Kinetic characterization of recombinant human acidic mammalian chitinase.
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.
Department of Chemical and Screening Sciences, Wyeth Research, 500 Arcola Road, Collegeville, Pennsylvania 19426, USA.
Yi-Te
YT
Chou
Shihua
S
Yao
Robert
R
Czerwinski
Margaret
M
Fleming
Rustem
R
Krykbaev
Dejun
D
Xuan
Huanfang
H
Zhou
Jonathan
J
Brooks
Lori
L
Fitz
James
J
Strand
Eleonora
E
Presman
Laura
L
Lin
Ann
A
Aulabaugh
Xinyi
X
Huang
D000818:Animals; D016466:CHO Cells; D002688:Chitinases / Q000378:metabolism*; D006224:Cricetinae; D003412:Cricetulus; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009994:Osmolar Concentration; D011994:Recombinant Proteins / Q000378:metabolism
D011994:Recombinant Proteins; C499254:CHIA protein, human; D002688:Chitinases