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  • Title: Kinetic isotope effects and electron transfer in the reduction of xanthine oxidoreductase with 4-hydroxypyrimidine. A comparison between oxidase and dehydrogenase forms.
    Author: Harris CM, Massey V.
    Journal: J Biol Chem; 1997 Sep 05; 272(36):22514-25. PubMed ID: 9278404.
    Abstract:
    Isolated from bovine milk, xanthine oxidase (XO) and xanthine dehydrogenase (XDH) are two interconvertible forms of the same protein, differing in the number of protein cysteines versus cystines. Most differences between XO and XDH are localized to the FAD center, the site at which the oxidizing substrates NAD and molecular oxygen react. A comparative study of the reduction of XO and XDH has been performed to assess differences in reactivity of the molybdopterin site, as well as subsequent electron-transfer events from molybdenum to 2Fe/2S and FAD centers. The compound 4-hydroxypyrimidine (4-OH-P) was chosen as reducing substrate because its higher Km value raised the possibility of binding weak enough to measure kinetically, and its high kcat value could allow detection of intramolecular electron-transfer reactions. As measured by stopped flow spectrophotometry, XO and XDH react with the first equivalent of 4-OH-P via similar mechanisms, differing in the magnitude of rate and dissociation constants. Using [2-2H]4-OH-P as substrate, a D(k/Kd) isotope effect of 1.9 to 2.3 suggests that movement of the hydrogen abstracted from substrate appreciably limits the rate of initial enzyme reduction from Mo(VI) to Mo(IV). Monitoring the visible spectrum of the enzymes, the first observed step is reduction of a single 2Fe/2S center and presumably re-oxidation of Mo(IV) to Mo(V). This suggests a common pathway for electron transfer involving reduction of a 2Fe/2S center prior to reduction of the second 2Fe/2S and FAD centers. Rates of the first electron transfer from molybdenum to the 2Fe/2S center are rapid, 290 s-1 with XO and 180 s-1 with XDH, and are consistent with rates measured by flash photolysis (Walker, M. C., Hazzard, J. T., Tollin, G., and Edmondson, D. E. (1991) Biochemistry 30, 5912-5917) allowing discrete observation of the electron-transfer reactions that occur during turnover. This step also exhibits a modest primary kinetic isotope effect of 1.5 to 1.6 when [2-2H]4-OH-P is used, possibly due to deprotonation of the molybdenum center prior to electron transfer. A second one-electron transfer, presumably oxidizing Mo(V) to Mo(VI), follows in a step coincident with product dissociation, consistent with a role for product release in controlling electron transfer events. The kinetics of this complex system are described and interpreted quantitatively in models that are consistent with all the data.
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