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Journal articles
Kinetic and Mechanistic Analysis of Trypanosoma cruzi Trans-Sialidase Reveals a Classical Ping-Pong Mechanism with Acid/Base Catalysis
Abstract : 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 R-(2,3)-sialyllactose or its derivatives with net retention of anomeric configuration. Through kinetic analyses in which the concentrations of two different donor aryl R-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 This is supported for both the native enzyme and a mutant (D59A) in which the putative acid/base catalyst has been replaced by the demonstration of the half-reaction in which a sialyl-enzyme intermediate is formed.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.
Cited literature [21 references]
https://hal-pasteur.archives-ouvertes.fr/pasteur-02554111
Contributor : Alejandro Buschiazzo <>
Submitted on : Saturday, April 25, 2020 - 12:19:08 AM
Last modification on : Friday, December 18, 2020 - 5:30:04 PM
Contributor : Alejandro Buschiazzo <>
Submitted on : Saturday, April 25, 2020 - 12:19:08 AM
Last modification on : Friday, December 18, 2020 - 5:30:04 PM
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