Mechanism of Nicotinate Dehydrogenase and its activity compared with Molybdoenzymes Possessing Sulfido, Oxo and Tellurido Terminals in Their Reductive Half-Reaction Active Site

Abstract

The active site of Nicotinate Dehydrogenase is somewhat similar to xanthine oxidoreductase (XOR) except the equatorial sulfido group in Xanthine oxidase is replaced by sellenido group in Nicotinate Dehydrogenase. In addition, the two enzymes were reported to vary in the binding pocket amino acid residue composition. Modeling the substrate binding step using acetaldehyde as a substrate and active site terminal containing selenium, oxygen or tellurium atom incorporated in place of sulfido terminal is mechanistically important. In order to probe the mechanistic route for the oxidation of acetaldehyde by molybdenium hydroxylases, Density functional theory with B3LYP level theory has been applied. Initially, a linear transit calculation is applied to obtain transition state structures. The exact positions of transition state structure was proved by maximum energy, inflection point in Mulliken charge profile and single negative frequency. Protonation of the carbonyl oxygen of acetaldehyde in the complexes resulted in stabilization of the transition state by around 420 kcal. But it retarded the way of dissociation of the transition state structure towards product bound complex, which was confirmed by decreased electron density and negative Mulliken charge on Hacetaldehyde along its way v 1 J J towards the terminals of the active sites, low extent Mo-Oz, elongation profile, low extent of Oeq-Cacetaldehyde shortening and higher frequency. The properties of Nicotinate Dehydrogenase were compared with complexes of active sites with oxo, sulfido and tellerido terminal to establish the mechanistic determinants for reaction with acetaldehyde. The differences in properties of the four complexes lie in the details of the Mo-X lengths and energy profile in the four transitions states, where X = 0, S, Se or Te. The complex with Oxo terminal required significantly larger relative deformation energy, with respect to the active site containing sulfido terminal in forming the transition state. This was confirmed by its high extent of Mo-X elongation, long bond distance between Oeq-Cacetaldehyde during nucleophillic attack, decreased mulliken charge on Hacetaldehyde along its way towards OMo, have highest frequency and shows considerably maximum energy barrier to the transition state. Generally activity of the terminals decreases down a group. Based on the similarities between Xanthine Oxidase and Nicotinate Dehydrogenase, Single point calculations were made on the proposed optimized transition state and intermediate structures, for developing the general mechanism for Nicotinate Dehydrogenas. The normalized energy profile of both the concerted and stepwise mechanisms of the reaction of its active site with acetaldehyde and nicotinate, have been obtained. It is found that the concerted mechanism has energy barrier of 16 . 32 kcal while the step wise route has a barrier of 425. 26 kcal when the substrate is acetaldehyde. Since the concerted mechanism has got very less energy barrier, it is the most plausible catalytic route for Nicotinate Dehydrogenase.