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Figure 1.
Figure 1. The schematic diagram showing the kinetic pathway
of the Gal-T1 (GT) enzyme and of lactose synthase reaction
where, in the presence of aLA, glucose (Glc) is the acceptor
substrate. The crystal structures of the representative
intermediates determined here along the reaction pathway,
together with a previously determined structure, are indicated
underneath the reaction scheme with the corresponding Figures
here, in blue and red, respectively. First the apo-enzyme exists
in an open conformation (Figure 2(a)), to which the manganese
ion (Mn2+) binds (Figure 3(a)), followed by the donor substrate,
UDP-Gal (Figure 3(b)). Upon UDP-Gal or UDP-sugar binding the
enzyme undergoes conformational changes from open to closed
(Figure 3(c)), creating the acceptor and aLA binding sites. aLA
and Glc bind together synergistically to GT-Mn2+-UDP-sugar
complex in the closed conformation, forming a ground state
pentenary complex (Figure 4). During the transition state the
sugar moiety is cleaved from UDP-sugar and exists as an
oxocarbenium ion, shown as Gal* (or GalNAc* in Figure 4), which
forms a disaccharide linkage with the acceptor sugar, Glc, and
is then released from the enzyme (GT) along with the aLA
molecule from the pentenary complex. Here, we have used
UDP-GalNAc as the donor substrate to crystallize the pentenary
complex (Figure 4), since due to the steric hindrance caused by
the side-chain of Tyr286 residue with the N-acetyl moiety of
UDP-GalNAc, the transfer of GalNAc from UDP-GalNAc to Glc is
very poor, thus enabling us to crystallize the pentenary complex.
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