Kinetic Characterization of Recombinant Human Acidic Mammalian Chitinase

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• 作者：Yi-Te Chou ; Shihua Yao ; Robert Czerwinski ; Margaret Fleming ; Rustem Krykbaev ; Dejun Xuan ; Huanfang Zhou ; Jonathan Brooks ; Lori Fitz ; James Strand ; Eleonora Presman ; Laura Lin ; Ann Aulabaugh ; and Xinyi
• 刊名：Biochemistry
• 出版年：2006
• 出版时间：April 11, 2006
• 年：2006
• 卷：45
• 期：14
• 页码：4444 - 4454
• 全文大小：276K
• 年卷期：v.45,no.14(April 11, 2006)
• ISSN：1520-4995

文摘

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 asthmaand allergic diseases. Inhibition of AMCase results in decreased airway inflammation and airway hyper-responsiveness in a mouse asthma model, suggesting that the AMCase activity is a part of the mechanismof Th2 cytokine-driven inflammatory response in asthma. In this paper, we report the first detailed kineticcharacterization of recombinant human AMCase. In contrast with mouse AMCase that has been reportedto have a major pH optimum at 2 and a secondary pH optimum around 3-6, human AMCase has onlyone 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 chitinaseactivity of human AMCase. HPLC characterization of cleavage of chitooligosaccharides (4-6-mers)suggests that human AMCase prefers the mages/gifchars/beta2.gif" BORDER=0 ALIGN="middle"> anomer of chitooligosaccharides as substrate. Human AMCasealso 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 againstfluorogenic substrates, chitobiose-4-methylumbelliferyl and chitotriose-4-methylumbelliferyl, and enhancesactivity against chitooligosaccharides. The physiological implications of these results are discussed.