Theoretical Studies of the Effect of Thio Substitution on Orotidine Monophosphate Decarboxylase Substrates

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Abstract
The effect of replacing carbonyl oxygens with sulfur in a series of orotidine 5‘-monophosphate decarboxylase (ODCase) substrates was studied computationally. Previous experimental results indicate that while 2-thio-orotidine 5‘-monophosphate (2-thio-OMP) is a poor substrate for ODCase, 4-thio-orotidine 5‘-monophosphate (4-thio-OMP) binds to ODCase, and the resultant kcat is measurable. Energetics calculations on 2-thio-1-methyl-orotate and 4-thio-1-methyl-orotate (as models for the 2- and 4-thio-OMPs) indicate that mechanisms involving proton transfer to the 2- or 4-site, regardless of substrate and regardless of whether the 2- or 4-position is a carbonyl or thiocarbonyl, are energetically favorable, as compared to direct decarboxylation without proton transfer. Proton transfer to the 4-site during decarboxylation is found to be energetically more favorable than 2-protonation. Each thiocarbonyl is also found to be more basic than its carbonyl counterpart. Therefore, if 2- or 4-proton transfer is the operative catalytic pathway, energetics alone would not explain why 2-thio-orotidine 5‘-monophosphate is a poor ODCase substrate. Conformational preferences for a series of ODCase substrates were also examined computationally. Specifically, the energies and Boltzmann probabilities of the conformers resulting from rotation about the C1‘−N1 bond (O4‘−C1‘−N1−C2 rotation from 0° to 360°) were calculated. It was found that a calculated preference for the syn versus the anti nucleoside conformation correlates to an experimentally better substrate: the OMP and 4-thio-OMP models show a preference for syn conformations, whereas the 2-thio-OMP (the only substrate of the three OMPs that is experimentally found to bind poorly) model shows a preference for an anti conformation. The same rough correlation was found for a series of ODCase inhibitors; that is, a preference for the syn conformation correlates to a better inhibitor. This result is of interest and points to the possibility that the ability for a substrate to bind well to ODCase may be related to its tendency to favor the syn conformation.