Effects of Ca2+-Activated Calmodulin on Neuronal Nitric Oxide Synthase Reductase Activity and Binding of Substrates:  pH Dependence of Kinetic Parameters†

Biochemistry ◽  
2002 ◽  
Vol 41 (1) ◽  
pp. 205-214 ◽  
Author(s):  
Kirsten R. Wolthers ◽  
Michael I. Schimerlik
Keyword(s):  
Nitric Oxide ◽  
Nitric Oxide Synthase ◽  
Kinetic Parameters ◽  
Reductase Activity ◽  
Ph Dependence ◽  
Neuronal Nitric Oxide Synthase ◽  
Oxide Synthase

scholarly journals Identification of the domains of neuronal nitric oxide synthase by limited proteolysis

1996 ◽  
Vol 314 (1) ◽  
pp. 55-62 ◽  
Author(s):  
Peter N. LOWE ◽  
Derek SMITH ◽  
David K. STAMMERS ◽  
Valentina RIVEROS-MORENO ◽  
Salvador MONCADA ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Nitric Oxide Synthase ◽  
Reductase Activity ◽  
Limited Proteolysis ◽  
Neuronal Nitric Oxide Synthase ◽  
Binding Region ◽  
Calmodulin Binding ◽  
N Terminus ◽  
Oxide Synthase ◽  
Brain Nitric Oxide

Nitric oxide synthase (EC 1.14.13.39) binds arginine and NADPH as substrates, and FAD, FMN, tetrahydrobiopterin, haem and calmodulin as cofactors. The protein consists of a central calmodulin-binding sequence flanked on the N-terminal side by a haem-binding region, analogous to cytochrome P-450, and on the C-terminal side by a region homologous with NADPH:cytochrome P-450 reductase. The structure of recombinant rat brain nitric oxide synthase was analysed by limited proteolyis. The products were identified by using antibodies to defined sequences, and by N-terminal sequencing. Low concentrations of trypsin produced three fragments, similar to those in a previous report [Sheta, McMillan and Masters (1994) J. Biol. Chem. 269, 15147–15153]: that of Mr approx. 135000 (N-terminus Gly-221) resulted from loss of the N-terminal extension (residues 1–220) unique to neuronal nitric oxide synthase. The fragments of Mr 90000 (haem region) and 80000 (reductase region, N-terminus Ala-728) were produced by cleavage within the calmodulin-binding region. With more extensive trypsin treatment, these species were shown to be transient, and three smaller, highly stable fragments of Mr 14000 (N-terminus Leu-744 within the calmodulin region), 60000 (N-terminus Gly-221) and 63000 (N-terminus Lys-856 within the FMN domain) were formed. The species of Mr approx. 60000 represents a domain retaining haem and nitroarginine binding. The two species of Mr 63000 and 14000 remain associated as a complex. This complex retains cytochrome c reductase activity, and thus is the complete reductase region, yet cleaved at Lys-856. This cleavage occurs within a sequence insertion relative to the FMN domain present in inducible nitric oxide synthase. Prolonged proteolysis treatment led to the production of a protein of Mr approx. 53000 (N-terminus Ala-953), corresponding to a cleavage between the FMN and FAD domains. The major products after chymotryptic digestion were similar to those with trypsin, although the pathway of intermediates differed. The haem domain was smaller, starting at residue 275, yet still retained the arginine binding site. These data have allowed us to identify stable domains representing both the arginine/haem-binding and the reductase regions.

scholarly journals Thermodynamic characterization of five key kinetic parameters that define neuronal nitric oxide synthase catalysis

FEBS Journal ◽  
2013 ◽  
Vol 280 (18) ◽  
pp. 4439-4453 ◽  
Author(s):  
Mohammad Mahfuzul Haque ◽  
Jesús Tejero ◽  
Mekki Bayachou ◽  
Zhi-Qiang Wang ◽  
Mohammed Fadlalla ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Nitric Oxide Synthase ◽  
Kinetic Parameters ◽  
Neuronal Nitric Oxide Synthase ◽  
Thermodynamic Characterization ◽  
Oxide Synthase
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