Biomaterial Database

Identification of a glycine-rich sequence as an NAD(P)H-binding site and tyrosine 128 as a dicumarol-binding site in rat liver NAD(P)H:quinone oxidoreductase by site-directed mutagenesis. 1992

Q Ma, and K Cui, and F Xiao, and A Y Lu, and C S Yang

Laboratory for Cancer Research, College of Pharmacy, Rutgers University, Piscataway, New Jersey 08855.

Site-directed mutagenesis was utilized to identify binding sites for NAD(P)H and dicumarol in rat liver NAD(P)H:quinone oxidoreductase (NQOR, EC 1.6.99.2). The mutant cDNA clones were generated by a procedure based on the polymerase chain reaction and were expressed in Escherichia coli. The mutant enzymes were purified to apparent homogeneity as judged by SDS-polyacrylamide gel electrophoresis and were found to contain 2 FADs/enzyme molecule identical with that of the wild-type NQOR. Purified mutant enzymes Y128D, G150F, G150V, S151F, and Y155D showed dramatic decreases in activities in the reduction of dichlorophenolindophenol in comparison with the activities of the wild-type enzyme, whereas the activities of F124L, T127V, T127E, Y128V, Y128F, S151A, and Y155V were similar to those of NQOR. Enzyme kinetic analysis revealed that the Km values of T127E, Y128D, G150F, G150V, S151F, and Y155D were, respectively, 4-, 2-, 13-, 5-, 26-, and 19-fold higher than the Km of NQOR for NADPH, and were, respectively, 2-, 3-, 7-, 3-, 20-, and 11-fold higher than that of NQOR for NADH. The kcat values of Y128D, G150F, and G150V were also much lower than those of NQOR, but the kcat values of other mutants were similar to those of the wild-type enzyme. The Km values of the mutants for dichlorophenolindophenol were the same or slightly higher than that of NQOR. The apparent inhibition constants (Ki) for dicumarol on Y128V and F124L were elevated 12 and 8 times, respectively. Similar, but smaller, changes on Ki for 4-hydroxycoumarin were also observed. This study demonstrated that residues Gly150, Ser151, and Tyr155 in the glycine-rich region of NQOR are essential for NADPH and NADH binding and Tyr128 is important for dicumarol binding. Based on the results of the study, it is proposed that the glycine-rich region of the enzyme, along with other residues around the region, forms a beta sheet-turn-alpha helix structure important for the binding of the pyrophosphate group of NADPH and NADH.

UI	MeSH Term	Description	Entries
D007700	Kinetics	The rate dynamics in chemical or physical systems.
D008099	Liver	A large lobed glandular organ in the abdomen of vertebrates that is responsible for detoxification, metabolism, synthesis and storage of various substances.	Livers
D008969	Molecular Sequence Data	Descriptions of specific amino acid, carbohydrate, or nucleotide sequences which have appeared in the published literature and/or are deposited in and maintained by databanks such as GENBANK, European Molecular Biology Laboratory (EMBL), National Biomedical Research Foundation (NBRF), or other sequence repositories.	Sequence Data, Molecular,Molecular Sequencing Data,Data, Molecular Sequence,Data, Molecular Sequencing,Sequencing Data, Molecular
D009249	NADP	Nicotinamide adenine dinucleotide phosphate. A coenzyme composed of ribosylnicotinamide 5'-phosphate (NMN) coupled by pyrophosphate linkage to the 5'-phosphate adenosine 2',5'-bisphosphate. It serves as an electron carrier in a number of reactions, being alternately oxidized (NADP+) and reduced (NADPH). (Dorland, 27th ed)	Coenzyme II,Nicotinamide-Adenine Dinucleotide Phosphate,Triphosphopyridine Nucleotide,NADPH,Dinucleotide Phosphate, Nicotinamide-Adenine,Nicotinamide Adenine Dinucleotide Phosphate,Nucleotide, Triphosphopyridine,Phosphate, Nicotinamide-Adenine Dinucleotide
D009838	Oligodeoxyribonucleotides	A group of deoxyribonucleotides (up to 12) in which the phosphate residues of each deoxyribonucleotide act as bridges in forming diester linkages between the deoxyribose moieties.	Oligodeoxynucleotide,Oligodeoxyribonucleotide,Oligodeoxynucleotides
D011485	Protein Binding	The process in which substances, either endogenous or exogenous, bind to proteins, peptides, enzymes, protein precursors, or allied compounds. Specific protein-binding measures are often used as assays in diagnostic assessments.	Plasma Protein Binding Capacity,Binding, Protein
D001728	Dicumarol	An oral anticoagulant that interferes with the metabolism of vitamin K. It is also used in biochemical experiments as an inhibitor of reductases.	Bishydroxycoumarin,Dicoumarin,Dicoumarol
D003067	Coenzymes	Small molecules that are required for the catalytic function of ENZYMES. Many VITAMINS are coenzymes.	Coenzyme,Enzyme Cofactor,Cofactors, Enzyme,Enzyme Cofactors,Cofactor, Enzyme
D003600	Cytosol	Intracellular fluid from the cytoplasm after removal of ORGANELLES and other insoluble cytoplasmic components.	Cytosols
D005998	Glycine	A non-essential amino acid. It is found primarily in gelatin and silk fibroin and used therapeutically as a nutrient. It is also a fast inhibitory neurotransmitter.	Aminoacetic Acid,Glycine, Monopotassium Salt,Glycine Carbonate (1:1), Monosodium Salt,Glycine Carbonate (2:1), Monolithium Salt,Glycine Carbonate (2:1), Monopotassium Salt,Glycine Carbonate (2:1), Monosodium Salt,Glycine Hydrochloride,Glycine Hydrochloride (2:1),Glycine Phosphate,Glycine Phosphate (1:1),Glycine Sulfate (3:1),Glycine, Calcium Salt,Glycine, Calcium Salt (2:1),Glycine, Cobalt Salt,Glycine, Copper Salt,Glycine, Monoammonium Salt,Glycine, Monosodium Salt,Glycine, Sodium Hydrogen Carbonate,Acid, Aminoacetic,Calcium Salt Glycine,Cobalt Salt Glycine,Copper Salt Glycine,Hydrochloride, Glycine,Monoammonium Salt Glycine,Monopotassium Salt Glycine,Monosodium Salt Glycine,Phosphate, Glycine,Salt Glycine, Monoammonium,Salt Glycine, Monopotassium,Salt Glycine, Monosodium