Reductase domain cysteines 1048 and 1114 are critical for catalytic activity of human endothelial cell nitric oxide synthase as probed by site-directed mutagenesis.
We examined whether highly conserved cysteine residues in the reductase domain of the constitutive isoform of nitric oxide synthase in human endothelial cells (ecNOS) are crucial for catalytic activity of the enzyme. Substitution of alanine for cysteines 976 (Cys-976), 991 (Cys-991), 1048 (Cys-1048), or 1114 (Cys-1114), located in the reductase domain of human ecNOS, was achieved by oligonucleotide-directed mutagenesis and expression in COS-7 cells. The specific activity of ecNOS was > 7-fold increased in wild-type and in mutants Cys-976 and Cys-991, but not in mutants Cys-1048 and Cys-1114. However, Western blot analysis indicated that expression of ecNOS protein was comparable in wild-type and in all mutants. NADPH concentration-dependent L-citrulline formation and NADPH oxidation during L-arginine metabolism were reduced in mutants Cys-1048 and Cys-1114 compared to wild-type. Similarly, NADPH cytochrome c reductase activity was increased in a time-dependent fashion in wild-type but not in mutants Cys-1048 and Cys-1114. These results indicate that Cys-1048 and Cys-1114 residues in the NADPH binding site of the reductase domain are critical for human ecNOS activity. The lack of utilization of NADPH in L-arginine metabolism and in cytochrome c reduction suggests that these active site cysteine residues may be responsible for binding of NADPH and/or for electron transfer in human ecNOS.
Zhang
J L
JL
Department of Medicine, University of Florida, Gainesville 32608-1197, USA.
Patel
J M
JM
Li
Y D
YD
Block
E R
ER
D000818:Animals; D001483:Base Sequence; D002384:Catalysis; D002460:Cell Line; D003545:Cysteine / Q000235:genetics / Q000378:metabolism*; D003574:Cytochrome c Group / Q000378:metabolism; D018076:DNA, Complementary; D004727:Endothelium / Q000201:enzymology*; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009249:NADP / Q000378:metabolism; D019001:Nitric Oxide Synthase / Q000235:genetics / Q000378:metabolism*; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000235:genetics / Q000378:metabolism*
D003574:Cytochrome c Group; D018076:DNA, Complementary; D009249:NADP; D010088:Oxidoreductases; D019001:Nitric Oxide Synthase; D003545:Cysteine