title: Cysteine 99 of endothelial nitric oxide synthase (NOS-III) is critical for tetrahydrobiopterin-dependent NOS-III stability and activity.
abstract: Tetrahydrobiopterin (BH4) is an essential cofactor for all three isoforms of nitric oxide synthase (NOS). However, its binding sites and functional roles remain elusive. Here, we demonstrated that cys-99 of human endothelial NOS (ecNOS) is critical for BH4 involvement in NOS catalytic activity and stability. Mutation of cys-99 to alanine in ecNOS resulted in loss of catalytic activity which could be restored to the level of wild type by adding a high concentration of exogenous BH4 to the crude extract. Purified C99A mutant was unstable and its maximal activity was only about 20% of the purified wild type activity. Comparison of BH4 concentration-dependent citrulline formation between C99A and the wild type revealed that the BH4 concentrations required for generating half-maximal citrulline were 10-fold higher for C99A. Purified C99A had no detectable BH4 and had a reduced heme content when compared to the purified wild type, but retained the ability of forming CO-ferrous heme complex and had the same Km value for L-arginine (approximately 4 microM) as the wild type. These findings indicate that Cys-99 is critically involved in BH4 binding. Mutation of this residue leads to reduced affinity for BH4 and the resultant enzyme instability and irreversible heme loss.
mesh_terms: D000818:Animals; D001483:Base Sequence; D001708:Biopterins / Q000031:analogs & derivatives* / Q000494:pharmacology; D002460:Cell Line; D003001:Cloning, Molecular; D003545:Cysteine; D017931:DNA Primers; D004795:Enzyme Stability; D006801:Humans; D007527:Isoenzymes / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D019001:Nitric Oxide Synthase / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D018411:Spodoptera; D014162:Transfection
chemical_list: D017931:DNA Primers; D007527:Isoenzymes; D011994:Recombinant Proteins; D001708:Biopterins; D019001:Nitric Oxide Synthase; C003402:sapropterin; D003545:Cysteine