Mutagenesis of acidic residues in the oxygenase domain of inducible nitric-oxide synthase identifies a glutamate involved in arginine binding.
The oxygenase domain of the mouse cytokine-inducible nitric-oxide synthase (iNOSox, amino acids 1-498) binds heme, tetrahydrobiopterin, and the substrate Arg and is the domain responsible for catalyzing nitric oxide synthesis and maintaining the enzyme's active dimeric structure. To further understand iNOSox structure-function, we carried out alanine point mutagenesis on 15 conserved acidic residues located within a region of iNOSox (amino acids 352-473) that shares sequence homology with the pterin-binding module in dihydrofolate reductases and may be important for iNOSox subunit dimerization and/or Arg binding. Five point mutants were identical or nearly identical to wild-type, while 10 exhibited a range of defects that included low heme content (2), heme ligand instability (2), defective dimerization (2), and poor Arg and/or tetrahydrobiopterin binding (4). Mutations that caused defective tetrahydrobiopterin binding were also associated with other defects. In contrast, two mutants (E371A and D376A) exhibited an exclusive defect in Arg binding. These mutants were dimeric, indicating that dimerization of iNOSox in Escherichia coli does not require Arg. In one case (E371A), the defect in Arg binding was absolute, as assessed by spectral perturbation, radioligand binding, and catalytic studies. We conclude that mutagenesis of conserved acidic residues within this region of iNOSox can lead to exclusive defects in dimerization and in Arg binding. Modeling considerations predict that the E371 carboxylate may participate in Arg binding by interacting with its guanidine moiety.
Gachhui
R
R
Department of Immunology, The Cleveland Clinic Research Institute, Ohio 44195, USA.
Ghosh
D K
DK
Wu
C
C
Parkinson
J
J
Crane
B R
BR
Stuehr
D J
DJ
D000409:Alanine; D000595:Amino Acid Sequence; D000818:Animals; D000975:Antioxidants / Q000378:metabolism; D001120:Arginine / Q000378:metabolism*; D001708:Biopterins / Q000031:analogs & derivatives / Q000378:metabolism; D002417:Cattle; D002850:Chromatography, Gel; D018698:Glutamic Acid / Q000378:metabolism*; D006418:Heme / Q000378:metabolism; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D019001:Nitric Oxide Synthase / Q000235:genetics / Q000378:metabolism*; D010105:Oxygenases / Q000235:genetics / Q000378:metabolism; D051381:Rats; D013329:Structure-Activity Relationship
D000975:Antioxidants; D001708:Biopterins; D018698:Glutamic Acid; D006418:Heme; D001120:Arginine; D010105:Oxygenases; D019001:Nitric Oxide Synthase; C003402:sapropterin; D000409:Alanine