Identification of an active-site residue in yeast invertase by affinity labeling and site-directed mutagenesis.
Deglycosylated yeast invertase is irreversibly inactivated by conduritol B epoxide (CBE), an active-site-directed reagent. The inactivated enzyme contained 0.8 mol of CBE/mol of invertase monomer suggesting that the inactivation results from the modification of a single amino acid residue. Peptic digestion of [3H]CBE-labeled invertase followed by reverse phase column chromatography yielded two labeled peptides, both located at the amino-terminal end of the enzyme. Sequence analyses of these peptides revealed that Asp-23 is the modified residue. The role of Asp-23 in the catalytic process was investigated by changing it to Asn using site-directed mutagenesis of the SCU2 gene. The mutant enzyme was basically inactive, confirming a role for Asp-23 in the catalytic process.
Reddy
V A
VA
Wadsworth Center for Laboratories and Research, New York State Department of Health, Albany.
Maley
F
F
D000118:Acetylglucosaminidase; D000345:Affinity Labels; D000595:Amino Acid Sequence; D001224:Aspartic Acid / Q000235:genetics; D001483:Base Sequence; D001665:Binding Sites; D002384:Catalysis; D002851:Chromatography, High Pressure Liquid; D006026:Glycoside Hydrolases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D006031:Glycosylation; D007294:Inositol / Q000031:analogs & derivatives* / Q000494:pharmacology; D007700:Kinetics; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; D008969:Molecular Sequence Data; D009154:Mutation; D010446:Peptide Fragments; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D014357:Trypsin; D043324:beta-Fructofuranosidase
D000345:Affinity Labels; D010446:Peptide Fragments; D001224:Aspartic Acid; D007294:Inositol; D006026:Glycoside Hydrolases; D043324:beta-Fructofuranosidase; D000118:Acetylglucosaminidase; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; D014357:Trypsin; C011423:conduritol epoxide