title: Standardization of α-L-iduronidase enzyme assay with Michaelis-Menten kinetics.
abstract: The lack of methodological uniformity in enzyme assays has been a long-standing difficulty, a problem for bench researchers, for the interpretation of clinical diagnostic tests, and an issue for investigational drug review. Illustrative of the problem, α-L-iduronidase enzyme catalytic activity is frequently measured with the substrate 4-methylumbelliferyl-α-L-iduronide (4MU-iduronide); however, final substrate concentrations used in different assays vary greatly, ranging from 25 μM to 1425 μM (Km ≈ 180 μM) making it difficult to compare results between laboratories. In this study, α-L-iduronidase was assayed with 15 different substrate concentrations. The resulting activity levels from the same specimens varied greatly with different substrate concentrations but, as a group, obeyed the expectations of Michaelis-Menten kinetics. Therefore, for the sake of improved comparability, it is proposed that α-L-iduronidase enzyme assays should be conducted either (1) under substrate saturating conditions; or (2) when concentrations are significantly below substrate saturation, with results standardized by arithmetic adjustment that considers Michaelis-Menten kinetics. The approach can be generalized to many other enzyme assays.
authors: Department of Genetics, Cell Biology and Development, PWB 13-146, 516 Delaware Str SE, Minneapolis, MN 55455, USA; Molecular, Cellular, Developmental Biology & Genetics Graduate Program, PWB 13-146, 516 Delaware Str SE, Minneapolis, MN 55455, USA; Gene Therapy Center, Department of Pediatrics, University of Minnesota, USA.
authors: Li
authors: L
authors: Ou
authors: Department of Biochemistry, Molecular Biology and Biophysics, 5-120 NHH, 312 Church Str SE, Minneapolis, MN 55455, USA; Gene Therapy Center, Department of Pediatrics, University of Minnesota, USA.
authors: Tyler L
authors: TL
authors: Herzog
authors: Department of Biochemistry, Molecular Biology and Biophysics, 5-120 NHH, 312 Church Str SE, Minneapolis, MN 55455, USA.
authors: Carrie M
authors: CM
authors: Wilmot
authors: Molecular, Cellular, Developmental Biology & Genetics Graduate Program, PWB 13-146, 516 Delaware Str SE, Minneapolis, MN 55455, USA; Gene Therapy Center, Department of Pediatrics, University of Minnesota, USA. Electronic address: whitley@umn.edu.
authors: Chester B
authors: CB
authors: Whitley
mesh_terms: D002138:Calibration; D057075:Enzyme Assays / Q000592:standards*; D006801:Humans; D006923:Hymecromone / Q000031:analogs & derivatives* / Q000737:chemistry / Q000592:standards; D007068:Iduronidase / Q000032:analysis* / Q000378:metabolism; D007700:Kinetics; D008059:Mucopolysaccharidosis I / Q000175:diagnosis / Q000201:enzymology* / Q000473:pathology; D011786:Quality Control
chemical_list: D006923:Hymecromone; C018343:4-methylumbelliferyl iduronide; D007068:Iduronidase
keywords: 4-methylumbelliferyl; Hurler syndrome; IDUA; Lineweaver–Burk plot; MPS; MU; Michaelis–Menten kinetics; mucopolysaccharidoses; mucopolysaccharidosis; α-L-iduronidase; α-L-iduronidase enzyme assay