title: An active carbonyl formed during glycosylphosphatidylinositol addition to a protein is evidence of catalysis by a transamidase.
abstract: Glycosylphosphatidylinositol (GPI) substitution is now recognized to be a ubiquitous method of anchoring a protein to membranes in eukaryotes. The structure of GPI and its biosynthetic pathways are known and the signals in a nascent protein for GPI addition have been elucidated. The enzyme(s) responsible for GPI addition with release of a COOH-terminal signal peptide has been considered to be a transamidase but has yet to be isolated, and evidence that it is a transamidase is indirect. The experiments reported here show that hydrazine and hydroxylamine, in the presence of rough microsomal membranes, catalyze the conversion of the pro form of the engineered protein miniplacental alkaline phosphatase (prominiPLAP) to mature forms from which the COOH-terminal signal peptide has been cleaved, apparently at the same site but without the addition of GPI. The products, presumable the hydrazide or hydroxamate of miniPLAP, have yet to be characterized definitively. However, our demonstration of enzyme-catalyzed cleavage of the signal peptide in the presence of the small nucleophiles, even in the absence of an energy source, is evidence of an activated carbonyl intermediate which is the hallmark of a transamidase.
authors.affiliation: Roche Institute of Molecular Biology, Roche Research Center, Nutley, New Jersey 07110-1199, USA.
authors.forename: S E
authors.initials: SE
authors.lastname: Maxwell
authors.forename: S
authors.initials: S
authors.lastname: Ramalingam
authors.forename: L D
authors.initials: LD
authors.lastname: Gerber
authors.forename: L
authors.initials: L
authors.lastname: Brink
authors.forename: S
authors.initials: S
authors.lastname: Udenfriend
mesh_terms: D000217:Acyltransferases / Q000378:metabolism*; D000469:Alkaline Phosphatase / Q000378:metabolism; D019881:Aminoacyltransferases; D002384:Catalysis; D002474:Cell-Free System; D017261:Glycosylphosphatidylinositols / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D006834:Hydrazines / Q000378:metabolism; D019811:Hydroxylamine; D006898:Hydroxylamines / Q000378:metabolism; D010920:Placenta / Q000201:enzymology; D014176:Protein Biosynthesis; D011499:Protein Processing, Post-Translational; D011506:Proteins / Q000378:metabolism*; D013379:Substrate Specificity
chemical_list: D017261:Glycosylphosphatidylinositols; D006834:Hydrazines; D006898:Hydroxylamines; D011506:Proteins; C029424:hydrazine; D019811:Hydroxylamine; D000217:Acyltransferases; D019881:Aminoacyltransferases; C023175:transamidases; D000469:Alkaline Phosphatase