title: The functional role of glutamine-280 and threonine-282 in human alpha-galactosidase.
abstract: Our previous study on chimeric mutants of alpha-galactosidase suggested that two peptide regions encoded by exons 1-2 and 6 of the enzyme gene contribute to substrate recognition (Ishii, S. et al. (1994) Biochim. Biophys. Acta 1204, 265-270). In this study, we constructed five single amino acid substitutions for functional analysis of the amino acid residues around glutamine-279, the mutation site detected in an atypical Fabry disease patient. Two mutants, Q280S (Gln280-->Ser; CAA-->TCA) and T282A (Thr282-->Ala; ACT-->GCT), showed increased Km and decreased thermostability as compared with normal enzyme. Circular dichroism spectrum was not modified. An additional chimeric mutation in the exon 1-2 region by substitution with the homologous sequence of alpha-N-acetylgalactosaminidase cDNA restored catalytic activity and thermostability in both mutants. These data indicated the functional significance of glutamine-280 and threonine-282 for expressing the activity and stability of alpha-galactosidase molecule, and also the presence of an intramolecular interaction between the two peptide regions encoded by exons 1-2 and 6.
authors.affiliation: Department of Clinical Genetics, Tokyo Metropolitan Institute of Medical Science, Japan.
authors.forename: S
authors.initials: S
authors.lastname: Ishii
authors.forename: R
authors.initials: R
authors.lastname: Kase
authors.forename: H
authors.initials: H
authors.lastname: Sakuraba
authors.forename: Y
authors.initials: Y
authors.lastname: Suzuki
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D002942:Circular Dichroism; D017931:DNA Primers / Q000235:genetics; D004795:Enzyme Stability / Q000235:genetics; D006801:Humans; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D013329:Structure-Activity Relationship; D000519:alpha-Galactosidase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*
chemical_list: D017931:DNA Primers; D011993:Recombinant Fusion Proteins; D000519:alpha-Galactosidase