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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 This is supported for both the native enzyme and a mutant (D59A) in which the putative acid/base catalyst has been replaced by the demonstration of the half-reaction in which a sialyl-enzyme intermediate is formed.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.
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Submitted on : Saturday, April 25, 2020 - 12:19:08 AM
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Iben Damager, Sabrina Buchini, Maria Amaya, Alejandro Buschiazzo, Pedro Alzari, et al.. Kinetic and Mechanistic Analysis of Trypanosoma cruzi Trans-Sialidase Reveals a Classical Ping-Pong Mechanism with Acid/Base Catalysis. Biochemistry, American Chemical Society, 2008, 47 (11), pp.3507-3512. ⟨10.1021/bi7024832⟩. ⟨pasteur-02554111⟩

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