title: Trypanosoma cruzi trans-sialidase operates through a covalent sialyl-enzyme intermediate: tyrosine is the catalytic nucleophile.
abstract: Modified sialic acid substrates have been used to label Trypanosoma cruzi trans-sialidase, demonstrating that the enzyme catalyses the transfer of sialic acid through a covalent glycosyl-enzyme intermediate, a mechanism common to most retaining glycosidases. Peptic digestion of labeled protein, followed by LC-MS/MS analysis of the digest, identified Tyr342 as the catalytic nucleophile. This is the first such example of a retaining glycosidase utilizing an aryl glycoside intermediate. It is suggested that this alternative choice of nucleophile is a consequence of the chemical nature of sialic acid. A Tyr/Glu couple is invoked to relay charge from a remote glutamic acid, thereby avoiding electrostatic repulsion with the sialic acid carboxylate group.
authors.affiliation: Department of Chemistry, University of British Columbia, 2036 Main Mall, Vancouver, B.C., Canada V6T 1Z1.
authors.forename: Andrew G
authors.initials: AG
authors.lastname: Watts
authors.forename: Iben
authors.initials: I
authors.lastname: Damager
authors.forename: Maria L
authors.initials: ML
authors.lastname: Amaya
authors.forename: Alejandro
authors.initials: A
authors.lastname: Buschiazzo
authors.forename: Pedro
authors.initials: P
authors.lastname: Alzari
authors.forename: Alberto C
authors.initials: AC
authors.lastname: Frasch
authors.forename: Stephen G
authors.initials: SG
authors.lastname: Withers
mesh_terms: D000818:Animals; D002384:Catalysis; D004789:Enzyme Activation; D006023:Glycoproteins / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D009439:Neuraminidase / Q000737:chemistry / Q000378:metabolism*; D021241:Spectrometry, Mass, Electrospray Ionization; D014349:Trypanosoma cruzi / Q000201:enzymology*; D014443:Tyrosine / Q000737:chemistry / Q000378:metabolism*
chemical_list: D006023:Glycoproteins; D014443:Tyrosine; C069715:trans-sialidase; D009439:Neuraminidase