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PDBsum entry 2a75

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Hydrolase PDB id
2a75
Jmol
Contents
Protein chain
632 a.a.
Ligands
SO4 ×2
FSI
Waters ×615

References listed in PDB file
Key reference
Title Structural and kinetic analysis of two covalent sialosyl-Enzyme intermediates on trypanosoma rangeli sialidase.
Authors A.G.Watts, P.Oppezzo, S.G.Withers, P.M.Alzari, A.Buschiazzo.
Ref. J Biol Chem, 2006, 281, 4149-4155. [DOI no: 10.1074/jbc.M510677200]
PubMed id 16298994
Abstract
Trypanosoma rangeli sialidase is a glycoside hydrolase (family GH33) that catalyzes the cleavage of alpha-2-->3-linked sialic acid residues from sialoglycoconjugates with overall retention of anomeric configuration. Retaining glycosidases usually operate through a ping-pong mechanism, wherein a covalent intermediate is formed between the carbohydrate and an active site carboxylic acid of the enzyme. Sialidases, instead, appear to use a tyrosine as the catalytic nucleophile, leaving the possibility of an essentially different catalytic mechanism. Indeed, a direct nucleophilic role for a tyrosine was shown for the homologous trans-sialidase from Trypanosoma cruzi, although itself not a typical sialidase. Here we present the three-dimensional structures of the covalent glycosyl-enzyme complexes formed by the T. rangeli sialidase with two different mechanism-based inactivators at 1.9 and 1.7A resolution. To our knowledge, these are the first reported structures of enzymatically competent covalent intermediates for a strictly hydrolytic sialidase. Kinetic analyses have been carried out on the formation and turnover of both intermediates, showing that structural modifications to these inactivators can be used to modify the lifetimes of covalent intermediates. These results provide further evidence that all sialidases likely operate through a similar mechanism involving the transient formation of a covalently sialylated enzyme. Furthermore, we believe that the ability to "tune" the inactivation and reactivation rates of mechanism-based inactivators toward specific enzymes represents an important step toward developing this class of inactivators into therapeutically useful compounds.
Figure 1.
A, structures of the fluorinated sialic acid derivatives 2,3-difluoro-N-acetyl-neuraminic acid (1) and 2,3-difluoro-2-keto-3-deoxy-d-glycero-d-galacto-nonulosonic acid (2) used as mechanism-based inactivators. B, a typical glycosidase-catalyzed reaction showing glycosylation (k[1]) and deglycosylation (k[2]) rate constants affected by the mechanism-based inactivators.
Figure 5.
3-Fluoro-KDN covalent complex with TrSA. Methionine 96 is observed in three alternate conformations, partially filling the cavity left by the absent N-acetyl group on the sialyl moiety. In the unbound enzyme (PDB code 1N1T) or bound to N-acetyl containing sialic acid derivatives, this Met is observed only in one conformation (corresponding to conformer A in this structure). The water network changes are also highlighted; W360 and W369 interact with important residues in the site. The refined 2mF[o] – DF[c] map contoured at 1σ is shown.
The above figures are reprinted by permission from the ASBMB: J Biol Chem (2006, 281, 4149-4155) copyright 2006.
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