Role of copper(II) in candidate human growth-related and time-keeping constitutive NADH oxidase (cCNOX) and molecular cloning of cCNOX
Abstract
The purpose of this research project is to identify the role of copper (II) in functions of constitutive nicotinamide adenine dinucleotide (NADH) oxidase (CNOX, ENOX1) and the biological clock. The constitutive NADH oxidase or CNOX is a human growth-related and time keeping protein located on the cell surface. In this study, a candidate CNOX (cCNOX) was cloned from a HeLa cDNA library. The open reading frame of the protein sequence included a NADH binding site, a disulfide-thiol interchange site and possible copper binding sites but excluded the anti-cancer drug binding site. The two enzymatic activities catalyzed by cCNOX, NADH oxidation and disulfide-thiol interchange, alternated to generate 24 min period oscillations with a complex pattern of five repeating maxima. The activities of the enzyme were inhibited by a CNOX-specific antibody but unaffected by the anti-cancer compounds. The oscillatory pattern of the reaction was shifted by addition of melatonin. Therefore, the candidate CNOX exhibited all properties attributed previously to CNOX protein and could function as the core oscillator of the biological clock. The study also found that copper (II) was necessary to sustain the activities of cCNOX. Removal of copper (II) from the protein caused the protein to lose enzymatic activity. Copper (II) alone in solution was capable of catalyzing NADH oxidation with a similar oscillation pattern of cCNOX. The period length was independent of temperature and pH. Application of a low frequency electromagnetic field to the NADH oxidation reactions catalyzed by either enzyme or copper (II) salt shifted the phase of the oscillations with unchanged period length. Through long term data recordings, a model of copper clock based on the regular oscillations of copper (II)-catalyzed NADH oxidation was finally constructed. As the cofactor of cCNOX enzyme, the content of copper bound to the protein was determined as two copper per one protein molecule. Site-directed mutagenesis was performed and finally two motifs, H260YSEH and H579VH, were decided as the copper binding sites in cCNOX. All of the results supported the hypothesis that the functions of cCNOX protein and regular oscillations of biological clock are inherent in the copper (II) ion bound with the protein as an enzyme cofactor.
Degree
Ph.D.
Advisors
Morre, Purdue University.
Subject Area
Biochemistry|Nutrition
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