The kinetic mechansim of bovine milk galactosyltransferase. The role of alpha-lactalbumin.

Crystal structures of guinea-pig, goat and bovine α-lactalbumin highlight the enhanced conformational flexibility of regions that are significant for its action in lactose synthase

“…This observation is consistent with the monosaccharide bridge model that has been proposed for the molecular mechanism of action of LA [6]. In this model, the galactosyl acceptor site of GT is adjacent to the site of interaction with LA, so that there is mutually exclusive binding between LA and monosaccharides extended at the C1(␤) or C2 carbon atoms of the pyranose ring, as well as between LA and glycoproteins and other oligosaccharide substrates [3,[31][32][33]. Binding at this site positions the cleft of LA, most likely the subsite F region, close to the galactosyl acceptor subsite of GT, providing favourable, stabilizing interactions for the bound monosaccharide.…”

“…Although circular dichroism spectroscopy suggests that structural alterations occur on the addition of either UDP-galactose or UDP-galactose and GlcNAc to a GT⋅Mn 2+ complex, there is no evidence that further changes take place when LA is added [36,37]. Large-scale conformational changes, arising from the association of the two proteins, appear to be inconsistent with LA's rapid equilibrium binding to GT [2,3,32]. Furthermore, alternative explanations for LA's modulatory effects, that invoke substantial structural changes in one or both components of the complex, seem unnecessary given that, in the absence of LA, GT is capable of carrying out the galactosylation of glucose at high concentrations of this acceptor substrate [2].…”

“…GT is unable to catalyze the biosynthesis of lactose (transfer of galactose to glucose) at physiological concentrations of glucose because of its poor affinity for this acceptor substrate. In the lactose synthase complex, reversible binding of the regulatory subunit, ␣-lactalbumin (LA), to GT promotes glucose binding (reflected in a 1000-fold decrease in the K m for glucose) and facilitates the biosynthesis of lactose [2,3].…”

A Chemoenzymatic Method Based on Easily Accessible Enzymes for Profiling Protein O-GlcNAcylation

“…Previously, GalT had already been applied in some pioneering studies of O-GlcNAcylation, where it transferred isotope-labeled UDP-Gal to nuclear-cytoplasmic O-GlcNAcylated proteins for detection. , Galactose was linked to both glucose and GlcNAc by GalT. It was reported that with α-lactalbumin, glucose became the preferred acceptor of the enzyme . However, without α-lactalbumin, the reaction rate for linking Gal to GlcNAc is much faster.…”

“…It was reported that with α-lactalbumin, glucose became the preferred acceptor of the enzyme. 34 However, without α-lactalbumin, the reaction rate for linking Gal to GlcNAc is much faster. Therefore, to increase the reaction efficiency toward O-GlcNAc, we did not add α-lactalbumin.…”