Studies on the formation of glutathionylcobalamin: Any free intracellular aquacobalamin is likely to be rapidly and irreversibly converted to glutathionylcobalamin

A decade ago Jacobsen and co-workers reported the first evidence for the presence of glutathionylcobalamin (GSCbl) in mammalian cells and suggested that it could in fact be a precursor to the formation of the two coenzyme forms of vitamin B₁₂, adenosylcobalamin and methylcobalamin (Pezacka et al. Biochem. Biophys. Res. Commun. 1990, 169, 443). It has also recently been proposed by McCaddon and co-workers that GSCbl may be useful for the treatment of Alzheimer's disease (McCaddon et al. Neurology 2002, 58, 1395). Aquacobalamin is one of the major forms of vitamin B₁₂ isolated from mammalian cells, and high concentrations of glutathione (1-10 mM) are also found in cells. We have now determined observed equilibrium constants, K_obs(GSCbl), for the formation of GSCbl from aquacobalamin and glutathione in the pH range 4.50-6.00. K_obs(GSCbl) increases with increasing pH, and this increase is attributed to increasing amounts of the thiolate forms (RS ^-) of glutathione. An estimate for the equilibrium constant for the formation of GSCbl from aquacobalamin and the thiolate forms of glutathione of ∼5 × 10⁹ M^-1 is obtained from the data. Hence, under biological conditions the formation of GSCbl from aquacobalamin and glutathione is essentially irreversible. The rate of the reaction between aquacobalamin/hydroxycobalamin and glutathione for 4.50 < pH < 11.0 has also been studied and the observed rate constant for the reaction was found to decrease with increasing pH. The data were fitted to a mechanism in which each of the 3 macroscopic forms of glutathione present in this pH region react with aquacobalamin, giving k₁ = 18.5 M^-1 s^-1, k ₂ = 28 ± 10 M^-1 s^-1, and k₃ = 163 ± 8 M^-1 s^-1. The temperature dependence of the observed rate constant at pH 7.40 (∼k₁) was also studied, and activation parameters were obtained typical of a dissociative process (ΔH‡ = 81.0 ± 0.5 kJ mol^-1 and ΔS‡ = 48 + 2 J K^-1 mol^-1). Formation of GSCbl from aquacobalamin is rapid; for example, at ∼5 mM concentrations of glutathione and at 37 °C, the half-life for formation of GSCbl from aquacobalamin and glutathione is 2.8 s. On the basis of our equilibrium and rate-constant data we conclude that, upon entering cells, any free (protein-unbound) aquacobalamin could be rapidly and irreversibly converted to GSCbl. GSCbl may indeed play an important role in vitamin B₁₂-dependent processes.