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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM033449-03
Application #
3283186
Study Section
Biochemistry Study Section (BIO)
Project Start
1984-04-01
Project End
1988-01-31
Budget Start
1986-04-01
Budget End
1988-01-31
Support Year
3
Fiscal Year
1986
Total Cost
Indirect Cost
Name
Albert Einstein College of Medicine
Department
Type
Schools of Medicine
DUNS #
009095365
City
Bronx
State
NY
Country
United States
Zip Code
10461
Argyrou, Argyrides; Vetting, Matthew W; Blanchard, John S (2004) Characterization of a new member of the flavoprotein disulfide reductase family of enzymes from Mycobacterium tuberculosis. J Biol Chem 279:52694-702
Argyrou, Argyrides; Blanchard, John S (2004) Flavoprotein disulfide reductases: advances in chemistry and function. Prog Nucleic Acid Res Mol Biol 78:89-142
Argyrou, Argyrides; Blanchard, John S (2004) Kinetic and chemical mechanism of Mycobacterium tuberculosis 1-deoxy-D-xylulose-5-phosphate isomeroreductase. Biochemistry 43:4375-84
Argyrou, Argyrides; Sun, Guangxing; Palfey, Bruce A et al. (2003) Catalysis of diaphorase reactions by Mycobacterium tuberculosis lipoamide dehydrogenase occurs at the EH4 level. Biochemistry 42:2218-28
Vetting, Matthew W; Roderick, Steven L; Yu, Michael et al. (2003) Crystal structure of mycothiol synthase (Rv0819) from Mycobacterium tuberculosis shows structural homology to the GNAT family of N-acetyltransferases. Protein Sci 12:1954-9
Argyrou, Argyrides; Blanchard, John S; Palfey, Bruce A (2002) The lipoamide dehydrogenase from Mycobacterium tuberculosis permits the direct observation of flavin intermediates in catalysis. Biochemistry 41:14580-90
Magnet, S; Lambert, T; Courvalin, P et al. (2001) Kinetic and mutagenic characterization of the chromosomally encoded Salmonella enterica AAC(6')-Iy aminoglycoside N-acetyltransferase. Biochemistry 40:3700-9
Argyrou, A; Blanchard, J S (2001) Mycobacterium tuberculosis lipoamide dehydrogenase is encoded by Rv0462 and not by the lpdA or lpdB genes. Biochemistry 40:11353-63
Patel, M P; Blanchard, J S (2001) Mycobacterium tuberculosis mycothione reductase: pH dependence of the kinetic parameters and kinetic isotope effects. Biochemistry 40:5119-26
Barlow, J N; Blanchard, J S (2000) Enzymatic synthesis of UDP-(3-deoxy-3-fluoro)-D-galactose and UDP-(2-deoxy-2-fluoro)-D-galactose and substrate activity with UDP-galactopyranose mutase. Carbohydr Res 328:473-80

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