The temperature dependence of activity and structure for the most prevalent mutant aldolase B associated with hereditary fructose intolerance

Hereditary fructose intolerance (HFI) is an autosomal recessive disorder in humans which is caused by mutations in the aldolase B gene. The most common HFI allele encodes an enzyme with an A149P substitution (AP-aldolase). A lysis method suitable for aggregation-prone proteins overexpressed in bacteria was developed. The enzyme’s structure and function is investigated as a function of temperature. Near-UV CD shows a qualitative difference in tertiary structure, whereas far-UV CD shows no difference in overall secondary structure, although both show increased temperature sensitivity for AP-aldolase compared to that seen with wild-type aldolase B. AP-aldolase exists as a dimer at all temperatures tested, unlike the tetrameric wild-type enzyme, thus providing a possible explanation for the loss in thermostability. AP-aldolase has sixfold lower activity than wild type at 10 °C, which decreases substantially at higher temperature. In addition to disruptions at the catalytic center, the kinetic constants toward different substrates suggest that there is a disruption at the C1-phosphate-binding site, which is not sensitive to temperature. The implications of these structural alterations are discussed with regard to the HFI disease.