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PMID:7488039

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Citation

Chen, PF, Tsai, AL and Wu, KK (1995) Cysteine 99 of endothelial nitric oxide synthase (NOS-III) is critical for tetrahydrobiopterin-dependent NOS-III stability and activity. Biochem. Biophys. Res. Commun. 215:1119-29

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.

Links

PubMed Online version:10.1006/bbrc.1995.2579

Keywords

Animals; Base Sequence; Biopterin/analogs & derivatives; Biopterin/pharmacology; Cell Line; Cloning, Molecular; Cysteine; DNA Primers; Enzyme Stability; Humans; Isoenzymes/chemistry; Isoenzymes/metabolism; Kinetics; Molecular Sequence Data; Mutagenesis, Site-Directed; Nitric Oxide Synthase/biosynthesis; Nitric Oxide Synthase/chemistry; Nitric Oxide Synthase/metabolism; Point Mutation; Polymerase Chain Reaction; Recombinant Proteins/biosynthesis; Recombinant Proteins/chemistry; Recombinant Proteins/metabolism; Spodoptera; Transfection

Significance

Annotations

Gene product Qualifier GO Term Evidence Code with/from Aspect Extension Notes Status

HUMAN:NOS3

enables

GO:0034617: tetrahydrobiopterin binding

ECO:0000314: direct assay evidence used in manual assertion

F

Seeded From UniProt

complete

HUMAN:NOS3

enables

GO:0034618: arginine binding

ECO:0000314: direct assay evidence used in manual assertion

F

Seeded From UniProt

complete

HUMAN:NOS3

enables

GO:0020037: heme binding

ECO:0000314: direct assay evidence used in manual assertion

F

Seeded From UniProt

complete

HUMAN:NOS3

involved_in

GO:0006809: nitric oxide biosynthetic process

ECO:0000314: direct assay evidence used in manual assertion

P

Seeded From UniProt

complete

HUMAN:NOS3

involved_in

GO:0006527: arginine catabolic process

ECO:0000314: direct assay evidence used in manual assertion

P

Seeded From UniProt

complete

HUMAN:NOS3

enables

GO:0004517: nitric-oxide synthase activity

ECO:0000314: direct assay evidence used in manual assertion

F

Seeded From UniProt

complete

HUMAN:NOS3

enables

GO:0046870: cadmium ion binding

ECO:0000303: author statement without traceable support used in manual assertion

F

Seeded From UniProt

complete

HUMAN:NOS3

enables

GO:0010181: FMN binding

ECO:0000303: author statement without traceable support used in manual assertion

F

Seeded From UniProt

complete

HUMAN:NOS3

enables

GO:0050660: flavin adenine dinucleotide binding

ECO:0000303: author statement without traceable support used in manual assertion

F

Seeded From UniProt

complete

HUMAN:NOS3

enables

GO:0050661: NADP binding

ECO:0000303: author statement without traceable support used in manual assertion

F

Seeded From UniProt

complete

HUMAN:NOS1

enables

GO:0034617: tetrahydrobiopterin binding

ECO:0000303: author statement without traceable support used in manual assertion

F

Seeded From UniProt

complete


See also

References

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