Sulfhydryl modification of the yeast Wbp1p inhibits oligosaccharyl transferase activity.
Chemical labeling of the multimeric Saccharomyces cerevisiae oligosaccharyl transferase indicates that the 48 kDa Wbp1p subunit is an integral component of the catalytically active enzyme. The enzyme was purified following chromatography on concanavalin A agarose, heparin agarose, Q-Sepharose, and hydroxyapatite media. The enzyme activity copurified with a tetrameric complex of polypeptide subunits. Two of the subunits have been identified as the yeast proteins Wbp1p and Swp1p by amino-terminal residue sequencing. A third subunit was identified as a variably glycosylated polypeptide near 64 kDa; preliminary amino acid sequencing showed no identity to known yeast proteins. Modification of a cysteine residue by the reagent methyl methanethiolsulfonate (MMTS) caused time-dependent and concentration-dependent inactivation of the enzyme. To identify the modified subunit of the transferase complex, the labeling reagent S-[(N-biotinoylamino)ethyl] methanethiolsulfonate (BMTS) was synthesized. Like MMTS, BMTS inactivated the oligosaccharyl transferase in a time-dependent manner. Additionally, incubation with the substrate (dolichylpyrophosphoryl)-N,N'-diacetylchitobiose [Dol-PP(GlcNAc)2] protected the enzyme from BMTS inactivation. When the purified enzyme complex was incubated with BMTS, Wbp1p alone was specifically labeled, thereby associating this subunit with catalysis and the binding of the dolichylpyrophosphoryl oligosaccharide substrate in the transferase reaction.
Pathak
R
R
Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena 91125, USA.
Hendrickson
T L
TL
Imperiali
B
B
D000595:Amino Acid Sequence; D001710:Biotin / Q000031:analogs & derivatives* / Q000494:pharmacology; D002240:Carbohydrate Sequence; D002384:Catalysis; D003545:Cysteine / Q000737:chemistry; D004229:Dithiothreitol / Q000494:pharmacology; D004795:Enzyme Stability; D006602:Hexosyltransferases; D007700:Kinetics; D046911:Macromolecular Substances; D008565:Membrane Proteins; D008698:Mesylates / Q000494:pharmacology*; D008741:Methyl Methanesulfonate / Q000031:analogs & derivatives* / Q000494:pharmacology; D008861:Microsomes / Q000201:enzymology; D008969:Molecular Sequence Data; D011104:Polyisoprenyl Phosphate Oligosaccharides / Q000378:metabolism / Q000494:pharmacology; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D013329:Structure-Activity Relationship; D013438:Sulfhydryl Compounds / Q000737:chemistry; D014166:Transferases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism
D046911:Macromolecular Substances; D008565:Membrane Proteins; D008698:Mesylates; D011104:Polyisoprenyl Phosphate Oligosaccharides; C092885:S-((N-biotinoylamino)ethyl)methanethiolsulfonate; D013438:Sulfhydryl Compounds; C014674:methyl methanethiosulfonate; C016418:dolichyl-diphosphate-di-N-acetylchitobiose; D001710:Biotin; D008741:Methyl Methanesulfonate; D014166:Transferases; D006602:Hexosyltransferases; C025620:dolichyl-diphosphooligosaccharide - protein glycotransferase; D003545:Cysteine; D004229:Dithiothreitol