The goal of the present application is to determine the precise chemical mechanisms and transition state structures of the two half-reactions catalyzed by glutathione reductase and NADH peroxidase. In the case of glutathione reductase, we are interested in determining the factors which result in the differential transition state stabilization of the reductive half reaction by the yeast, spinach and human erythrocyte enzymes. The structure of human erythrocyte glutathione reductase has been determined, and we will interpret the data to be obtained within a precisely defined structural framework. We also propose to analyze the rate-limiting nature and transition state structure of proton transfers occurring in the oxidative half-reaction catalyzed by glutathione reductase. For NADH peroxidase, we propose to examine the chemical mechanism of peroxide cleavage, and investigate a unique, non-redox role for the bound cofactor, FAD. The methods that will be used to probe these questions include: determination of steady-state kinetic parameters for various nucleotide and reducible substrates, analysis of steady-state and pre-steady-state primary and secondary deuterium and tritium kinetic isotope effects, determination of solvent equilibrium and kinetic isotope effects, determination of enzyme-bound flavin and disulfide-dithiol redox potentials, and stopped-flow identification of flavin intermediates. The methodologies developed and information obtained for glutathione reductase and NADH peroxidase will be applicable to other flavoprotein reductases. Six enzymes may be considered to be members of this family: glutathione reductase trypanathione reductase, thioredoxin reductase, mercuric reductase, lipoamide dehydrogenase and NADH peroxidase. All these enzymes serve physiologically important roles, including maintenance of intracellular thiol redox poise, providing reducing equivalents for deoxyribonucleoside synthesis, heavy metal detoxication, and removal of injurious peroxides. While they each exhibit non-overlapping specificity for their respective reducible substrates, they share an impressive and extensive number of kinetic, stereochemical and physiocochemical similarities, including a high degree of primary sequence homology, especially int he active site region. A long term objective is to understand the structural basis for the functional differences observed within this family.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM033449-08
Application #
3283190
Study Section
Biochemistry Study Section (BIO)
Project Start
1984-04-01
Project End
1993-01-31
Budget Start
1992-02-01
Budget End
1993-01-31
Support Year
8
Fiscal Year
1992
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; 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; 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; 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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