Glycobiology Advance Access originally published online on June 9, 2004
Glycobiology 2004 14(10):895-907; doi:10.1093/glycob/cwh108
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Glycobiology vol. 14 no. 10 © Oxford University Press 2004; all rights reserved.
NMR spectroscopic and molecular modeling investigations of the trans-sialidase from Trypanosoma cruzi
Institute for Glycomics, Griffith University (Gold Coast Campus), PMB 50 Gold Coast Mail Centre, Queensland, 9726, Australia
Received on April 2, 2004; revised on May 21, 2004; accepted on June 2, 2004
Nuclear magnetic resonance (NMR) spectroscopy was used to investigate the transfer of sialic acid from a range of sialic acid donor compounds to acceptor molecules, catalyzed by Trypanosoma cruzi trans-sialidase (TcTS). We demonstrate here that NMR spectroscopy is a powerful tool to monitor the trans-sialidase enzyme reaction for a variety of donor and acceptor molecules. The hydrolysis or transfer reactions that are catalyzed by TcTS were also investigated using a range of N-acetylneuraminosyl-based donor substrates and asialo acceptor molecules. These studies showed that the synthetic N-acetylneuraminosyl donor 4-methylumbelliferyl 
-D-N-acetylneuraminide (MUN) is hydrolyzed by the enzyme 
3–5 times faster than either the disaccharide Neu5Ac(2,3)Galß1Me or the trisaccharide Neu5Ac(2,3)Lacß1Me. In the transfer reaction, we show that Neu5Ac(2,3)Lacß1Me is the most favorable substrate for TcTS and is a better substrate than the naturally-occurring N-acetylneuraminosyl donor 1-acid glycoprotein. In the case of MUN as the donor molecule, the transfer of Neu5Ac to different acceptors is significantly slower than when other N-acetylneuraminosyl donors are used. We hypothesize that when MUN is bound by the enzyme, the orientation and steric bulk of the umbelliferyl aglycon moiety may restrict the access for the correct positioning of an acceptor molecule. AutoDock studies support our hypothesis and show that the umbelliferyl aglycon moiety undergoes a strong pi-stacking interaction with Trp-312. The binding properties of TcTS towards acceptor (lactose) and donor substrate (Neu5Ac) molecules have also been investigated using saturation transfer difference (STD) NMR experiments. These experiments, taken together with other published data, have clearly demonstrated that lactose in the absence of other coligands does not bind to the TcTS active site or other binding domains. However, in the presence of the sialic acid donor, lactose (an asialo acceptor) was observed by NMR spectroscopy to interact with the enzyme's active site. The association of the asialo acceptor with the active site is an absolute requirement for the transfer reaction to proceed.
1 To whom correspondence should be addressed; e-mail: m.vonitzstein{at}griffith.edu.au