ray structural results described above apply only to the oxidized form of the protein, i.e., the form containing Cu . The reduced form of the enzyme containing Cu is also stable and fully active as an SOD. If, as is likely, the mechanism of CuZnSOD-catalyzed superoxide disproportionation is Mechanism I (Reactions 5.96-5.97), the structure of the reduced form is of critical importance in understanding the enzymatic mechanism. Unfortunately, that structure is not yet available. 

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