Aguilera Begoña, Ghauharali-van der Vlugt Karen, Helmond Mariette T J, Out Jos M M, Donker-Koopman Wilma E, Groener Johanna E M, Boot Rolf G, Renkema G Herma, van der Marel Gijs A, van Boom Jacques H, Overkleeft Hermen S, Aerts Johannes M F G
Department of Biochemistry, Academic Medical Center, University of Amsterdam, Meibergdreef 15, 1105 AZ Amsterdam, The Netherlands.
J Biol Chem. 2003 Oct 17;278(42):40911-6. doi: 10.1074/jbc.M301804200. Epub 2003 Jul 30.
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.
壳三糖苷酶是一种几丁质酶，在人体中由富含脂质的组织巨噬细胞大量表达。在患有溶酶体脂质贮积症、结节病、地中海贫血和内脏利什曼病的患者血清中，其酶活性显著升高。在戈谢病患者疾病进展和治疗纠正过程中监测血清壳三糖苷酶活性，有助于深入了解病理性巨噬细胞的身体负担变化。然而，通过酶法准确定量壳三糖苷酶水平因明显的底物抑制而变得复杂，这使得无法使用饱和底物浓度。因此，我们更详细地研究了壳三糖苷酶的催化特性。结果表明，在底物浓度过高时酶活性的抑制可以完全由底物分子的转糖基化来解释。讨论了壳三糖苷酶水解和转糖基化能力的潜在生理后果。对壳三糖苷酶转糖苷酶活性的新认识导致设计了一种新的底物分子，4-甲基伞形酮基-（4-脱氧）壳二糖。使用这种不能作为转糖基化受体的底物，壳三糖苷酶表现出正常的米氏动力学，从而使酶活性测量的灵敏度和重现性有了显著提高。这种新型便捷的壳三糖苷酶酶法应该有助于准确监测接受昂贵酶替代疗法的戈谢病患者。
