TY - JOUR
T1 - Unusual role of Tyr588 of neuronal nitric oxide synthase in controlling substrate specificity and electron transfer
AU - Sato, Yuko
AU - Sagami, Ikuko
AU - Matsui, Toshitaka
AU - Shimizu, Toru
N1 - Funding Information:
This work was supported in part by the Ogura Science Foundation to Y.S., and by Grants-in-Aid from the Ministry of Education, Science, Sports and Culture of Japan to I.S. (12680624) and T.S. (09480130). Abbreviations used: NO, nitric oxide; NHA, NG-hydroxy-L-Arg; NOS, nitric-oxide synthase; P450, cytochrome P450; H4B, (6R)-5,6,7,8-tetrahydro-L-biopterin; CaM, calmodulin; nNOS, neuronal NOS; iNOS, inducible NOS; eNOS, endothelial NOS; DTT, dithiothreitol. 1To whom correspondence should be addressed at Institute for Chemical Reaction Science, Tohoku University, 2-1-1 Katahira, Aoba-ku, Sendai 980-8577, Japan. Fax: +81-22-217-5604/5664. E-mail: shimizu@icrs.tohoku.ac.jp.
PY - 2001
Y1 - 2001
N2 - Nitric oxide (NO) is synthesized from L-Arg via NG-hydroxyl-L-Arg (NHA) in the heine active site of nitric oxide synthase (NOS). According to the crystal structure of other NOS isoforms, the carboxylate group of L-Arg hydrogen bonds to the hydroxyl group of the conserved Tyr588 residue in the heme distal site of neuronal NOS (nNOS). Indeed, the nNOS mutations Tyr588His, Tyr588Ser, and Tyr588Phe markedly increased the dissociation constants for L-Arg and NHA by 2.2-8.2-fold and 1.5-3.9-fold, respectively. Similarly, Tyr588His and Tyr588Ser mutations markedly decreased the L-Arg-driven NO formation rates by 50 and 30% than that of the wild type, respectively. However, the catalytic activities of the same mutants using NHA were higher than that of the wild type by up to 136%. As a result, the turnover ratio of NHA to L-Arg was 4.12 for the Tyr588Ser mutant, compared with 1.07 for the wild-type enzyme. Intriguingly, heme reduction rates for the Tyr588 mutants were much lower than for wild type by two orders of magnitude.
AB - Nitric oxide (NO) is synthesized from L-Arg via NG-hydroxyl-L-Arg (NHA) in the heine active site of nitric oxide synthase (NOS). According to the crystal structure of other NOS isoforms, the carboxylate group of L-Arg hydrogen bonds to the hydroxyl group of the conserved Tyr588 residue in the heme distal site of neuronal NOS (nNOS). Indeed, the nNOS mutations Tyr588His, Tyr588Ser, and Tyr588Phe markedly increased the dissociation constants for L-Arg and NHA by 2.2-8.2-fold and 1.5-3.9-fold, respectively. Similarly, Tyr588His and Tyr588Ser mutations markedly decreased the L-Arg-driven NO formation rates by 50 and 30% than that of the wild type, respectively. However, the catalytic activities of the same mutants using NHA were higher than that of the wild type by up to 136%. As a result, the turnover ratio of NHA to L-Arg was 4.12 for the Tyr588Ser mutant, compared with 1.07 for the wild-type enzyme. Intriguingly, heme reduction rates for the Tyr588 mutants were much lower than for wild type by two orders of magnitude.
KW - Arginine
KW - Electron transfer
KW - Heme reduction
KW - NADPH oxidation
KW - Nitric-oxide synthase
KW - Site-directed mutagenesis
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U2 - 10.1006/bbrc.2001.4356
DO - 10.1006/bbrc.2001.4356
M3 - Article
C2 - 11237702
AN - SCOPUS:0034817107
VL - 281
SP - 621
EP - 626
JO - Biochemical and Biophysical Research Communications
JF - Biochemical and Biophysical Research Communications
SN - 0006-291X
IS - 3
ER -