Kinetic and mechanistic analysis of Trypanosoma cruzi trans-sialidase reveals a classical ping-pong mechanism with acid/base catalysis.
The trans-sialidase from Trypanosoma cruzi catalyzes the transfer of a sialic acid moiety from sialylated donor substrates to the terminal galactose moiety of lactose and lactoside acceptors to yield alpha-(2,3)-sialyllactose or its derivatives with net retention of anomeric configuration. Through kinetic analyses in which the concentrations of two different donor aryl alpha-sialoside substrates and the acceptor substrate lactose were independently varied, we have demonstrated that this enzyme follows a ping-pong bi-bi kinetic mechanism. This is supported for both the native enzyme and a mutant (D59A) in which the putative acid/base catalyst has been replaced by the demonstration of the half-reaction in which a sialyl-enzyme intermediate is formed. Mass spectrometric analysis of the protein directly demonstrates the formation of a covalent intermediate, while the observation of release of a full equivalent of p-nitrophenol by the mutant in a pre-steady state burst provides further support. The active site nucleophile is confirmed to be Tyr342 by trapping of the sialyl-enzyme intermediate using the D59A mutant and sequencing of the purified peptic peptide. The role of D59 as the acid/base catalyst is confirmed by chemical rescue studies in which activity is restored to the D59A mutant by azide and a sialyl azide product is formed.
Damager
Iben
I
Department of Chemistry, University of British Columbia, 2036 Main Mall, Vancouver, British Columbia, V6T1Z1 Canada.
Buchini
Sabrina
S
Amaya
Maria F
MF
Buschiazzo
Alejandro
A
Alzari
Pedro
P
Frasch
Alberto C
AC
Watts
Andrew
A
Withers
Stephen G
SG
D000409:Alanine / Q000235:genetics; D000818:Animals; D001224:Aspartic Acid / Q000235:genetics; D001386:Azides / Q000737:chemistry; D002384:Catalysis; D006023:Glycoproteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D019158:N-Acetylneuraminic Acid / Q000737:chemistry; D009439:Neuraminidase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D009596:Nitrophenols / Q000737:chemistry; D013379:Substrate Specificity / Q000235:genetics; D014349:Trypanosoma cruzi / Q000201:enzymology* / Q000235:genetics; D014443:Tyrosine / Q000737:chemistry
D001386:Azides; D006023:Glycoproteins; D009596:Nitrophenols; D001224:Aspartic Acid; D014443:Tyrosine; C069715:trans-sialidase; D009439:Neuraminidase; D019158:N-Acetylneuraminic Acid; D000409:Alanine; C024836:4-nitrophenol