THE FUNCTIONAL-ROLE OF GLUTAMINE-280 AND THREONINE-282 IN HUMAN ALPHA-GALACTOSIDASE

Our previous study on chimeric mutants of alpha-galactosidase suggeste d that two peptide regions encoded by exons 1-2 and 6 of the enzyme ge ne contribute to substrate recognition (Ishii, S. et al. (1994) Biochi m. Biophys. Acta 1204, 265-270). In this study, we constructed five si ngle amino acid substitutions for functional analysis of the amino aci d residues around glutamine-279, the mutation site detected in an atyp ical Fabry disease patient. Two mutants, Q280S (Gln280 --> Ser; CAA -- > TCA) and T282A (Thr282 --> Ala; ACT --> GCT), showed increased K-m a nd decreased thermostability as compared with normal enzyme. Circular dichroism spectrum was not modified. An additional chimeric mutation i n the exon 1-2 region by substitution with the homologous sequence of alpha-N-acetylgalac tosaminidase cDNA restored catalytic activity and thermostability in both mutants. These data indicated the functional s ignificance of glutamine-280 and threonine-282 for expressing the acti vity and stability of alpha-galactosidase molecule, and also the prese nce of an intramolecular interaction between the two peptide regions e ncoded by exons 1-2 and 6.