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PDBsum entry 5cql
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References listed in PDB file
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Key reference
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Title
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Glycosyltransfer in mutants of putative catalytic residue glu303 of the human abo(h) a and b blood group glycosyltransferases gta and gtb proceeds through a labile active site.
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Authors
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R.J.Blackler,
S.M.Gagnon,
R.Polakowski,
N.L.Rose,
R.B.Zheng,
J.A.Letts,
A.R.Johal,
B.Schuman,
S.N.Borisova,
M.M.Palcic,
S.V.Evans.
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Ref.
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Glycobiology, 2017,
27,
370-380.
[DOI no: ]
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PubMed id
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Note: In the PDB file this reference is
annotated as "TO BE PUBLISHED". The citation details given above have
been manually determined.
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Abstract
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The homologous glycosyltransferases α-1,3-N-acetylgalactosaminyltransferase
(GTA) and α-1,3-galactosyltransferase (GTB) carry out the final synthetic step
of the closely related human ABO(H) blood group A and B antigens. The catalytic
mechanism of these model retaining enzymes remains under debate, where Glu303
has been suggested to act as a putative nucleophile in a double displacement
mechanism, a local dipole stabilizing the intermediate in an orthogonal
associative mechanism or a general base to stabilize the reactive oxocarbenium
ion-like intermediate in an SNi-like mechanism. Kinetic analysis of GTA and GTB
point mutants E303C, E303D, E303Q and E303A shows that despite the enzymes
having nearly identical sequences, the corresponding mutants of GTA/GTB have up
to a 13-fold difference in their residual activities relative to wild type.
High-resolution single crystal X-ray diffraction studies reveal, surprisingly,
that the mutated Cys, Asp and Gln functional groups are no more than 0.8 Å
further from the anomeric carbon of donor substrate compared to wild type.
However, complicating the analysis is the observation that Glu303 itself plays a
critical role in maintaining the stability of a strained "double-turn"
in the active site through several hydrogen bonds, and any mutation other than
E303Q leads to significantly higher thermal motion or even disorder in the
substrate recognition pockets. Thus, there is a remarkable juxtaposition of the
mutants E303C and E303D, which retain significant activity despite disrupted
active site architecture, with GTB/E303Q, which maintains active site
architecture but exhibits zero activity. These findings indicate that
nucleophilicity at position 303 is more catalytically valuable than active site
stability and highlight the mechanistic elasticity of these enzymes.
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