Effects of Asp-369 and Arg-372 mutations on heme environment and function in human endothelial nitric-oxide synthase.
Eight polar amino acid residues in the putative substrate-binding region from Thr-360 to Val-379 in human endothelial nitric-oxide synthase (eNOS) (Thr-360, Arg-365, Cys-368, Asp-369, Arg-372, Tyr-373, Glu-377, and Asp-378) were individually mutated. Only two of these residues, Asp-369 and Arg-372, were found to be essential for enzyme activity. A further series of mutants was generated by replacing these two residues with various amino acids and the mutant proteins were expressed in a baculovirus system. Mutant eNOS had a very low L-citrulline formation activity with the exception of D369E and R372K, which retained 27% and 44% of the wild-type enzyme activity, respectively. Unlike the wild-type enzyme, all mutants except D369E, R372K, and R372M had a low spin heme (Soret peak at 416 nm). All the Asp-369 mutants had higher Kd values for L-arginine (1-10 mM) than wild-type eNOS (0.4 microM) and an unstable heme-CO complex, and except for D369E, had a very low (6R)-5,6,7, 8-tetrahydro-L-biopterin (BH4) content. In contrast, each of Arg-372 mutants retained a considerable amount of BH4, had a moderate reduction in L-arginine affinity, and had a more stable heme-CO complex. 1-Phenylimidazole did not bind to wild-type eNOS heme, but bound to all Asp-369 and Arg-372 mutants (Kd ranged from 10 to 65 microM) except R372K. Heme spin-state changes caused by binding of 3, 5-lutidine appeared to depend on both charge and size of the side chains of residues 369 and 372. Furthermore, all Asp-369 and Arg-372 mutants were defective in dimer formation. These results suggest that residues Asp-369 and Arg-372 in eNOS play a critical role in oxygenase domain active-site structure and activity.
Chen
P F
PF
Vascular Biology Research Center and Division of Hematology, Department of Internal Medicine, University of Texas Health Science Center, Houston, Texas 77225, USA. pfchen@heart.med.uth.tmc.edu
Berka
V
V
Tsai
A L
AL
Wu
K K
KK
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001120:Arginine; D001224:Aspartic Acid; D002248:Carbon Monoxide / Q000378:metabolism; D020134:Catalytic Domain; D017931:DNA Primers; D006418:Heme / Q000378:metabolism*; D006801:Humans; D007093:Imidazoles / Q000494:pharmacology; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D019001:Nitric Oxide Synthase / Q000737:chemistry* / Q000378:metabolism*; D052248:Nitric Oxide Synthase Type I; D052247:Nitric Oxide Synthase Type II; D052250:Nitric Oxide Synthase Type III; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013053:Spectrophotometry
D017931:DNA Primers; D007093:Imidazoles; D011994:Recombinant Proteins; D001224:Aspartic Acid; D006418:Heme; C037738:1-phenylimidazole; D002248:Carbon Monoxide; D001120:Arginine; C496320:NOS1 protein, human; C496319:NOS2 protein, human; C496314:NOS3 protein, human; D019001:Nitric Oxide Synthase; D052248:Nitric Oxide Synthase Type I; D052247:Nitric Oxide Synthase Type II; D052250:Nitric Oxide Synthase Type III