Active site of bovine galactosyltransferase: kinetic and fluorescence studies

Bovine galactosyltransferase has been shown to have two metal binding sites. The functional properties of these metal binding sites have been established by using kinetic, spectroscopic, and affinity chromatographic approaches. Metal site I, which is involved in maintaining the structural integrity of the protein, must be liganded prior to other substrates binding and prior to a second metal binding to site II, which is shown to be associated with UDP-galactose binding. Both metal sites can bind a variety of metals; however, calcium and its fluorescent analogue europium bind only to site II. Fluorescent resonance energy transfer measurements between europium in site II and cobalt in site I indicate a distance of 18 +/- 3 A between the two sites. Chemical modification studies with S-mercuric-N-dansylcysteine indicate that one (of a total of three exposed sulfhydryl groups) can be specifically dansylated and that this sulfhydryl group is in or near the UDP-galactose binding site. Resonance energy transfer measurements between this introduced sulfhydryl group and cobalt in metal site I give a distance of 19 +/- 3 A between these points, consistent with the interpretation that the UDP-galactose binding site, which is associated with metal site II, is located some distance from the structural metal site (site I).