Specificity and mechanism of metal ion activation in UDP-galactose:beta -galactoside-alpha -1,3-galactosyltransferase.
UDP-galactose:beta-galactosyl-alpha1,3-galactosyltransferase (alpha3GT) catalyzes the synthesis of galactosyl-alpha-1,3-beta-galactosyl structures in mammalian glycoconjugates. In humans the gene for alpha3GT is inactivated, and its product, the alpha-Gal epitope, is the target of a large fraction of natural antibodies. alpha3GT is a member of a family of metal-dependent-retaining glycosyltransferases that includes the histo blood group A and B enzymes. Mn(2+) activates the catalytic domain of alpha3GT (alpha3GTcd), but the affinity reported for this ion is very low relative to physiological levels. Enzyme activity over a wide range of metal ion concentrations indicates a dependence on Mn(2+) binding to two sites. At physiological metal ion concentrations, Zn(2+) gives higher levels of activity and may be the natural cofactor. To determine the role of the cation, metal activation was perturbed by substituting Co(2+) and Zn(2+) for Mn(2+) and by mutagenesis of a conserved D(149)VD(151) sequence motif that is considered to act in cation binding in many glycosyltransferases. The aspartates of this motif were found to be essential for activity, and the kinetic properties of a Val(150) to Ala mutant with reduced activity were determined. The results indicate that the cofactor is involved in binding UDP-galactose and has a crucial influence on catalytic efficiency for galactose transfer and for the low endogenous UDP-galactose hydrolase activity. It may therefore interact with one or more phosphates of UDP-galactose in the Michaelis complex and in the transition state for cleavage of the UDP to galactose bond. The DXD motif conserved in many glycosyltransferases appears to have a key role in metal-mediated donor substrate binding and phosphate-sugar bond cleavage.
Zhang
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Department of Biochemistry and Molecular Biology, University of Miami School of Medicine, Miami, Florida 33101, USA.
Wang
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PG
Brew
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D000818:Animals; D001483:Base Sequence; D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D002384:Catalysis; D002417:Cattle; D002942:Circular Dichroism; D017931:DNA Primers; D004789:Enzyme Activation; D005700:Galactosyltransferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008670:Metals / Q000378:metabolism*; D008969:Molecular Sequence Data; D016296:Mutagenesis; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity
D017931:DNA Primers; D008670:Metals; D011994:Recombinant Proteins; D005700:Galactosyltransferases; C047006:N-acetyllactosaminide alpha-1,3-galactosyltransferase