Kinetic characterization of recombinant human acidic mammalian chitinase.

Abstract

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 hyper-responsiveness 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.

• Authors: 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, Xinyi Huang
• Journal: Biochemistry (Biochemistry) Vol. 45 Issue 14 Pg. 4444-54 (Apr 11 2006) ISSN: 0006-2960 [Print] United States
• PMID: 16584180 (Publication Type: Journal Article)

Chemical References

• Recombinant Proteins
• CHIA protein, human
• Chitinases

Topics

• Animals
• CHO Cells
• Chitinases (metabolism)
• Cricetinae
• Cricetulus
• Humans
• Hydrogen-Ion Concentration
• Kinetics
• Osmolar Concentration
• Recombinant Proteins (metabolism)