title: The carboxyl terminus of coffee bean alpha-galactosidase is critical for enzyme activity.
abstract: The role of the carboxyl (C)-terminal region of coffee bean alpha-galactosidase (alpha-GAL) has been studied by expressing C-terminal deletion mutants in the methylotrophic yeast strain Pichia pastoris. A previous study of human alpha-galactosidase determined that enzyme activity increased when up to 10 amino acid residues were deleted. Deleting 11 residues reduced activity, and deleting 12 residues abolished activity. In our studies, alpha-GAL activity is reduced when one or two amino acids are deleted, as is enzyme secretion directed by P. pastoris signal sequences. The pH profile is similar to that of the wild-type enzyme. Deleting 3 or more residues from the C-terminal end results in a complete loss of both enzyme secretion and activity. The C-terminus of alpha-GAL seems to play an important role in overall enzyme conformation and may directly affect the proper conformation of the active site.
authors.affiliation: Lindsley F. Kimball Research Institute of the New York Blood Center, 310 East 67th Street, New York, New York, 10021, USA.
authors.forename: E
authors.initials: E
authors.lastname: Maranville
authors.forename: A
authors.initials: A
authors.lastname: Zhu
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D020134:Catalytic Domain / Q000235:genetics; D003069:Coffee / Q000201:enzymology* / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics; D006801:Humans; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D007700:Kinetics; D008969:Molecular Sequence Data; D010843:Pichia / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017384:Sequence Deletion; D017386:Sequence Homology, Amino Acid; D012689:Sequence Homology, Nucleic Acid; D000519:alpha-Galactosidase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
chemical_list: D003069:Coffee; D018076:DNA, Complementary; D011994:Recombinant Proteins; D000519:alpha-Galactosidase