Galactose oxidase mechanism

Figure 5. Proposed mechanism of alcohol oxidation for galactose oxidase.

The alcohol dehydrogenases were already described in Chapter 3. These enzymes are cofactor dependent and in the active site hydrogen transfer takes place from NADH or NADPH. In the reverse way they can, however, be applied for the oxidation of alcohols in some cases (see below). Oxidases are very appealing for biocatalytic purposes, because they use oxygen as the only oxidant without the need for a cofactor. Oxidases usually have flavins (glucose oxidase, alcohol oxidase) or copper (examples galactose oxidase, laccase and tyrosinase) in the active site [18]. The mechanism for glucose oxidase (GOD) is denoted in... 

Fig. 21. Proposed catalytic mechanism for substrate oxidation by galactose oxidase. (A) Substrate binding displaces Tyr-495 phenolate which serves as a general base for abstracting the hydroxylic proton. (B) Stererospecihc pro- hydrogen abstraction by the Tyr-Cys phenoxyl radical. (C) Inner sphere electron transfer reducing Cu(II) to Cu(I). (D) Dissociation of the aldehyde product.

Chemically binding enzymes to nylon net is very simple and gives strong mechanically resistant membranes (135). The nylon net is first activated by methylation and then quickly treated with lysine. Finally, the enzyme is chemically bound with GA. The immobilized disks are fixed direcdy to the sensor surface or stored in a phosphate buffer. GOD, ascorbate oxidase, cholesterol oxidase, galactose oxidase, urease, alcohol oxidase (135), and lactate oxidase (142) have been immobilized by this procedure and the respective enzyme electrode performance has been established. 

With galactose oxidase, our understanding of the catalytic mechanism is less advanced than for amine oxidases but all the essential foundations for continued advances are in place high resolution X-ray structures of native and mutational variant forms, eomplete with advanced spectroscopic... 

Whittaker, M. M., Ballou, D. P., and Whittaker, J. W., 1998, Kinetic isotope effects as probes of die mechanism of galactose oxidase. Biochemistry 37 8426fi8436. 

These data have led to the development of a catalytic mechanism, shown in Scheme 6, that has been further refined by kinetic isotope effect (KIE) experiments. Substrate binds to Cu(II), replacing bound solvent. The metal coordination facilitates the deprotonation of the substrate hydroxyl group. The proton is transferred to Tyr495, which dissociates from copper. The temperature and pH dependence of the visible absorption and circular dichroism spectra indicate that galactose oxidase exists as an equilibrium of the Tyr495-Cu(II) form (TyroN) and the protonated Tyr495 state. 

The second half-reaction of galactose oxidase turnover involves reduction of O2 to form peroxide, H2O2. Although much less is known about this mechanistic step, it is thought that O2 binds to Cu(I). Both substrate-derived protons are transferred to the peroxide product, as determined from the large substrate (alcohol) KIE on O2 reduction evidently hydrogen transfer is fully rate limiting for O2 reduction. Additional work is necessary to develop a more detailed mechanism of O2 reduction. 

Scheme 6 Catal)ftic mechanism of galactose oxidase. (Reprinted with permission from Ref 52. 2003 American Chemical Society)...

Figure 2, Proposed reaction mechanism for galactose oxidase.

We start the discussion of the mechanism of galactose oxidase by noting the following. From experimental heats of formation one can calculate that the net reaction catalyzed by the enzyme, with methanol as substrate ... 

Stack and co-woikers [25] have synthesized model complexes that resemble both the spectroscopic characteristics and the catalytic activity of galactose oxidase. For these complexes, EXAFS and edge XAS experiments indicate that the radical is most likely located axially in the non-square planar coordination of the copper. Calculations by Rothlisberger and Carloni [26] on these model systems confirm this fact. We also recommend the chapter herein by that group, in which the fuU reaction mechanism of GO has been investigated using Car-ParineUo MD methods. 

To summarize this section, the theoretical calculations [2] strongly support the mechanism proposed for galactose oxidase. It was shown that the proton transfer step proposed to initiate the oxidation of the substrate is very fast and just slightly exothermic. The rate-limiting hydrogen atom transfer step has a calculated barrier of feasible 13.6 kcal/mol. The proposed short-lived ketyl radical intermediate has been localized, and it was argued that the subsequent... 

Figure 7. Calculated, IMOMM(B3LYP/MM3), potential energy surface for the mechanism of galactose oxidase.

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