Cysteine 99 of endothelial nitric oxide synthase (NOS-III) is critical for tetrahydrobiopterin-dependent NOS-III stability and activity.
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.
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
D017931:DNA Primers; D007527:Isoenzymes; D011994:Recombinant Proteins; D001708:Biopterins; D019001:Nitric Oxide Synthase; C003402:sapropterin; D003545:Cysteine