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. 2016 Aug 8;37(10):1097–1105. doi: 10.1002/humu.23047

Figure 4.

Figure 4

Fumarylacetoacetate hydrolase (FAH) enzyme kinetic assays show that the FAH p.R142G variant is unable to break down fumarylacetoacetate (FAA), the hepto‐ and nephrotoxic substrate in tyrosinemia type 1. A: FAA and succinylacetoacetate (SAA) have similar chemical structures; the only difference is the presence of a C–C double bond in FAA (highlighted in yellow). Because of the shape of the catalytic pocket, only the extended transconformations of FAA and SAA can be accommodated. It appears that FAA is bound more tightly by FAH and the rate of dissociation is much slower than for SAA, which may reflect differences in physiological substrate preference. As FAA is the reactive substrate that causes liver damage, the ability of FAH to process FAA (and not SAA) is thus a clinically relevant molecular assay for predicting hepatotoxicity potential for sequence variants in this enzyme. B: Enzyme kinetic assay for FAH activity measures the decrease in absorbance of FAA (330 nm) over time. Lentiviral constructs (green fluorescent protein control, human FAH, and p.R142G variant FAH) were used to transduce the Huh7 hepatocellular carcinoma cell line. Huh7 cells transduced with the wild‐type human FAH construct were able to break down FAA as evidenced by the decrease in absorbance at 330 nm over time. Huh7 cells transduced with the p.R142G variant lentivirus had a decrease in absorbance that was identical to that of the green fluorescent protein control, indicating that the variant was a functional null allele and is therefore incapable of breaking down FAA. Huh7 cells transduced with three times the amount of p.R142G‐lentivirus also did not show a measurable decrease in absorbance at 330 nm compared with the green fluorescent protein‐lentivirus control. C: Western blot analysis of FAH (∼46 kDa) showing that the two lentivirus constructs expressed equivalent levels of protein in Huh7 cells transduced at the same multiplicity of infection. Lane 1 is the green fluorescent protein control. Lanes 2 and 3 are the R142G variant FAH lentivirus. Lane 4 is the wild‐type human FAH lentivirus.