Kinetics of CO and NO ligation with the Cys(331)-->Ala mutant of neuronal nitric-oxide synthase.
Nitric-oxide synthases (NOS) catalyze the conversion of l-arginine to NO, which then stimulates many physiological processes. In the active form, each NOS is a dimer; each strand has both a heme-binding oxygenase domain and a reductase domain. In neuronal NOS (nNOS), there is a conserved cysteine motif (CX(4)C) that participates in a ZnS(4) center, which stabilizes the dimer interface and/or the flavoprotein-heme domain interface. Previously, the Cys(331) --> Ala mutant was produced, and it proved to be inactive in catalysis and to have structural defects that disrupt the binding of l-Arg and tetrahydrobiopterin (BH(4)). Because binding l-Arg and BH(4) to wild type nNOS profoundly affects CO binding with little effect on NO binding, ligand binding to the mutant was characterized as follows. 1) The mutant initially has behavior different from native protein but reminiscent of isolated heme domain subchains. 2) Adding l-Arg and BH(4) has little effect immediately but substantial effect after extended incubation. 3) Incubation for 12 h restores behavior similar but not quite identical to that of wild type nNOS. Such incubation was shown previously to restore most but not all catalytic activity. These kinetic studies substantiate the hypothesis that zinc content is related to a structural rather than a catalytic role in maintaining active nNOS.
Scheele
J S
JS
Department of Medicine, University of Freiburg, D-79106 Freiburg, Germany.
Bruner
E
E
Zemojtel
T
T
Martásek
P
P
Roman
L J
LJ
Masters
B S
BS
Sharma
V S
VS
Magde
D
D
D000818:Animals; D001120:Arginine / Q000378:metabolism; D001708:Biopterins / Q000031:analogs & derivatives* / Q000378:metabolism; D002248:Carbon Monoxide / Q000378:metabolism*; D003545:Cysteine / Q000235:genetics; D007700:Kinetics; D009474:Neurons / Q000201:enzymology*; D009569:Nitric Oxide / Q000378:metabolism*; D019001:Nitric Oxide Synthase / Q000378:metabolism*; D017354:Point Mutation; D051381:Rats
D001708:Biopterins; D009569:Nitric Oxide; D002248:Carbon Monoxide; D001120:Arginine; D019001:Nitric Oxide Synthase; C003402:sapropterin; D003545:Cysteine