Transglycosidase activity of chitotriosidase: improved enzymatic assay for the human macrophage chitinase.
Chitotriosidase is a chitinase that is massively expressed by lipid-laden tissue macrophages in man. Its enzymatic activity is markedly elevated in serum of patients suffering from lysosomal lipid storage disorders, sarcoidosis, thalassemia, and visceral Leishmaniasis. Monitoring of serum chitotriosidase activity in Gaucher disease patients during progression and therapeutic correction of their disease is useful to obtain insight in changes in body burden on pathological macrophages. However, accurate quantification of chitotriosidase levels by enzyme assay is complicated by apparent substrate inhibition, which prohibits the use of saturating substrate concentrations. We have therefore studied the catalytic features of chitotriosidase in more detail. It is demonstrated that the inhibition of enzyme activity at excess substrate concentration can be fully explained by transglycosylation of substrate molecules. The potential physiological consequences of the ability of chitotriosidase to hydrolyze as well as transglycosylate are discussed. The novel insight in transglycosidase activity of chitotriosidase has led to the design of a new substrate molecule, 4-methylumbelliferyl-(4-deoxy)chitobiose. With this substrate, which is no acceptor for transglycosylation, chitotriosidase shows normal Michaelis-Menten kinetics, resulting in major improvements in sensitivity and reproducibility of enzymatic activity measurements. The novel convenient chitotriosidase enzyme assay should facilitate the accurate monitoring of Gaucher disease patients receiving costly enzyme replacement therapy.
Aguilera
Begoña
B
Department of Biochemistry, Academic Medical Center, University of Amsterdam, Meibergdreef 15, 1105 AZ Amsterdam, The Netherlands.
Ghauharali-van der Vlugt
Karen
K
Helmond
Mariette T J
MT
Out
Jos M M
JM
Donker-Koopman
Wilma E
WE
Groener
Johanna E M
JE
Boot
Rolf G
RG
Renkema
G Herma
GH
van der Marel
Gijs A
GA
van Boom
Jacques H
JH
Overkleeft
Hermen S
HS
Aerts
Johannes M F G
JM
D002384:Catalysis; D002624:Chemistry, Clinical / Q000379:methods; D002688:Chitinases / Q000032:analysis* / Q000097:blood / Q000737:chemistry; D018076:DNA, Complementary / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D005776:Gaucher Disease / Q000175:diagnosis; D006026:Glycoside Hydrolases / Q000097:blood* / Q000378:metabolism; D006031:Glycosylation; D006596:Hexosaminidases / Q000097:blood* / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008264:Macrophages / Q000201:enzymology*; D008954:Models, Biological; D009097:Multienzyme Complexes / Q000097:blood* / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry; D013997:Time Factors; D014166:Transferases / Q000097:blood* / Q000378:metabolism
D018076:DNA, Complementary; D009097:Multienzyme Complexes; D011994:Recombinant Proteins; C023176:transglycosidase enzyme system; D014166:Transferases; D006026:Glycoside Hydrolases; D006596:Hexosaminidases; C086400:chitotriosidase; D002688:Chitinases