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.
Keywords: 4-methylumbelliferyl; Hurler syndrome; IDUA; Lineweaver–Burk plot; MPS; MU; Michaelis–Menten kinetics; mucopolysaccharidoses; mucopolysaccharidosis; α-L-iduronidase; α-L-iduronidase enzyme assay.
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