Structure and Function in Galactosyltransferase

The region(s) of bovine galactosyltransferase that interacts with the lactose synthase regulatory protein a-lactalbumin was investigated using trace 3H acetylation to probe the effects of a-lactalbumin on the reactivities of the individual amino groups of galactosyltransferase. In the presence of Mn2+, a-lactalbumin was found to reduce the reactivities of lysines 93 and 181 and to increase the reactivities of one or more of lysines 230, 237, and 241. The addition of N-acetylglucosamine (20 mM), which enhances complex formation between the two proteins, did not significantly alter the pattern of perturbation. These results indicate that the NH2-terminal region of the catalytic domain of galactosyltransferase, and possibly part of the proline-rich “stem” region, is affected by the association with a-lactalbumin and is therefore implicated in the binding of acceptor substrates. In a separate study only cysteines 176, 266, and 342 of galactosyltransferase were found to react with [3H]iodoacetic acid under denaturing conditions. From their lack of reactivity it is deduced that the remaining two cysteines, residues 134 and 247, are joined in a disulfide linkage. From these results and those of a previous study of UDPgalactose binding (Yadav, S., and Brew, K. (1990) J. Biol. Chem. 265, 14163-14169) it appears that the soluble form of galactosyltransferase is composed of two domains, the NH2-terminal 150 residues containing the disulfide bond, which functions in a-lactalbumin and acceptor binding, and the COOHterminal region, which is involved in UDP-galactose binding.