Kinetic characterization of recombinant human acidic mammalian chitinase
Kinetic characterization of recombinant human acidic mammalian chitinase
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DOI：
10.1021/bi0525977
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发表时间：
2006-04-11
期刊：
影响因子：
2.9
通讯作者：
Huang, XY
中科院分区：
文献类型：
--
作者：
Chou, YT;Yao, SH;Huang, XY
人酸性哺乳动物几丁质酶（AMCase）是18糖基水解酶家族的成员，是Th 2介导的炎症反应的重要蛋白质之一，与哮喘和过敏性疾病有关。在小鼠哮喘模型中，抑制AMCase导致气道炎症和气道高反应性降低，这表明AMCase活性是哮喘中Th 2型丝氨酸驱动的炎症反应机制的一部分。在本文中，我们报告的第一个详细的动力学特性的重组人AMCase。据报道，小鼠AMCase的主要最适pH值为2，次要最适pH值为3-6，而人AMCase的k（cat）/K-m最适pH值只有一个，介于pH 4和5之间。稳态动力学显示，人AMCase具有“低”内在转糖苷酶活性，这导致观察到明显的底物抑制。这种缓慢的转糖基化可能提供了一种反馈调节人AMCase几丁质酶活性的体内机制。壳寡糖（4-6-聚体）裂解的HPLC表征表明，人AMCase偏好壳寡糖的β异头体作为底物。人AMCase似乎也主要通过二糖单元从非还原端切割壳寡糖。离子强度调节人AMCase对荧光底物（壳二糖-4-甲基伞形酮基和壳三糖-4-甲基伞形酮基）的酶活性和底物裂解模式，并增强对壳寡糖的活性。这些结果的生理意义进行了讨论。
Human acidic mammalian chitinase (AMCase), a member of the family 18 glycosyl hydrolases, is one of the important proteins involved in Th2-mediated inflammation and has been implicated in asthma and allergic diseases. Inhibition of AMCase results in decreased airway inflammation and airway hyperresponsiveness in a mouse asthma model, suggesting that the AMCase activity is a part of the mechanism of Th2 cytokine-driven inflammatory response in asthma. In this paper, we report the first detailed kinetic characterization of recombinant human AMCase. In contrast with mouse AMCase that has been reported to have a major pH optimum at 2 and a secondary pH optimum around 3-6, human AMCase has only one pH optimum for k(cat)/K-m between pH 4 and 5. Steady state kinetics shows that human AMCase has "low" intrinsic transglycosidase activity, which leads to the observation of apparent substrate inhibition. This slow transglycosylation may provide a mechanism in vivo for feedback regulation of the chitinase activity of human AMCase. HPLC characterization of cleavage of chitooligosaccharides (4-6-mers) suggests that human AMCase prefers the beta anomer of chitooligosaccharides as substrate. Human AMCase also appears to cleave chitooligosaccharides from the nonreducing end primarily by disaccharide units. Ionic strength modulates the enzymatic activity and substrate cleavage pattern of human AMCase against fluorogenic substrates, chitobiose-4-methylumbelliferyl and chitotriose-4-methylumbelliferyl, and enhances activity against chitooligosaccharides. The physiological implications of these results are discussed.