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J Biol Chem, Vol. 273, Issue 51, 34164-34170, December 18, 1998

Effects of Asp-369 and Arg-372 Mutations on Heme Environment and Function in Human Endothelial Nitric-oxide Synthase

From the Vascular Biology Research Center and Division of Hematology, Department of Internal Medicine, University of Texas Health Science Center, Houston, Texas 77225

Eight polar amino acid residues in the putative substrate-binding region from Thr-360 to Val-379 in human endothelial nitric-oxide synthase (eNOS) (Thr-360, Arg-365, Cys-368, Asp-369, Arg-372, Tyr-373, Glu-377, and Asp-378) were individually mutated. Only two of these residues, Asp-369 and Arg-372, were found to be essential for enzyme activity. A further series of mutants was generated by replacing these two residues with various amino acids and the mutant proteins were expressed in a baculovirus system. Mutant eNOS had a very low L-citrulline formation activity with the exception of D369E and R372K, which retained 27% and 44% of the wild-type enzyme activity, respectively. Unlike the wild-type enzyme, all mutants except D369E, R372K, and R372M had a low spin heme (Soret peak at 416 nm). All the Asp-369 mutants had higher K_d values for L-arginine (1-10 mM) than wild-type eNOS (0.4 µM) and an unstable heme-CO complex, and except for D369E, had a very low (6R)-5,6,7,8-tetrahydro-L-biopterin (BH₄) content. In contrast, each of Arg-372 mutants retained a considerable amount of BH₄, had a moderate reduction in L-arginine affinity, and had a more stable heme-CO complex. 1-Phenylimidazole did not bind to wild-type eNOS heme, but bound to all Asp-369 and Arg-372 mutants (K_d ranged from 10 to 65 µM) except R372K. Heme spin-state changes caused by binding of 3,5-lutidine appeared to depend on both charge and size of the side chains of residues 369 and 372. Furthermore, all Asp-369 and Arg-372 mutants were defective in dimer formation. These results suggest that residues Asp-369 and Arg-372 in eNOS play a critical role in oxygenase domain active-site structure and activity.

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