Galactose oxidase catalytic mechanism

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.

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... 

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. 

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. 

Since both alcoholic oxidation and O2 reduction are two-electron processes, the catalytic reaction is conceptually equivalent to a transfer of the elements of dihydrogen between the two substrates. Biological hydrogen transfer generally involves specialized organic redox factors (e.g., flavins, nicotinamide, quinones), with well-characterized reaction mechanisms. Galactose oxidase does not contain any of these conventional redox factors and instead utilizes a very different type of active site, a free radical-coupled copper complex, to perform this chemistry. The new type of active site structure implies that the reaction follows a novel biochemical redox mechanisms based on free radicals and the two-electron reactivity of the metalloradical complex. 

Figure 16 Proposed catalytic mechanism for galactose oxidase.Reproduced from M. J. McPherson M. R. Parsons R. K. Spooner C. M. Wilmot, In Handbook of Metalloproteins, A. Messerschmidt, R. Huber, T. Poulos, K. Wieghardt, Eds. John Wiley Sons Chichester, 2001 Vol. 2, pp 1272-1283, with permission from John Wiley Sons.

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

This CuBr2-bpy/TEMPO-based catalytic system can be considered as the first synthetic functional model of galactose oxidase, as both the achieved chemoselectivity, and the proposed reaction mechanism, resemble that of the biological copper enzyme. Nevertheless, this functional model is not able to compete with the natural enzyme in terms of catalytic efficiency. Indeed, the rate of turnover is only 0.006 s while a TOE of 800 s is reached by GOase for its native substrate. The objective of future research investigations is therefore to enhance the proficiency of the catalyst to obtain an economically interesting system for industrial applications. 

Mechanism. Since its discovery in 1959 (2), galactose oxidase has attracted considerable interest in the scientific community because of its enigmatic catalytic mechanism. Various aspects of early research have been discussed in two excellent reviews (J, 4), The most puzzling property of this enzyme has been the ability of the monomeric protein to carry out two-electron redox reactions without any apparent involvement of a cofactor. Although a number of elegant explanations of this phenomena have been proposed (3, 5), the mechanism still remains elusive. On the... 

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