Binding kinetics of calmodulin with target peptides of three nitric oxide synthase isozymes.
Binding kinetics of calmodulin with target peptides of three nitric oxide synthase isozymes.
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DOI：
10.1016/j.jinorgbio.2011.06.003
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发表时间：
2011-09
影响因子：
3.9
通讯作者：
Tsai, Ah-Lim
中科院分区：
文献类型：
--
作者：
Wu, Gang;Berta, Vladimir;Tsai, Ah-Lim
关键词：
一氧化氮合酶（NOS）还原酶结构域向加氧酶结构域的有效电子传递依赖于钙调素（CaM）的结合。先前仅通过表面等离子体共振（SPR）（Biochemistry 35，8742 - 8747，1996）确定了CaM与NOS靶肽结合的速率常数，这表明CaM与NOS的结合是缓慢的，并且不支持在先前和本研究中测量的NOS中的快速电子转移。为了解决这一矛盾，采用荧光停流法测定了Alexa 350标记的T34C/T110W CaM（Alexa-CaM）与三种NOS同工酶靶肽的结合率。所有三种靶肽在4.5 ° C下均表现出快速kon常数：nNOS 726 - 749为6.6 × 108 M − 1s − 1，eNOS 492 - 511为2.9 × 108 M − 1s − 1，iNOS 507 - 531为6.1 × 108 M − 1s − 1，比先前通过SPR测定的值快3 - 4个数量级。通过用ETDA螯合Ca2+来测量NOS靶肽从Alexa-CaM/肽复合物的解离速率：nNOS 726 - 749为3.7 s − 1，eNOS 492 - 511为4.5 s − 1，iNOS 507 - 531为0.063 s − 1。我们的数据表明，钙调素与NOS的结合是快速和动力学胜任有效的电子转移，不太可能在NOS催化限速。只有iNOS507 - 531能够结合载脂蛋白Alexa-CaM，但与其结合全Alexa-CaM的构象非常不同。
Efficient electron transfer from reductase domain to oxygenase domain in nitric oxide synthase (NOS) is dependent on the binding of calmodulin (CaM). Rate constants for the binding of CaM to NOS target peptides was only determined previously by surface plasmon resonance (SPR) (Biochemistry 35, 8742–8747, 1996) suggesting that the binding of CaM to NOSs is slow and does not support the fast electron transfer in NOSs measured in previous and this studies. To resolve this contradiction, the binding rates of holo Alexa 350 labeled T34C/T110W CaM (Alexa-CaM) to target peptides from three NOS isozymes were determined using fluorescence stopped-flow. All three target peptides exhibited fast kon constants at 4.5 °C: 6.6 × 108 M−1s−1 for nNOS726–749, 2.9 × 108 M−1s−1 for eNOS492–511 and 6.1 × 108 M−1s−1 for iNOS507–531, 3 – 4 orders of magnitude faster than those determined previously by SPR. Dissociation rates of NOS target peptides from Alexa-CaM/peptide complexes were measured by Ca2+ chelation with ETDA: 3.7 s−1 for nNOS726–749, 4.5 s−1 for eNOS492–511, and 0.063 s−1 for iNOS507–531. Our data suggest that the binding of CaM to NOS is fast and kinetically competent for efficient electron transfer and is unlikely rate-limiting in NOS catalysis. Only iNOS507–531 was able to bind apo Alexa-CaM, but in a very different conformation from its binding to holo Alexa-CaM.
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