A site-directed mutagenesis analysis of tNOX functional domains.
Constitutive NADH oxidase proteins of the mammalian cell surface exhibit two different activities, oxidation of hydroquinones (or NADH) and protein disulfide-thiol interchange which alternate to yield oscillatory patterns with period lengths of 24 min. A drug-responsive tNOX (tumor-associated NADH oxidase) has a period length of about 22 min. The tNOX cDNA has been cloned and expressed. These two proteins are representative of cycling oxidase proteins of the plant and animal cell surface. In this report, we describe a series of eight amino acid replacements in tNOX which, when expressed in Escherichia coli, were analyzed for enzymatic activity, drug response and period length. Replacement sites selected include six cysteines that lie within the processed plasma membrane (34 kDa) form of the protein, and amino acids located in putative drug and adenine nucleotide (NADH) binding domains. The latter, plus two of the cysteine replacements, resulted in a loss of enzymatic activity. The recombinant tNOX with the modified drug binding site retained activity but the activity was no longer drug-responsive. The four remaining cysteine replacements were of interest in that both activity and drug response were retained but the period length for both NADH oxidation and protein disulfide-thiol interchange was increased from 22 min to 36 or 42 min. The findings confirm the correctness of the drug and adenine nucleotide binding motifs within the tNOX protein and imply a potential critical role of cysteine residues in determining the period length.
Chueh
Pin-Ju
PJ
Department of Medicinal Chemistry and Molecular Pharmacology, Purdue University, West Lafayette, IN 47907, USA.
Morré
Dorothy M
DM
Morré
D James
DJ
D000409:Alanine / Q000737:chemistry; D001665:Binding Sites; D002211:Capsaicin / Q000494:pharmacology; D003545:Cysteine / Q000737:chemistry; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004926:Escherichia coli / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D009243:NAD / Q000737:chemistry; D009247:NADH, NADPH Oxidoreductases / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics*; D011994:Recombinant Proteins / Q000737:chemistry; D013997:Time Factors
D004791:Enzyme Inhibitors; D011994:Recombinant Proteins; D009243:NAD; D009247:NADH, NADPH Oxidoreductases; C431909:tumor-associated NADH oxidase; D003545:Cysteine; D000409:Alanine; D002211:Capsaicin