Proton Transfer from the C₅-proR/proS Positions of l-Dihydroorotate:  General-Base Catalysis, Isotope Effects, and Internal Return¹

Rate constants for C 5 - proR / proS -hydron exchange from l -dihydroorotate were determined by 1 H NMR and detritiation in various oxygen-containing and amine buffers at 37 °C and ionic strength 1.0 M in aqueous solution. Thermodynamically unfavorable proton transfer from the C 5 - proR and -proS positions (p K a = 20−21) to oxygen-containing and amine bases shows general-base catalysis with a Brønsted β value of 0.84 ± 0.05 and 0.81 ± 0.08, respectively, which is consistent with a late, enolate-like transition state. General-base catalysis is detectable because there is a 160- or 85-fold negative deviation for the C 5 - proR and -proS protons, respectively, from this correlation for deuterioxide ion. Deviations of ( k H / k T ) obsd and ( k D / k T ) obsd from the Swain−Schaad equation are consistent with internal return of the transferred hydron to a free C 5 -carbanion/enolate intermediate from the conjugate general acid. This corresponds to an Eigen-type mechanism for hydron transfer, in which both proton transfer and diffusional separation of the C 5 -carbanion/enolate·conjugate general-acid complex are partially rate-limiting, and a modest intrinsic barrier for C 5 -hydron exchange. It is concluded that the C 5 -carbanion/enolate can have a significant lifetime in aqueous solution and on dihydroorotate dehydrogenases (EC 1.3.1.14; EC 1.3.99.11).