Mutations at Lysine 525 of Inducible Nitric-oxide Synthase Affect Its Ca2+-independent Activity

Shiow-Ju Lee,Kathy Beckingham,James T Stull

doi:10.1074/jbc.m003935200

Abstract

Calmodulin binding to inducible nitric-oxide synthase may play an important role in its Ca(2+)-independent activity. Studies of inducible nitric-oxide synthase chimeras containing the calmodulin binding sequence of neuronal or endothelial nitric-oxide synthases show that the calmodulin binding sequence of inducible nitric-oxide synthase is necessary but not sufficient for the Ca(2+)-independent activity. The mutations at lysine 525 located at the C terminus of the calmodulin binding sequence of inducible nitric-oxide synthase were examined for the effects on the Ca(2+)-independent activity with chimeras containing the oxygenase or reductase domains of inducible or neuronal nitric-oxide synthases. Results show that the Ca(2+)-independent binding of calmodulin is not solely responsible for maximal Ca(2+)-independent activity of inducible nitric-oxide synthase. Lysine 525 of inducible nitric-oxide synthase may also play an important role in coordinating the maximal Ca(2+)-independent activity.

Highlights

All NOS1 isoforms contain three similar functional domains, an oxygenase domain in the N-terminal half and a reductase domain in the C-terminal half with an intervening CaM-binding domain
The mutations at lysine 525 located at the C terminus of the calmodulin binding sequence of inducible nitric-oxide synthase were examined for the effects on the Ca2؉-independent activity with chimeras containing the oxygenase or reductase domains of inducible or neuronal nitric-oxide synthases
Studies of the interactions of CaM mutants with nNOS indicated that CaM binding and stimulation of NO synthesis are distinct and separate events, CaM binding to the CaM-binding sequence is necessary for catalysis [16, 17]

Summary

Introduction

All NOS1 isoforms contain three similar functional domains, an oxygenase domain in the N-terminal half and a reductase domain in the C-terminal half with an intervening CaM-binding domain. Dimerization necessary for NOS activity occurs mainly through the intersubunit interactions of the oxygenase domains to form the active sites and is independent of the reductase domain [1]. NOS chimeras, containing the CaM-binding region and the oxygenase domain or the reductase domain of iNOS with different affinities for CaM, confer significant Ca2ϩ-independent activity. The maximal Ca2ϩ-independent activity of iNOS may be conferred by a range of structural features in all three domains of the enzyme, the CaM-binding site and oxygenase and reductase domains. Mutation or phosphorylation of the CaM-binding regions of nNOS or eNOS decreases Ca2ϩ/CaM binding affinity (12, 18 –20) and enzyme activity [18, 20]. The mutations at Lys-525 affect iNOS activity in the absence of Ca2ϩ but not through Ca2ϩ-independent binding of CaM in the CaMNOS complex

Methods

Results

Conclusion