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Autoinhibition of neuronal nitric oxide synthase: distinct effects of reactive nitrogen and oxygen species on enzyme activity

Kotsonis, Peter, Frey, Armin, Fröhlich, Lothar G., Hofmann, Heinrich, Reif, Andreas, Wink, David A., Feelisch, Martin and Schmidt, Harald H.H.W. (1999) Autoinhibition of neuronal nitric oxide synthase: distinct effects of reactive nitrogen and oxygen species on enzyme activity Biochemical Journal, 340, pp. 745-752. (PMID:10359660).

Record type: Article

Abstract

Nitric oxide (NO) synthases (NOSs), which catalyse the oxidation of L-arginine to L-citrulline and an oxide of nitrogen, possibly NO or nitroxyl (NO-), are subject to autoinhibition by a mechanism that has yet to be fully elucidated. In the present study we investigated the actions of NO and other NOS-derived products as possible autoregulators of enzyme activity. With the use of purified NOS-I, L-arginine turnover was found to operate initially at Vmax (0-15 min, phase I) although, despite the presence of excess substrate and cofactors, prolonged catalysis (15-90 min, phase II) was associated with a rapid decline in L-arginine turnover. Taken together, these observations suggested that one or more NOS products inactivate NOS. Indeed, exogenously applied reactive nitrogen oxide species (RNSs) decreased Vmax during phase I, although with different potencies (NO->NO> ONOO-) and efficacies (NO>NO-=ONOO-). The NO scavengers oxyhaemoglobin (HbO2; 100 microM) and 1H-imidazol-1 - yloxy - 2 - (4-carboxyphenyl) - 4,5 - dihydro - 4,4,5,5 - tetramethyl - 3 -oxide (CPTIO; 10 microM) and the ONOO- scavenger GSH (7 mM) had no effect on NOS activity during phase I, except for an endogenous autoinhibitory influence of NO and ONOO-. However, superoxide dismutase (SOD; 300 units/ml), which is thought either to increase the half-life of NO or to convert NO- to NO, lowered Vmax in an NO-dependent manner because this effect was selectively antagonized by HbO2 (100 microM). This latter observation demonstrated the requirement of SOD to reveal endogenous NO-mediated autoinhibition. Importantly, during phase II of catalysis, NOS became uncoupled and began to form H2O2 because catalase, which metabolizes H2O2, increased enzyme activity. Consistent with this, exogenous H2O2 also inhibited NOS activity during phase I. Thus during catalysis NOS is subject to complex autoinhibition by both enzyme-derived RNS and H2O2, differentially affecting enzyme activity.

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More information

Published date: 15 June 1999
Keywords: autoregulation, hydrogen peroxide, NOS catalysis
Organisations: Clinical & Experimental Sciences

Identifiers

Local EPrints ID: 337882
URI: http://eprints.soton.ac.uk/id/eprint/337882
ISSN: 1470-8728
PURE UUID: d8305519-f612-42a4-8e02-5ed78dca3f37
ORCID for Martin Feelisch: ORCID iD orcid.org/0000-0003-2320-1158

Catalogue record

Date deposited: 22 Jun 2012 15:47
Last modified: 18 Jul 2017 06:00

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Contributors

Author: Peter Kotsonis
Author: Armin Frey
Author: Lothar G. Fröhlich
Author: Heinrich Hofmann
Author: Andreas Reif
Author: David A. Wink
Author: Martin Feelisch ORCID iD
Author: Harald H.H.W. Schmidt

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