Kinetic and mechanistic analysis of Trypanosoma cruzi trans-sialidase reveals a classical ping-pong mechanism with acid/base catalysis

Iben Damager, Sabrina Buchini, Maria F Amaya, Alejandro Buschiazzo, Pedro Alzari, Alberto C Frasch, Andrew G Watts, Stephen G Withers

Research output: Contribution to journalArticlepeer-review

52 Citations (Scopus)


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 This is supported for both the native enzyme and a mutant (D59A) in which the putative acid/base catalyst has been replaced by the demonstration of the half-reaction in which a sialyl-enzyme intermediate is formed.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.
Original languageEnglish
Pages (from-to)3507-3512
Number of pages6
Issue number11
Early online date20 Feb 2008
Publication statusPublished - 18 Mar 2008

Fingerprint Dive into the research topics of 'Kinetic and mechanistic analysis of <em>Trypanosoma cruzi</em> trans-sialidase reveals a classical ping-pong mechanism with acid/base catalysis'. Together they form a unique fingerprint.

Cite this