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 B(12), 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 B(12) 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 approximately 5 x 10(9) 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 k(1) = 18.5 M(-1) s(-1), k(2) = 28 +/- 10 M(-1) s(-1), and k(3) = 163 +/- 8 M(-1) s(-1). The temperature dependence of the observed rate constant at pH 7.40 ( approximately k(1)) was also studied, and activation parameters were obtained typical of a dissociative process (DeltaH++ = 81.0 +/- 0.5 kJ mol(-1) and DeltaS++ = 48 +/- 2 J K(-1) mol(-1)). Formation of GSCbl from aquacobalamin is rapid; for example, at approximately 5 mM concentrations of glutathione and at 37 degrees 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 B(12)-dependent processes.
The selenoprotein thioredoxin reductase (TrxR1) is an essential antioxidant enzyme known to reduce many compounds in addition to thioredoxin, its principle protein substrate. Here we found that TrxR1 reduced ubiquinone-10 and thereby regenerated the antioxidant ubiquinol-10 (Q10), which is important for protection against lipid and protein peroxidation. The reduction was timeand dose-dependent, with an apparent K m of 22 M and a maximal rate of about 12 nmol of reduced Q10 per milligram of TrxR1 per minute. TrxR1 reduced ubiquinone maximally at a physiological pH of 7.5 at similar rates using either NADPH or NADH as cofactors. The reduction of Q10 by mammalian TrxR1 was selenium dependent as revealed by comparison with Escherichia coli TrxR or selenium-deprived mutant and truncated mammalian TrxR forms. In addition, the rate of reduction of ubiquinone was significantly higher in homogenates from human embryo kidney 293 cells stably overexpressing thioredoxin reductase and was induced along with increasing cytosolic TrxR activity after the addition of selenite to the culture medium. These data demonstrate that the selenoenzyme thioredoxin reductase is an important selenium-dependent ubiquinone reductase and can explain how selenium and ubiquinone, by a combined action, may protect the cell from oxidative damage.
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