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

Abstract

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.

Nuclear quantum effects and kinetic isotope effects in enzyme reactions

“…In particular, a large experimental 13 C KIE of 1.0494 for the carboxylate carbon [164], which may be compared to the computed value of 1.050, is in agreement with the notion that decarboxylation is rate limiting. Interestingly, a mechanism involving pre-protonation of the O2 OMP oxygen has been substantiated based on an inverse 18 O2 KIE [163]. However, our PI-FEP simulated 18 O2 KIE also exhibited an inverse effect for the direct decarboxylation mechanism, suggesting that the observed inverse oxygen KIE is due to changes in H-bonding rather than protonation.…”

“…10 17 [151]. The catalytic mechanism of ODC has been subject to numerous investigations by both experimental [154][155][156][157][158][159][160][161][162][163][164] and computational [156,[165][166][167][168][169][170][171][172] means. In particular, Rishavy and Cleland employed 13 C and 15 N KIEs to elucidate the mechanism [164], and concluded that the most likely pathway involves a direct decarboxylation with an anionic intermediate [162,173].…”

Experimental and Computational Evidence of Metal-O2 Activation and Rate-Limiting Proton-Coupled Electron Transfer in a Copper Amine Oxidase

“…Competitive oxygen-18 kinetic isotope effects ( 18 O KIEs) have been determined on a number of stoichiometric and catalytic reactions. − These probes are particularly useful when combined with DFT calculations, which can provide information regarding transition states that would not otherwise be accessible. ,− The 18 O KIE is defined by the ratio of rate constants, k cat / K M ( 16,16 O 2 ) to k cat / K M ( 16,18 O 2 ). Therefore, it reflects bonding changes that occur in steps beginning with O 2 encounter and the enzyme leading up to and including the first irreversible step.…”

Structural determinants for the inhibitory ligands of orotidine-5′-monophosphate decarboxylase

“…20,21 This transition-state intermediate appears during the early stage of the reaction, and electrostatic stress may play a role in the process of decarboxylation, although other mechanisms using computational and kinetic isotope methods suggest alternatives. 20,21,22,23,24 We have recently revealed that under suitable conditions ODCase can facilitate interesting reactions other than decarboxylation, such as the transformation of 6-cyano-UMP ( 9 ) into BMP ( 4 ). 25,26 Based on the catalytic promiscuity exhibited by ODCase, Wittmann et al proposed the possibility of a covalent mechanism as a unifying means of addressing various biochemical reactions undertaken by ODCase.…”