1VZT

ROLES OF INDIVIDUAL RESIDUES OF ALPHA-1,3 GALACTOSYLTRANSFERASES IN SUBSTRATE BINDING AND CATALYSIS

Replaces:  1O7R

Summary for 1VZT

• Entry DOI: 10.2210/pdb1vzt/pdb
• Related: 1FG5 1G8O 1G93 1GWV 1GWW 1GX0 1GX4 1K4V 1O7O 1O7Q
• Descriptor: N-ACETYLLACTOSAMINIDE ALPHA-1,3-GALACTOSYLTRANSFERASE, MANGANESE (II) ION, URIDINE-5'-DIPHOSPHATE, ... (5 entities in total)
• Functional Keywords: transferase, alpha-1, 3-galactosyltransferase-udp complex, glycosyltransferase, glycoprotein, transmembrane, nucleotide-binding protein, xenotransplantation
• Biological source: BOS TAURUS (BOVINE)
• Cellular location: Golgi apparatus, Golgi stack membrane; Single-pass type II membrane protein: P14769
• Total number of polymer chains: 2
• Total formula weight: 69084.49

Authors

Zhang, Y.,Swaminathan, G.J.,Deshpande, A.,Natesh, R.,Xie, X.,Acharya, K.R.,Brew, K. (deposition date: 2004-05-26, release date: 2005-05-26, Last modification date: 2023-12-13)

Primary citation

Zhang, Y.,Swaminathan, G.J.,Deshpande, A.,Boix, E.,Natesh, R.,Xie, Z.,Acharya, K.R.,Brew, K.
Roles of Individual Enzyme-Substrate Interactions by Alpha-1,3-Galactosyltransferase in Catalysis and Specificity.
Biochemistry, 42:13512-, 2003

PubMed Abstract: 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.

PubMed: 14621997
DOI: 10.1021/BI035430R

Experimental method

X-RAY DIFFRACTION (2 Å)