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

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• 作者：Kirsten R. Wolthers and Michael I. Schimerlik
• 刊名：Biochemistry
• 出版年：2002
• 出版时间：January 8, 2002
• 年：2002
• 卷：41
• 期：1
• 页码：205 - 214
• 全文大小：107K
• 年卷期：v.41,no.1(January 8, 2002)
• ISSN：1520-4995

文摘

The pH dependence of basal and calmodulin- (CaM-) stimulated neuronal nitric oxide synthase(nNOS) reduction of 2,6-dichloroindophenol (DCIP) and cytochrome c³⁺ was investigated. The wave-shaped log V versus pH profile revealed that optimal DCIP reduction occurred when a group, pK_a of7.6-7.8, was ionized. The (V/K)_NADPH and (V/K)_DCIP versus pH profiles increased with the protonation ofa group with a pK_a of 6.5 or 5.9 and the ionization of two groups with the same pK_a of 7.5 or 7.0,respectively. (V/K)_DCIP decreased with the ionization of a group, pK_a of 9.0. Similar V, (V/K)_NADPH, and(V/K)_DCIP versus pH profiles for DCIP reduction were obtained with and without CaM, indicating thatCaM does not influence ionizable groups involved in catalysis or substrate binding. In contrast, CaMaffected the pH dependence of cytochrome c³⁺ reduction. The wave-shaped log V versus pH profile forbasal cytochrome c³⁺ reduction revealed that ionization of a group, pK_a of 8.6, increased catalysis. LogV for CaM-stimulated cytochrome c³⁺ reduction displayed a bell-shaped pH dependence with the protonationof a group with a pK_a of 6.4 and the ionization of a group with a pK_a of 9.3, resulting in a loss of activity.The log(V/K)_cytc versus pH profiles with and without CaM were bell-shaped with the ionization of agroup at pK_a of 7.1 or 7.6 (CaM) or pK_a of 9.4 or 9.6 (CaM), increasing and decreasing (V/K)_cytc. Theseresults suggest that CaM may change the nature of the rate-limiting catalytic steps or ionizable groupsinvolved in cytochrome c³⁺ reduction.