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Characterization of neuronal nitric-...

Wolthers, Kirsten Rene.

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Characterization of neuronal nitric-oxide synthase reductase activity.

Record Type:	Language materials, printed : Monograph/item
Title/Author:	Characterization of neuronal nitric-oxide synthase reductase activity./
Author:	Wolthers, Kirsten Rene.
Description:	236 p.
Notes:	Adviser: Michael I. Schimerlik.
Contained By:	Dissertation Abstracts International62-05B.
Subject:	Chemistry, Biochemistry. -
Online resource:	http://pqdd.sinica.edu.tw/twdaoapp/servlet/advanced?query=3015238
ISBN:	0493252142

Characterization of neuronal nitric-oxide synthase reductase activity.
Wolthers, Kirsten Rene.

Characterization of neuronal nitric-oxide synthase reductase activity. - 236 p.

Adviser: Michael I. Schimerlik.

Thesis (Ph.D.)--Oregon State University, 2001.

During catalysis the flavoprotein domain of neuronal nitric-oxide synthase (nNOS) shuttles NADPH-derived reducing equivalents from FAD to FMN and then to the P450-heme enabling heme-based oxygen activation and subsequent NO-synthesis. The binding of Ca<super>2+</super>-activated calmodulin (Ca<super>2+</super>-CaM) to nNOS alleviates inhibition of flavin mediated electron transfer within the diflavin domain, which is demonstrated by the increase in the rate of 2,6-dichloroindolphenol (DCIP) reduction by 2 to 3 fold and that of cytochrome c<super>3+</super> by 10 to 20 fold. To investigate the effect of the Ca<super> 2+</super>-CaM on the nNOS reductase activity, the steady-state kinetics of basal and CaM-stimulated reduction of these two substrates was studied. Parallel initial velocity patterns indicated that both substrates are reduced in a ping-pong mechanism. Product and dead-end inhibition data with DCIP as the electron acceptor were consistent with a di iso ping-pong bi-bi mechanism. In contrast, product and dead-end inhibition studies with cytochrome c<super> 3+</super> as the second substrate were consistent with an iso (two-site) ping-pong mechanism. Ca<super>2+</super>-CaM did not alter the proposed kinetic mechanisms; however, it did effect to varying degrees the (<italic>k</italic> cat/<italic>K</italic>m) for the various substrates. The pH-dependence of basal and CaM-stimulated reduction of DCIP revealed that ionizable groups involved in the binding of substrates and catalysis are not altered by Ca<super>2+</super>-CaM. However, the activated cofactor does influence catalytic rate constants and/or ionizable groups involved in cytochrome c<super> (A-side) hydrogen from NADPH. Primary deuterium isotope effects (NADP(D)) and solvent isotope effects (SKIE) suggests that of the two half reactions, the reductive half reaction involving NADPH oxidation limits the overall reaction rate, but that hydride transfer to FAD is not the slow step. A small value of <super>D</super>(<italic>V</italic>/<italic>K</italic>)NADPH (1.2–1.6) suggests hydride transfer is not the rate-limiting step within the reductive half-reaction. Large solvent kinetic isotope effects (SKIE) were observed on (<italic>V</italic>/<italic>K</italic>)cytc for basal and CaM-stimulated reduction of cytochrome c<super>3+</super> suggesting that proton uptake from the solvent limits the rate of the oxidative half-reaction. A small SKIE on <italic>V</italic> and (<italic>V</italic>/<italic>K</italic>) NADPH indicates that proton uptake does not limit the overall reaction rate. Proton inventory analysis revealed multiple transition-state protons contributed to the observed SKIE.

ISBN: 0493252142Subjects--Topical Terms:

1017722
Chemistry, Biochemistry.

Characterization of neuronal nitric-oxide synthase reductase activity.
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020 $a 0493252142
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040 $a UnM $c UnM
100 1 $a Wolthers, Kirsten Rene. $3 1252828
245 1 0 $a Characterization of neuronal nitric-oxide synthase reductase activity.
300 $a 236 p.
500 $a Adviser: Michael I. Schimerlik.
500 $a Source: Dissertation Abstracts International, Volume: 62-05, Section: B, page: 2323.
502 $a Thesis (Ph.D.)--Oregon State University, 2001.
520 $a During catalysis the flavoprotein domain of neuronal nitric-oxide synthase (nNOS) shuttles NADPH-derived reducing equivalents from FAD to FMN and then to the P450-heme enabling heme-based oxygen activation and subsequent NO-synthesis. The binding of Ca<super>2+</super>-activated calmodulin (Ca<super>2+</super>-CaM) to nNOS alleviates inhibition of flavin mediated electron transfer within the diflavin domain, which is demonstrated by the increase in the rate of 2,6-dichloroindolphenol (DCIP) reduction by 2 to 3 fold and that of cytochrome c<super>3+</super> by 10 to 20 fold. To investigate the effect of the Ca<super> 2+</super>-CaM on the nNOS reductase activity, the steady-state kinetics of basal and CaM-stimulated reduction of these two substrates was studied. Parallel initial velocity patterns indicated that both substrates are reduced in a ping-pong mechanism. Product and dead-end inhibition data with DCIP as the electron acceptor were consistent with a di iso ping-pong bi-bi mechanism. In contrast, product and dead-end inhibition studies with cytochrome c<super> 3+</super> as the second substrate were consistent with an iso (two-site) ping-pong mechanism. Ca<super>2+</super>-CaM did not alter the proposed kinetic mechanisms; however, it did effect to varying degrees the (<italic>k</italic> cat/<italic>K</italic>m) for the various substrates. The pH-dependence of basal and CaM-stimulated reduction of DCIP revealed that ionizable groups involved in the binding of substrates and catalysis are not altered by Ca<super>2+</super>-CaM. However, the activated cofactor does influence catalytic rate constants and/or ionizable groups involved in cytochrome c<super> (A-side) hydrogen from NADPH. Primary deuterium isotope effects (NADP(D)) and solvent isotope effects (SKIE) suggests that of the two half reactions, the reductive half reaction involving NADPH oxidation limits the overall reaction rate, but that hydride transfer to FAD is not the slow step. A small value of <super>D</super>(<italic>V</italic>/<italic>K</italic>)NADPH (1.2–1.6) suggests hydride transfer is not the rate-limiting step within the reductive half-reaction. Large solvent kinetic isotope effects (SKIE) were observed on (<italic>V</italic>/<italic>K</italic>)cytc for basal and CaM-stimulated reduction of cytochrome c<super>3+</super> suggesting that proton uptake from the solvent limits the rate of the oxidative half-reaction. A small SKIE on <italic>V</italic> and (<italic>V</italic>/<italic>K</italic>) NADPH indicates that proton uptake does not limit the overall reaction rate. Proton inventory analysis revealed multiple transition-state protons contributed to the observed SKIE.
590 $a School code: 0172.
650 4 $a Chemistry, Biochemistry. $3 1017722
690 $a 0487
710 2 0 $a Oregon State University. $3 625720
773 0 $t Dissertation Abstracts International $g 62-05B.
790 $a 0172
790 1 0 $a Schimerlik, Michael I., $e advisor
791 $a Ph.D.
792 $a 2001
856 4 0 $u http://pqdd.sinica.edu.tw/twdaoapp/servlet/advanced?query=3015238