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 di...

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Bibliographic Details
Published in:The Journal of biological chemistry 1990-07, Vol.265 (19), p.10817-10820
Main Authors: Reddy, V.A. (New York State Department of Health, Albany, NY), Maley, F
Format: Article
Language:English
Subjects:
Acetylglucosaminidase
ACIDE ASPARTIQUE
ACIDO ASPARTICO
actine site amino acid
affinity labelling
Affinity Labels
amino acid residue
Amino Acid Sequence
ASPARTIC ACID
Aspartic Acid - genetics
Base Sequence
beta -fructofuranosidase
BINDING SITE
Binding Sites
Catalysis
Chromatography, High Pressure Liquid
Exact sciences and technology
FRUCTOFURANOSIDASA
FRUCTOFURANOSIDASE
Glycoside Hydrolases - antagonists & inhibitors
Glycoside Hydrolases - genetics
Glycoside Hydrolases - metabolism
Glycosylation
Inositol - analogs & derivatives
Inositol - pharmacology
Kinetics
Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase
Mathematical analysis
Mathematics
Molecular Sequence Data
Mutation
Peptide Fragments
Potential theory
SACCHAROMYCES CEREVISIAE
Saccharomyces cerevisiae - enzymology
Saccharomyces cerevisiae - genetics
Sciences and techniques of general use
site-directed mutagenesis
Trypsin
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Description
Summary: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.
ISSN:0021-9258
1083-351X
DOI:10.1016/s0021-9258(19)38518-7