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 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 This is supported for both the native enzyme and a mutant (D59A) in which the putative acid/base catalyst has been replaced by the demonstration of the half-reaction in which a sialyl-enzyme intermediate is formed.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.

Galactosyl-Lactose Sialylation Using Trypanosoma cruzi trans-Sialidase as the Biocatalyst and Bovine κ-Casein-Derived Glycomacropeptide as the Donor Substrate

“…Conversely, the V max determined for lactose was higher than that determined for ␤3=-GL, ␤4=-GL, and ␤6=-GL ( Table 2). The k cat value for lactose, being similar to that reported previously (15), was higher than the k cat values for the GL substrates. The higher affinity of the GL substrates compared to that for lactose suggests stronger binding of the former to TcTS, e.g., with stronger and/or more interactions with amino acid residues in the acceptor binding site.…”

“…TcTS-mediated trans-sialylation was shown to proceed via a ping-pong mechanism (14,15). In the first step, a relatively long-lived sialo-enzyme intermediate is formed through a covalent bond to the nucleophile Tyr342 (16).…”

Evidence of Ternary Complex Formation in Trypanosoma cruzi trans-Sialidase Catalysis

“…Many examples of enzymes that do not fall into the strict classification of sequential or ping-pong mechanisms but lie somewhere in between these two systems have been reported (70,71). Furthermore, a hybrid ping-pong sequential mechanism fits all of the data published to date (11,15,25,69,72).…”

“…The occurrence of such an intermediate was further observed in several enzymes of the GH33 family (12,13). Based on structural (11) and kinetic studies (14), Tyr-342 is considered to be the catalytic nucleophile residue of TcTS (11), whereas Asp-59 is proposed to act as a general acid/base catalyst in a double displacement reaction (14) that follows a classical ping-pong mechanism (15) in which the sialosyl-aglycone may leave the active site to allow entry of an acceptor substrate. According to this hypothesis, however, a water molecule could attack the sialosyl-enzyme intermediate before the acceptor substrate reaches the binding site, which would result in hydrolysis rather than in an efficient sugar transfer.…”

Synthesis of oseltamivir conjugates with lactose analogs for inhibition studies on Trypanosoma cruzi trans-sialidase

“…9 The TcTS works by a bisubstrate ping pong mechanism with formation of a covalent TcTS-sialosyl intermediate with Tyr 342, and acid-base catalysis. 10 The active site of TcTS shows some conserved features of microbial sialidases including the presence of an arginine triad which interacts with the negatively charged carboxylate group of sialic acid. In the absence of a suitable carbohydrate acceptor, TcTS functions as a sialidase, catalyzing sialoside hydrolysis.…”