title: Stabilization of the predominant disease-causing aldolase variant (A149P) with zwitterionic osmolytes.
abstract: Hereditary fructose intolerance (HFI) is a disease of carbohydrate metabolism that can result in hyperuricemia, hypoglycemia, liver and kidney failure, coma, and death. Currently, the only treatment for HFI is a strict fructose-free diet. HFI arises from aldolase B deficiency, and the most predominant HFI mutation is an alanine to proline substitution at position 149 (A149P). The resulting aldolase B with the A149P substitution (AP-aldolase) has activity that is <100-fold that of the wild type. The X-ray crystal structure of AP-aldolase at both 4 and 18 °C reveals disordered adjacent loops of the (α/β)(8) fold centered around the substitution, which leads to a dimeric structure as opposed to the wild-type tetramer. The effects of osmolytes were tested for restoration of structure and function. An initial screen of osmolytes (glycerol, sucrose, polyethylene glycol, 2,4-methylpentanediol, glutamic acid, arginine, glycine, proline, betaine, sarcosine, and trimethylamine N-oxide) reveals that glycine, along with similarly structured compounds, betaine and sarcosine, protects AP-aldolase structure and activity from thermal inactivation. The concentration and functional moieties required for thermal protection show a zwitterion requirement. The effects of osmolytes in restoring structure and function of AP-aldolase are described. Testing of zwitterionic osmolytes of increasing size and decreasing fractional polar surface area suggests that osmolyte-mediated AP-aldolase stabilization occurs neither primarily through excluded volume effects nor through transfer free energy effects. These data suggest that AP-aldolase is stabilized by binding to the native structure, and they provide a foundation for developing stabilizing compounds for potential therapeutics for HFI.
authors.affiliation: Program in Molecular and Cellular Biology and Biochemistry, Boston University, 5 Cummington Street, Boston, Massachusetts 02215, United States.
authors.forename: Jack D
authors.initials: JD
authors.lastname: Stopa
authors.forename: Sushil
authors.initials: S
authors.lastname: Chandani
authors.forename: Dean R
authors.initials: DR
authors.lastname: Tolan
mesh_terms: D004795:Enzyme Stability; D005633:Fructose Intolerance / Q000201:enzymology* / Q000235:genetics; D005634:Fructose-Bisphosphate Aldolase / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D014644:Genetic Variation; D006801:Humans; D020125:Mutation, Missense; D009930:Organic Chemicals / Q000737:chemistry*; D011487:Protein Conformation
chemical_list: D009930:Organic Chemicals; D005634:Fructose-Bisphosphate Aldolase