NAD(H) functions as the coenzyme of a large number of enzymes. In addition to its well documented roles in oxidation-reduction reactions, NAD(H) is also involved in many aspects of metabolic regulation which have only recently been recognized. An understanding of the structure of these enzymes will be important for the understanding of those biochemical processes associated with these enzymes. However, among all NAD(H) dependent enzymes, only the structure of a few simple oxido-reduction enzymes have been investigated. In this application we propose to investigate the active site of two NAD(H) dependent enzymes, bovine heart mitochondrial NADH dehydrogenase, and NADH-NAD+ transhydrogenase by affinity labeling techniques. Both of these enzymes are important enzymes involved in the cellular energy production process. The mitochondrial NAD(H) dehydrogenase has also been shown to take part in mechanisms inducing cardiac oxygen toxicity and 1- methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) toxicity related to Parkinson Disease. Although many researchers have shown great interest in these enzymes, no structural information is yet available for either enzyme because of the heterogenity of these enzyme preparations. In this proposed project, the active sites of these enzymes will be identified by labeling with radioactive derivatives of six affinity probes of NAD+. The peptides modified by these probes will be characterized by microsequencing, amino acid sequences of analysis, and fast atom bombardment mass spectrometry. The amino acid sequences of the modified peptides of three enzymes will be compared to those at the active site regions of well characterized NAD(H)- dependent enzymes. This investigation will identify important regions of these two enzymes. The information generated through this study will be valuable for further understanding the molecular mechanism of the reactions these enzymes catalyze. Furthermore, this structural information will be very useful for future structure-function study of these two enzymes by molecular cloning and site specific mutagenesis experiments.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM037297-03
Application #
3292603
Study Section
Biochemistry Study Section (BIO)
Project Start
1988-04-01
Project End
1991-09-30
Budget Start
1990-04-01
Budget End
1991-09-30
Support Year
3
Fiscal Year
1990
Total Cost
Indirect Cost
Name
City of Hope/Beckman Research Institute
Department
Type
DUNS #
City
Duarte
State
CA
Country
United States
Zip Code
91010
Chen, S; Deng, P S; Swiderek, K et al. (1995) Interaction of flavones and their bromoacetyl derivatives with NAD(P)H:quinone acceptor oxidoreductase. Mol Pharmacol 47:419-24
Chen, S; Clarke, P E; Martino, P A et al. (1994) Mouse liver NAD(P)H:quinone acceptor oxidoreductase: protein sequence analysis by tandem mass spectrometry, cDNA cloning, expression in Escherichia coli, and enzyme activity analysis. Protein Sci 3:1296-304
Chen, S; Deng, P S; Bailey, J M et al. (1994) A two-domain structure for the two subunits of NAD(P)H:quinone acceptor oxidoreductase. Protein Sci 3:51-7
Chen, S; Hwang, J; Deng, P S (1993) Inhibition of NAD(P)H:quinone acceptor oxidoreductase by flavones: a structure-activity study. Arch Biochem Biophys 302:72-7
Chen, S; Liu, X F (1992) Suggested mechanism for the modulation of the activity of NAD(P)H:quinone acceptor oxidoreductase (DT-diaphorase) by menadione: interpretation of the effect of menadione on 5'-[p-(Fluorosulfonyl)benzoyl]adenosine labeling of rat liver NAD(P)H:quinone acce Mol Pharmacol 42:545-48
Chen, H H; Ma, J X; Forrest, G L et al. (1992) Expression of rat liver NAD(P)H:quinone-acceptor oxidoreductase in Escherichia coli and mutagenesis in vitro at Arg-177. Biochem J 284 ( Pt 3):855-60
Deng, P S; Zhao, S H; Iyanagi, T et al. (1991) Photodependent inhibition of rat liver NAD(P)H:quinone acceptor oxidoreductase by (A)-2-azido-NAD+ and (A)-8-azido-NAD. Biochemistry 30:6942-8
Rutherfurd, K J; Chen, S A; Shively, J E (1991) Isolation and amino acid sequence analysis of bovine adrenal 3 beta-hydroxysteroid dehydrogenase/steroid isomerase. Biochemistry 30:8108-16
Rutherfurd, K J; Chen, S A; Shively, J E (1991) Affinity labeling of bovine adrenal 3 beta-hydroxysteroid dehydrogenase/steroid isomerase by 5'-[p-(fluorosulfonyl)benzoyl]adenosine. Biochemistry 30:8116-23
Deng, P S; Hatefi, Y; Chen, S (1990) N-arylazido-beta-alanyl-NAD+, a new NAD+ photoaffinity analogue. Synthesis and labeling of mitochondrial NADH dehydrogenase. Biochemistry 29:1094-8

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