Mutation of arginine 228 to lysine enhances the glucosyltransferase activity of bovine beta-1,4-galactosyltransferase I

Biochemistry. 2005 Mar 8;44(9):3202-10. doi: 10.1021/bi0479454.


Beta-1,4-galactosyltransferase I (beta4Gal-T1) normally transfers Gal from UDP-Gal to GlcNAc in the presence of Mn(2+) ion (Gal-T activity) and also transfers Glc from UDP-Glc to GlcNAc (Glc-T activity), albeit at only 0.3% efficiency. In addition, alpha-lactalbumin (LA) enhances this Glc-T activity more than 25 times. Comparison of the crystal structures of UDP-Gal- and UDP-Glc-bound beta4Gal-T1 reveals that the O4 hydroxyl group in both Gal and Glc moieties forms a hydrogen bond with the side chain carboxylate group of Glu317. The orientation of the O4 hydroxyl of glucose causes a steric hindrance to the side chain carboxylate group of Glu317, accounting for the enzyme's low Glc-T activity. In this study, we show that mutation of Arg228, a residue in the vicinity of Glu317, to lysine (R228K-Gal-T1) results in a 15-fold higher Glc-T activity, which is further enhanced by LA to nearly 25% of the Gal-T activity of the wild type. The kinetic parameters indicate that the main effect of the mutation of Arg228 to lysine is on the k(cat) of Glc-T, which increases 3-4-fold, both in the absence and in the presence of LA; simultaneously, the k(cat) for the Gal-T reaction is reduced 30-fold. The crystal structure of R228K-Gal-T1 complexed with LA, UDP-Gal, and Mn(2+) determined at 1.9 A resolution shows that the Asp318 side chain exhibits a minor alternate conformation, compared to that in the wild type. This alternate conformation now causes a steric hindrance to the O4 hydroxyl group of the Gal moiety of UDP-Gal, probably causing the dissociation of UDP-Gal and the reduced k(cat) of the Gal-T reaction.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Animals
  • Arginine / genetics*
  • Cattle
  • Crystallization
  • Crystallography, X-Ray
  • Enzyme Activation / genetics
  • Galactosyltransferases / genetics*
  • Galactosyltransferases / metabolism*
  • Glucosyltransferases / metabolism
  • Kinetics
  • Lactalbumin / metabolism
  • Lysine / genetics*
  • Macromolecular Substances / metabolism
  • Manganese / chemistry
  • Mutagenesis, Site-Directed*
  • Uridine Diphosphate Galactose / metabolism
  • Uridine Diphosphate Glucose / metabolism


  • Macromolecular Substances
  • Uridine Diphosphate Galactose
  • Manganese
  • Lactalbumin
  • Arginine
  • Galactosyltransferases
  • Glucosyltransferases
  • beta-1,4-galactosyltransferase I
  • Lysine
  • Uridine Diphosphate Glucose

Associated data

  • PDB/1YRO