Structure and function in galactosyltransferase. Sequence locations of alpha-lactalbumin binding site, thiol groups, and disulfide bond.
The region(s) of bovine galactosyltransferase that interacts with the lactose synthase regulatory protein alpha-lactalbumin was investigated using trace 3H acetylation to probe the effects of alpha-lactalbumin on the reactivities of the individual amino groups of galactosyltransferase. In the presence of Mn2+, alpha-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 alpha-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 UDP-galactose 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 Cys134-Cys247 disulfide bond, which functions in alpha-lactalbumin and acceptor binding, and the COOH-terminal region, which is involved in UDP-galactose binding.
Yadav
S P
SP
Department of Biochemistry and Molecular Biology, University of Miami, Florida 33136.
Brew
K
K
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry; D000818:Animals; D001665:Binding Sites; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D003545:Cysteine / Q000378:metabolism; D004220:Disulfides / Q000378:metabolism; D005700:Galactosyltransferases / Q000378:metabolism*; D007768:Lactalbumin / Q000378:metabolism*; D008969:Molecular Sequence Data; D013329:Structure-Activity Relationship; D013438:Sulfhydryl Compounds / Q000378:metabolism*
D000596:Amino Acids; D004220:Disulfides; D013438:Sulfhydryl Compounds; D007768:Lactalbumin; D005700:Galactosyltransferases; D003545:Cysteine