Specificity and mechanism of metal ion activation in UDP-galactose :beta-Galactoside-alpha-1,3-galactosyltransferase

UDP-galactose:beta -galactosyl-alpha1,3-galactosyltransferase (alpha 3GT) c atalyzes the synthesis of galactosyl-alpha -1,3-beta -galactosyl structures in mammalian glycoconjugates. In humans the gene for a3GT is inactivated, and its product, the alpha -Gal epitope, is the target of a large fraction of natural antibodies. alpha 3GT is a member of a family of metal-dependent -retaining glycosyltransferases that includes the histo blood group A and B enzymes. Mn2+ activates the catalytic domain of alpha 3GT (alpha 3GTcd), b ut the affinity reported for this ion is very low relative to physiological levels. Enzyme activity over a wide range of metal ion concentrations indi cates a dependence on Mn2+ binding to two sites. At physiological metal ion concentrations, Zn2+ gives higher levels of activity and may be the natura l cofactor. To determine the role of the cation, metal activation was pertu rbed by substituting Co2+ and Zn2+ for Mn2+ and by mutagenesis of a conserv ed (DVD151)-V-149 sequence motif that is considered to act in cation bindin g 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 U DP-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 i n many glycosyltransferases appears to have a key role in metal-mediated do nor substrate binding and phosphate-sugar bond cleavage.