1o7q

File:1o7q.jpg

spinqualitylabelsall models 1o7q, resolution 1.30Å Show:Asymmetric Unit Biological Assembly Drag the structure with the mouse to rotate   Export Animated Image

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

OverviewOverview

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.

About this StructureAbout this Structure

1O7Q is a Single protein structure of sequence from Bos taurus with , and as ligands. Active as Transferred entry: 2.4.1.87, with EC number 2.4.1.151 Known structural/functional Site: . Full crystallographic information is available from OCA.

ReferenceReference

Roles of individual enzyme-substrate interactions by alpha-1,3-galactosyltransferase in catalysis and specificity., Zhang Y, Swaminathan GJ, Deshpande A, Boix E, Natesh R, Xie Z, Acharya KR, Brew K, Biochemistry. 2003 Nov 25;42(46):13512-21. PMID:14621997

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