FUNCTIONAL ANALYSIS OF THE CATALYTIC DOMAIN OF BETA-1,4GALACTOSYLTRANSFERASE

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We have continued our work on the structural analyses of glycosyltransferases that function in the biosynthesis of glycoproteins and glycolipids. The beta-1,4-galactosyltransferase (beta-1,4GT) is localized in the trans Golgi, where in the presence of manganese, it catalyzes the transfer of galactose from UDP-galactose to the non-reducing terminal N-acetylglucosamine residue of glycoproteins and glycolipids. From the kinetic data it has been suggested that two metal ions are required, one during catalysis and the other being essential for the structural integrity of the protein. In the previous year we have separately expressed the N-and C-terminal fragments of the catalytic domain, residues 130-257, and 258-402, respectively, in E.coli. By binding analyses of the renatured fragments we showed that the major binding region for the sugar acceptor lies in the N-terminal portion while the nucleotide sugar donor binding is localized to the C-terminal half of the catalytic domain. The disulfide bond between Cys134 and Cys247 is required during enzymatic activity and is not essential for binding to the substrates. (Boeggeman et al., Glycoconjugate J 1995:12;865-78). Crystal structure analysis of the Taq polymerase has revealed conserved sequence motifs which are essential for divalent manganese binding and catalysis. Two such sites are localized within the N-(DYD and DVD) and C-terminal (EDDD) halves of the catalytic domain of a1,4GT, respectively. We have used site directed mutagenesis to analyze Mn2+ binding regions of beta-1,4GT. Our results demonstrate that mutation of Asp residue at positions 242, 244, 252 and 254, within the N-terminal fragment, abrogate the enzymatic activity of beta-1,4GT. Mutation of E317 and Asp residue at positions 318, 319 and 320, also destroyed the enzymatic activity. Furthermore, kinetic analyses show 50 fold increase in the apparent Km for UDP-galactose of the mutant D320N compared to D244N and the wild type beta-1,4GT. These results demonstrate that mutation of D320, located within the C-terminal portion, influences only the binding to UDP-galactose substrate and not to N-acetylglucosamine, while as mutation of D244, located within the N-terminal portion, that abrogates the enzymatic activity, has no influence on the binding of either UDP-galactose or N-acetylglucosamine.