A substantial oxygen isotope effect at O2 in the OMP decarboxylase reaction: mechanistic implications.

Orotidine-5'-monophosphate decarboxylase (OMP decarboxylase, ODCase) catalyzes the decarboxylation of orotidine-5'-monophosphate (OMP) to uridine-5'-monophosphate (UMP). Despite extensive enzymological, structural, and computational studies, the mechanism of ODCase remains incompletely characterized. Herein, carbon kinetic isotope effects were measured for both the natural abundance substrate and a substrate mixture synthesized for the purpose of carrying out the remote double label isotope effect procedure, with O2 of the substrate as the remote position. The carbon kinetic isotope effect on enzymatic decarboxylation of this substrate mix was measured to be 1.0199 +/- 0.0007, compared to the value of 1.0289 +/- 0.0009 for natural abundance OMP, revealing an (18)O2 isotope effect of 0.991 +/- 0.001. This value equates to an intrinsic isotope effect of approximately 0.983, using a calculated commitment factor derived from previous isotope effect data. The measured (18)O2 isotope effect requires a mechanism with one or more enzymatic processes, including binding and/or chemistry, that contribute to this substantial inverse isotope effect. (18)O2 kinetic isotope effects were calculated for four proposed mechanisms: decarboxylation preceded by proton transfer to 1) O2; 2) O4; and 3) C5; and 4) decarboxylation without a preceding protonation step. A mechanism involving no pre-decarboxylation step does not appear to have any steps with the necessary substantial inverse (18)O2 effect, thus calling into question any mechanism involving simple direct decarboxylation. Protonation at O2, O4, or C5 are all calculated to proceed with inverse (18)O2 effects, and could contribute to the experimentally measured value. Recent crystal structures indicate that O2 of the substrate appears to be involved in an intricate bonding arrangement involving the substrate phosphoryl group, an enzyme Gln side chain, and a bound water molecule; this interaction likely contributes to the observed isotope effect.

Design of LFA-1 antagonists based on a 2,3-dihydro-1H-pyrrolizin-5(7aH)-one scaffold.

A new class of lymphocyte function-associated antigen-1 (LFA-1) antagonists is described. Elaboration of the 2,3-dihydro-1H-pyrrolizin-5(7aH)-one scaffold resulted in the synthesis of potent inhibitors of the LFA-1/ICAM-1 interaction. Along with the in vitro activity, we present the X-ray crystal structure of the complex of compound 9b, in a novel binding mode to the I-domain of LFA-1.

Determination of the absolute configuration and solution conformation of a novel disubstituted pyrrolidine acid A by vibrational circular dichroism.

Compound A, a novel disubstituted pyrrolidine acid, is a member of a new class of agents that are potentially useful for the treatment of diabetes and dyslipidemia. The absolute configuration of this compound was determined by using vibrational circular dichroism (VCD). The results are in agreement with the assignments based on both X-ray analysis and the stereo-selective chemical synthesis. During VCD analysis, the solution conformation for a portion of compound A in CDCl(3) was also established. The compound is found to associate as an H-bonded carboxylic acid "dimer" in CDCl(3) solution, and VCD calculations on a model dimer fragment were required to establish the absolute configuration.

Theoretical studies of the effect of thio substitution on orotidine monophosphate decarboxylase substrates.

[reaction: see text] 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 k(cat) 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 degrees to 360 degrees ) 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.

The gas phase proton affinity of uracil: measuring multiple basic sites and implications for the enzyme mechanism of orotidine 5'-monophosphate decarboxylase.

We have shown for the first time experimentally that the O2 and O4 sites of uracil have different proton affinities, and as implied in previous computational studies, the O4 is more basic and would be energetically preferred in an orotate ribose 5'-monophosphate decarboxylase catalysis mechanism involving proton transfer to oxygen.