Endothelial nitric-oxide synthase. Expression in Escherichia coli, spectroscopic characterization, and role of tetrahydrobiopterin in dimer formation.
Bovine endothelial nitric-oxide synthase (eNOS) expressed in Escherichia coli does not have the post-translational modifications found in the native enzyme and is free of tetrahydrobiopterin (BH4). In the presence of BH4, eNOS has an absorption maximum at 400 nm that shifts to 395 nm when the substrate L-arginine is added. The low-spin component of the spectrum of the BH4-free protein is decreased by the addition of BH4 without a corresponding increase in the high-spin component. Addition of BH4 decreases the low-spin population of eNOS even in the presence of excess L-arginine. These results indicate that BH4 directly modulates the heme environment. BH4-free eNOS is completely inactive, but catalytic activity is recovered when BH4 (EC50 approximately 200 nM) is added. The spectroscopically determined binding constants for L-arginine are approximately 1.9 microM in the presence and approximately 4.0 microM in the absence of BH4. The BH4-supplemented enzyme has an activity of 90-120 nmol of citrulline.min-1.mg-1 and Km values of 3 and 14 microM for L-arginine and N-hydroxy-L-arginine, respectively. Of particular interest is the finding by SDS-polyacrylamide gel electrophoresis that BH4-free eNOS exists in a monomer-dimer equilibrium very similar to that observed with the BH4-reconstituted protein. Addition of BH4, increases the percent of the dimer by only approximately 5%. The results establish that BH4 influences the heme environment and stabilizes the protein with respect to heme loss but is not required for dimer formation.
Rodríguez-Crespo
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Department of Pharmaceutical Chemistry, School of Pharmacy, University of California, San Francisco 94143-0446, USA.
Gerber
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Ortiz de Montellano
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PR
D000818:Animals; D001120:Arginine / Q000378:metabolism; D001708:Biopterins / Q000031:analogs & derivatives* / Q000494:pharmacology; D002417:Cattle; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004730:Endothelium, Vascular / Q000201:enzymology*; D006418:Heme; D006639:Histidine; D007700:Kinetics; D046911:Macromolecular Substances; D008970:Molecular Weight; D019001:Nitric Oxide Synthase / Q000096:biosynthesis* / Q000737:chemistry* / Q000302:isolation & purification; D011489:Protein Denaturation; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D015183:Restriction Mapping; D016324:Sequence Tagged Sites; D013053:Spectrophotometry; D013379:Substrate Specificity
D046911:Macromolecular Substances; D011994:Recombinant Proteins; D001708:Biopterins; D006418:Heme; D006639:Histidine; D001120:Arginine; D019001:Nitric Oxide Synthase; C003402:sapropterin