Haque Mohammad Mahfuzul, Panda Koustubh, Tejero Jesús, Aulak Kulwant S, Fadlalla Mohammed Adam, Mustovich Anthony T, Stuehr Dennis J
Department of Pathobiology, Lerner Research Institute, Cleveland Clinic, 9500 Euclid Avenue, Cleveland, OH 44195, USA.
Proc Natl Acad Sci U S A. 2007 May 29;104(22):9254-9. doi: 10.1073/pnas.0700332104. Epub 2007 May 21.
In mammals, endothelial nitric oxide synthase (eNOS) has the weakest activity, being one-tenth and one-sixth as active as the inducible NOS (iNOS) and the neuronal NOS (nNOS), respectively. The basis for this weak activity is unclear. We hypothesized that a hinge element that connects the FMN module in the reductase domain but is shorter and of unique composition in eNOS may be involved. To test this hypothesis, we generated an eNOS chimera that contained the nNOS hinge and two mutants that either eliminated (P728IeNOS) or incorporated (I958PnNOS) a proline residue unique to the eNOS hinge. Incorporating the nNOS hinge into eNOS increased NO synthesis activity 4-fold, to an activity two-thirds that of nNOS. It also decreased uncoupled NADPH oxidation, increased the apparent K(m)O(2) for NO synthesis, and caused a faster heme reduction. Eliminating the hinge proline had similar, but lesser, effects. Our findings reveal that the hinge is an important regulator and show that differences in its composition restrict the activity of eNOS relative to other NOS enzymes.
在哺乳动物中，内皮型一氧化氮合酶（eNOS）的活性最弱，分别只有诱导型一氧化氮合酶（iNOS）和神经元型一氧化氮合酶（nNOS）活性的十分之一和六分之一。这种低活性的原因尚不清楚。我们推测，连接还原酶结构域中FMN模块的一个铰链元件可能与此有关，该元件在eNOS中较短且组成独特。为了验证这一假设，我们构建了一个包含nNOS铰链的eNOS嵌合体以及两个突变体，其中一个突变体消除了（P728IeNOS）eNOS铰链特有的脯氨酸残基，另一个突变体引入了（I958PnNOS）该脯氨酸残基。将nNOS铰链整合到eNOS中可使一氧化氮合成活性提高4倍，达到nNOS活性的三分之二。它还减少了未偶联的NADPH氧化，增加了一氧化氮合成的表观K(m)O(2)，并使血红素还原更快。消除铰链脯氨酸也有类似但较小的影响。我们的研究结果表明，该铰链是一个重要的调节因子，并表明其组成差异限制了eNOS相对于其他一氧化氮合酶的活性。
