Glycobiology, 2002, Vol. 12, No. 7 395-407
© 2002 Oxford University Press
Studies on the metal binding sites in the catalytic domain of ß1,4-galactosyltransferase
2 Structural Glycobiology Section and 3 Intramural Research Support Program-SAIC, Laboratory of Experimental and Computational Biology, NCI-CCR, Building 469, Room 221, Frederick, MD, 21702-1201, USA
The catalytic domain of bovine ß1,4-galactosyltransferase (ß4Gal-T1) has been shown to have two metal binding sites, each with a distinct binding affinity. Site I binds Mn2+ with high affinity and does not bind Ca2+, whereas site II binds a variety of metal ions, including Ca2+. The catalytic region of ß4Gal-T1 has DXD motifs, associated with metal binding in glycosyltransferases, in two separate sequences: D242YDYNCFVFSDVD254 (region I) and W312GWGGEDDD320 (region II). Recently, the crystal structure of ß4Gal-T1 bound with UDP, Mn2+, and 
-lactalbumin was determined in our laboratory. It shows that in the primary metal binding site of ß4Gal-T1, the Mn2+ ion, is coordinated to five ligands, two supplied by the phosphates of the sugar nucleotide and the other three by Asp254, His347, and Met344. The residue Asp254 in the D252VD254 sequence in region I is the only residue that is coordinated to the Mn2+ ion. Region II forms a loop structure and contains the E317DDD320 sequence in which residues Asp318 and Asp319 are directly involved in GlcNAc binding. This study, using site-directed mutagenesis, kinetic, and binding affinity analysis, shows that Asp254 and His347 are strong metal ligands, whereas Met344, which coordinates less strongly, can be substituted by alanine or glutamine. Specifically, substitution of Met344 to Gln has a less severe effect on the catalysis driven by Co2+. Glu317 and Asp320 mutants, when partially activated by Mn2+ binding to the primary site, can be further activated by Co2+ or inhibited by Ca2+, an effect that is the opposite of what is observed with the wild-type enzyme.
1 To whom correspondence should be addressed; E-mail: qasba{at}helix.nih.gov
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