Kinetic mechanistic studies of wild-type leucine-rich repeat kinase 2: characterization of the kinase and GTPase activities.
Kinetic mechanistic studies of wild-type leucine-rich repeat kinase 2: characterization of the kinase and GTPase activities.
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DOI：
10.1021/bi901851y
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发表时间：
2010-03-09
期刊：
影响因子：
2.9
通讯作者：
Stein, Ross L.
中科院分区：
文献类型：
--
作者：
Liu, Min;Dobson, Brittany;Glicksman, Marcie A.;Yue, Zhenyu;Stein, Ross L.
最近的研究已经在最常见的家族型和一些散发性帕金森病（PD）中发现了富含亮氨酸的重复激酶2基因（LRRK2）的突变。LRRK2是一个大而复杂的蛋白，具有激酶和GTPase活性。一些LRRK2突变体增强激酶活性，并可能通过毒性功能获得机制促进帕金森病。鉴于LRRK2在帕金森病发病机制中的作用，了解其两种酶性质的动力学机制对于发现LRRK2激酶抑制剂至关重要，这将有助于治疗帕金森病。在本报告中，我们利用从小鼠脑中纯化的LRRK2蛋白，首先表征了LRRK2催化的两种肽底物磷酸化的动力学机制：plk衍生肽（PLK-peptide）和LRRKtide。我们发现LRRK2遵循一个快速平衡随机机制来磷酸化plk -肽，ATP或plk -肽是第一个与酶结合的底物，通过核苷酸类似物AMP和AMP- pnp、产物ADP和肽底物类似物的初始速度和抑制机制研究证明了这一点。第一底物的结合对第二底物的结合亲和力没有影响。当LRRKtide为磷酸化受体时，得出了相同的机制结论。接下来，我们对LRRK2的GTPase活性进行了表征，kcat为0.2±0.02 s−1，Km为210±29µM。在D2O摩尔分数为0.86的条件下，LRRK2的GTP酶活性在kcat上的SKIE值为0.97±0.04，表明产物解离步骤是LRRK2催化GTP水解过程中kcat控制的限速步骤。令人惊讶的是，GTP、GDP或GMP的结合对激酶活性没有影响，尽管GMP和GDP抑制GTPase活性。最后，我们通过筛选LRRK2激酶抑制剂，鉴定出一个化合物LDN-73794。我们的研究表明，该化合物是ATP结合的竞争性抑制剂，在不影响GTPase活性的情况下抑制激酶活性。
Recent studies have identified mutations in the leucine-rich repeat kinase2 gene (LRRK2) in the most common familial forms and some sporadic forms of Parkinson’s disease (PD). LRRK2 is a large and complex protein that possesses kinase and GTPase activities. Some LRRK2 mutants enhance kinase activity and possibly contribute to PD through a toxic gain-of-function mechanism. Given the role of LRRK2 in the pathogenesis of PD, understanding the kinetic mechanism of its two enzymatic properties is critical for the discovery of inhibitors of LRRK2 kinase that would be therapeutically useful to treat PD. In this report, by using LRRK2 protein purified from murine brain, first we characterize kinetic mechanisms for the LRRK2-catalyzed phosphorylation of two peptide substrates: PLK-derived peptide (PLK-peptide) and LRRKtide. We found that LRRK2 follows a rapid equilibrium random mechanism for the phosphorylation of PLK-peptide with either ATP or PLK-peptide being the first substrate binding to the enzyme, as evidenced by initial velocity and inhibition mechanism studies by nucleotide analogues AMP and AMP-PNP, product ADP, and analogue of peptide substrate. The binding of the first substrate has no effect on the binding affinity of the second substrate. Identical mechanistic conclusions were drawn when LRRKtide was the phosphoryl acceptor. Next, we characterize GTPase activity of LRRK2 with a kcat of 0.2 ± 0.02 s−1 and Km of 210 ± 29 µM. SKIE of 0.97 ± 0.04 was measured on kcat for the GTPase activity of LRRK2 in a D2O molar fraction of 0.86 and suggested that the product dissociation step is rate limiting of the steps governed by kcat in the LRRK2-catalyzed GTP hydrolysis. Surprisingly, binding of GTP, GDP or GMP has no effect on kinase activity, although GMP and GDP inhibit the GTPase activity. Finally, we have identified a compound LDN-73794 through screen of LRRK2 kinase inhibitors. Our study revealed that this compound is a competitive inhibitor of the binding of ATP and inhibits the kinase activity without affecting the GTPase activity.
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影响因子：
2.2
作者：
Mata, IF;Kachergus, JM;Farrer, MJ
通讯作者：
Farrer, MJ
影响因子：
4.8
作者：
Greggio, Elisa;Zambrano, Ibardo;Cookson, Mark R.
通讯作者：
Cookson, Mark R.
影响因子：
2.9
作者：
Ito, Genta;Okai, Takuro;Iwatsubo, Takeshi
通讯作者：
Iwatsubo, Takeshi
影响因子：
3.7
作者：
Guo, Luxuan;Gandhi, Payal N.;Chen, Shu G.
通讯作者：
Chen, Shu G.
DOI：
10.1073/pnas.0507360102
发表时间：
2005-11-15
影响因子：
11.1
作者：
West, AB;Moore, DJ;Dawson, TM
通讯作者：
Dawson, TM