Cysteine 99 of endothelial nitric oxide synthase (NOS-III) is critical for tetrahydrobiopterin-dependent NOS-III stability and activity

Biochem Biophys Res Commun. 1995 Oct 24;215(3):1119-29. doi: 10.1006/bbrc.1995.2579.

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.

Publication types

  • Comparative Study
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Animals
  • Base Sequence
  • Biopterins / analogs & derivatives*
  • Biopterins / 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

Substances

  • DNA Primers
  • Isoenzymes
  • Recombinant Proteins
  • Biopterins
  • Nitric Oxide Synthase
  • sapropterin
  • Cysteine