Montgomery H J, Romanov V, Guillemette J G
Department of Chemistry and the Guelph Waterloo Centre for Graduate Work in Chemistry and Biochemistry, University of Waterloo, Waterloo, Ontario N2L 3G1, Canada.
J Biol Chem. 2000 Feb 18;275(7):5052-8. doi: 10.1074/jbc.275.7.5052.
Neuronal nitric-oxide synthase (NOS) and endothelial NOS are constitutive NOS isoforms that are activated by binding calmodulin in response to elevated intracellular calcium. In contrast, the inducible NOS isoform binds calmodulin at low basal levels of calcium in resting cells. Primary sequence comparisons show that each constitutive NOS isozyme contains a polypeptide segment within its reductase domain, which is absent in the inducible NOS enzyme. To study a possible link between the presence of these additional polypeptide segments in constitutive NOS enzymes and their calcium-dependent calmodulin activation, three deletion mutants were created. The putative inhibitory insert was removed from the FMN binding regions of the neuronal NOS holoenzyme and from two truncated neuronal NOS reductase enzymes in which the calmodulin binding region was either included or deleted. All three mutant enzymes showed reduced incorporation of FMN and required reconstitution with exogenous FMN for activity. The combined removal of both the calmodulin binding domain and the putative inhibitory insert did not result in a calmodulin-independent neuronal NOS reductase. Thus, although the putative inhibitory element has an effect on the calcium-dependent calmodulin activation of neuronal NOS, it does not have the properties of the typical autoinhibitory domain found in calmodulin-activated enzymes.
神经元型一氧化氮合酶（NOS）和内皮型NOS是组成型NOS同工型，它们通过在细胞内钙升高时结合钙调蛋白而被激活。相比之下，诱导型NOS同工型在静息细胞中钙水平较低时结合钙调蛋白。一级序列比较表明，每种组成型NOS同工酶在其还原酶结构域内都含有一个多肽片段，而诱导型NOS酶中不存在该片段。为了研究组成型NOS酶中这些额外多肽片段的存在与其钙依赖性钙调蛋白激活之间的可能联系，构建了三个缺失突变体。从神经元型NOS全酶的FMN结合区域以及两个截短的神经元型NOS还原酶中去除了假定的抑制性插入片段，这两个截短的神经元型NOS还原酶中钙调蛋白结合区域要么包含在内，要么被删除。所有三种突变酶都显示出FMN掺入减少，并且需要用外源性FMN重建才能具有活性。同时去除钙调蛋白结合结构域和假定的抑制性插入片段并没有产生不依赖钙调蛋白的神经元型NOS还原酶。因此，尽管假定的抑制元件对神经元型NOS的钙依赖性钙调蛋白激活有影响，但它不具有钙调蛋白激活酶中典型的自抑制结构域的特性。
