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The present work investigates the hydroxylation reaction of two enzymes (aldehyde oxidoreductase and xanthine oxidase), which contain slightly different molybdopterin cofactors, using a quantum-mechanical/molecular-mechanical (QM/MM) approach. Herein, a small part of the complete model consisting of enzyme, substrate, and solvent, is described by quantum-mechanical methods (mostly density functional theory) and the rest is treated with molecular-mechanical methods (the CHARMM force field).
For the reaction from acetaldehyde to acetic acid, catalyzed by aldehyde oxidoreductase (AOR), five different reaction mechanisms were investigated, which had only been partially discussed in the literature so far. Based on the calculated QM/MM energies, a Lewis base catalyzed mechanism could be favored, not only confirming the function of Glu869 postulated on the basis of experimental results, but also coming up with significantly lower reaction barriers than the alternative pathways. To corroborate these results, free-energy barriers for the rate-determining steps of each mechanism were computed. This leads to appreciable corrections of the barriers, which however do not cause any qualitative changes in the proposed reaction mechanism.
Concerning the conversion of xanthine to uric acid by xanthine oxidase (XO), seven different setups were studied, covering different tautomers of xanthine, different protonation states, and different orientations of the substrate. Due to the much more specific binding of the substrate (compared to AOR), its rearrangement within the binding pocket is impossible. All setups therefore follow a multi-step mechanism in XO, consisting of an initial activation, followed by a nucleophilic attack of the cofactor toward the substrate and a final hydride transfer. The seven investigated variants differ by their ability to stabilize the occuring intermediates. Additional calculations were performed to establish an intrinsic reactivity and a common energy scale.
Based on these results, the consequences of variations in the cofactor (oxo, sulfido and selenido form), in the substrate (2-oxo-6-methylpurin instead of xanthine) and in one specific active-site residue of the binding pocket (Glu802 →Gln mutation) were explored through additional calculations. The results obtained are compatible with the corresponding experimental findings.
The investigations on both enzymes, AOR and XO, together with the available experimental evidence, provide a consistent mechanistic picture, enabling a thorough explanation of the reactivity of this family of enzymes.
