The retaining glycosyltransferase, α-1,3-galactosyltransferase α3GT), is mutationally inactivated in humans, leading to the presence of circulating antibodies against its product, the α-Gal epitope. α3GT catalyzes galactose transfer from UDP-Gal to β-linked galactosides, such as lactose, and in the absence of an acceptor substrate, to water at a lower rate. We have used site-directed mutagenesis to investigate the roles in catalysis and specificity of residues in α3GT that form H-bonds as well as other interactions with substrates. Mutation of the conserved Glu 317 to Gln weakens lactose binding and reduces the kcat for galactosyltransfer to lactose and water by 2400 and 120, respectively. The structure is not perturbed by this substitution, but the orientation of the bound lactose molecule is changed. The magnitude of these changes does not support a previous proposal that Glu317 is the catalytic nucleophile in a double displacement mechanism and suggests it acts in acceptor substrate binding and in stabilizing a cationic transition state for cleavage of the bond between UDP and C1 of the galactose. Cleavage of this bond also linked to a conformational change in the C-terminal region of α3GT that is coupled with UDP binding. Mutagenesis indicates that His280, which is projected to interact with the 2-OH of the galactose moiety of UDP-Gal, is a key residue in the stringent donor substrate specificity through its role in stabilizing the bound UDP-Gal in a suitable conformation for catalysis. Mutation of Gln247, which forms multiple interactions with acceptor substrates, to Glu reduces the catalytic rate of galactose transfer to lactose but not to water. This mutation is predicted to perturb the orientation or environment of the bound acceptor substrate. The results highlight the importance of H-bonds between enzyme and substrates in this glycosyltransferase, in arranging substrates in appropriate conformations and orientation for efficient catalysis. These factors are manifested in increases in catalytic rate rather than substrate affinity.
|Publication status||Published - 25 Nov 2003|