Specific hetero-oligosaccharides on glycoproteins and glycolipids play important roles in cell-cell and cell-matrix interactions, affect the stability and structure of proteins, and modulate cellular interactions with viruses, toxins, and other proteins; they are also epitopes that are recognized by the immune system (1). The range and types of carbohydrate structures present on a cell vary in different tissues and species as a reflection of the specificity of glycosyltransferases, enzymes that catalyze the transfer of a specific monosaccharide from an activated derivative (such as UDP-galactose) into a defined linkage with a specific acceptor (2, 3). The carbohydrate chains of glycoconjugates have an enormous potential for variation because of the range of different sugars and the large number of alternative glycosidic linkages. Consequently, they are carriers of large amounts of biological information that originates from the specificity of glycosyltransferases. In addition to their biosynthetic roles, some glycosyltransferases also function directly in cellular interactions and regulation (4, 5). The limited information currently available regarding the structural basis of molecular recognition and catalysis by these important enzymes precludes a clear understanding of the molecular basis of their various biological functions. High resolution structural data are also needed to inform the design of inhibitors and to facilitate the engineering of new catalysts for the enzymatic synthesis of natural and novel glycoconjugates for therapeutic uses (4, 6, 7).

The retaining glycosyltransferase, alpha-1,3-galactosyltransferase (alpha3GT), is mutationally inactivated in humans, leading to the presence of circulating antibodies against its product, the alpha-Gal epitope. alpha3GT catalyzes galactose transfer from UDP-Gal to beta-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 alpha3GT 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 k(cat) 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 Glu(317) 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 alpha3GT that is coupled with UDP binding. Mutagenesis indicates that His(280), 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 Gln(247), 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.