Flavoprotein reductases catalyze the pyridine nucleotide dependent reduction of biochemically important disulfide compounds to their corresponding dithiols, thus regulating the redox level of these sulfur compounds. This investigation details approaches to the study of the chemical mechanism of glutathione reductase and NADH peroxidase. In the case of glutathione reductase, primary and secondary deuterium kinetic isotope effects on pyridine nucleotide oxidation will be measured using NADPH and NADPH analogs. The intrinsic isotope effect on pyridine nucleotide oxidation will be determined by comparing the primary tritium and deuterium kinetic isotope effects. The effects of pH on the kinetic parameters and on the primary isotope effects will be used to identify the enzymic groups involved in the binding of substrates and in catalysis. In the case of NADH peroxidase, the only flavin containing peroxidase, experiments are described which will determine the kinetic mechanism and stereochemistry of pyridine nucleotide oxidation. Primary and secondary deuterium kinetic isotope effects on pyridine nucleotide oxidation will be measured using NADH and NADH analogs. The intrinsic isotope effect will be measured as described above, and compare to that obtained for glutathione reductase. The effect of pH on the kinetic parameters and on the primary isotope effects will be examined, as will the Vmax activation by anions. Stopped-flow spectral studies will be performed to identify flavin intermediates in the oxidative half-reaction. Both of these enzymes serve protective functions in vivo: glutathione reductase being responsibe for maintaining glutathione in the reduced state to serve its function in detoxification and peroxide removal via glutathione peroxidase, while NADH peroxidase directly removes hydrogen peroxide from the cellular milieu. Since the three-dimensional structure of glutathione reductase has been solved, and these two enzymes appear to use identical catalytic functionalities to perform their respective chemical transformations, it should be possible to use the static picture obtained by crystallographic analysis to interpret and evaluate the kinetic events which result in enzyme catalysis.
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