Mutation of Glu-361 in human endothelial nitric-oxide synthase selectively abolishes L-arginine binding without perturbing the behavior of heme and other redox centers.
Nitric oxide (NO) and L-citrulline are formed from the oxidation of L-arginine by three different isoforms of NO synthase (NOS). Defining amino acid residues responsible for L-arginine binding and oxidation is a primary step toward a detailed understanding of the NOS reaction mechanisms and designing strategies for the selective inhibition of the individual isoform. We have altered Glu-361 in human endothelial NOS to Gln or Leu by site-directed mutagenesis and found that these mutations resulted in a complete loss of L-citrulline formation without disruption of the cytochrome c reductase and NADPH oxidase activities. Optical and EPR spectroscopic studies demonstrated that the Glu-361 mutants had similar spectra either in resting state or reduced CO-complex as the wild type. The heme ligand, imidazole, could induce a low spin state in both wild-type and Glu-361 mutants. However, unlike the wild-type enzyme, the low spin imidazole complex of Glu-361 mutants was not reversed to a high spin state by addition of either L-arginine, acetylguanidine, or 2-aminothiazole. Direct L-arginine binding could not be detected in the mutants either. These results strongly indicate that Glu-361 in human endothelial NOS is specifically involved in the interaction with L-arginine. Mutation of this residue abolished the L-arginine binding without disruption of other functional characteristics.
Chen
P F
PF
Department of Internal Medicine, Vascular Biology Research Center and Division of Hematology, University of Texas Health Science Center, Houston, Texas 77030, USA.
Tsai
A L
AL
Berka
V
V
Wu
K K
KK
D000595:Amino Acid Sequence; D001120:Arginine / Q000378:metabolism; D001665:Binding Sites; D004578:Electron Spin Resonance Spectroscopy; D004730:Endothelium, Vascular / Q000201:enzymology; D005971:Glutamates / Q000737:chemistry; D006418:Heme / Q000378:metabolism; D006420:Hemeproteins / Q000737:chemistry; D006801:Humans; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009249:NADP / Q000378:metabolism; D019001:Nitric Oxide Synthase / Q000737:chemistry*; D010084:Oxidation-Reduction; D013329:Structure-Activity Relationship
D005971:Glutamates; D006420:Hemeproteins; D006418:Heme; D009249:NADP; D001120:Arginine; D019001:Nitric Oxide Synthase