| Abstract: | Lactose synthase was found to show anomeric preference for beta-D-glucose. This information was utilized in the design of methyl, ethyl, propyl, butyl, and pentyl N-acetyl-beta-D-glucosaminides, which were subsequently demonstrated to be substrates for galactosyltransferase with apparent Km values in the low millimolar range. alpha-Lactalbumin competitively inhibits the transferase activity against these N-acetylglucosamine derivatives. This pattern of inhibition has also been observed when the dimer, trimer, and tetramer of N-acetylglucosamine and ovomucoid served as the galactose acceptor. The data suggest that the binding of alpha-lactalbumin and the N-acetylglucosamine derivatives is mutually exclusive. This assertion is further supported by the inability of methyl and butyl N-acetyl-beta-D-glucosaminides to facilitate retention of galactosyltransferase on a column of alpha-lactalbumin immobilized onto Sepharose. Free N-acetylglucosamine, on the other hand, does cause retention of the transferase under the same conditions. Thus, alpha-lactalbumin must bind to a region on galactosyltransferase in close proximity to the monosaccharide binding site and exert its substrate-specifying action through competitive and mutually exclusive binding with the N-acetylglucosamine analogues accompanied by an increased affinity for glucose. In short, our substrate analogue studies have revealed that the association-dissociation modulation of galactosyltransferase activity is effected through a topographical blockade of glycoprotein binding by alpha-lactalbumin. |
