Functional domains of bovine beta-1,4 galactosyltransferase.
A number of N- and C-terminal deletion and point mutants of bovine beta-1,4 galactosyltransferase (beta-1,4GT) were expressed in E. coli to determine the binding regions of the enzyme that interact with N-acetylglucosamine (NAG) and UDP-galactose. The N-terminal truncated forms of the enzyme between residues 1-129, do not show any significant difference in the apparent Kms towards NAG or linear oligosaccharide acceptors e.g. for chitobiose and chitotriose, or for the nucleotide donor UDP-galactose. Deletion or mutation of Cys 134 results in the loss of enzymatic activity, but does not affect the binding properties of the protein either to NAG- or UDP-agarose. From these columns the protein can be eluted with 15 mM NAG and 50 mM EDTA, like the enzymatically active protein, TL-GT129, that contains residues 130-402 of bovine beta-1,4GT. Also the N-terminus fragment, TL-GT129NAG, that contains residues 130-257 of the beta-1,4GT, binds to, and elutes with 15 mM NAG and 50 mM EDTA from the NAG-agarose column as efficiently as the enzymatically active TL-GT129. Unlike TL-GT129, the TL-GT129NAG binds to UDP-columns less efficiently and can be eluted from the column with only 15 mM NAG. The C-terminus fragment GT-257UDP, containing residues 258-402 of beta-1,4GT, binds tightly to both NAG- and UDP-agarose columns. A small fraction, 5-10% of the bound protein, can be eluted from the UDP-agarose column with 50 mM EDTA alone. The results show that the binding behaviour of N- and C-terminal fragments of beta-1,4GT towards the NAG- and UDP-agarose columns differ, the former binds preferentially to NAG-columns, while the latter binds to UDP-agarose columns via Mn2+.
Boeggeman
E E
EE
Laboratory of Mathematical Biology, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892-8105, USA.
Balaji
P V
PV
Qasba
P K
PK
D000117:Acetylglucosamine / Q000737:chemistry*; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002417:Cattle; D002872:Chromosome Deletion; D004926:Escherichia coli; D005822:Genetic Vectors; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009237:N-Acetyllactosamine Synthase / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D011994:Recombinant Proteins / Q000096:biosynthesis; D012995:Solubility; D014531:Uridine Diphosphate Galactose / Q000737:chemistry*
D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D014531:Uridine Diphosphate Galactose; D009237:N-Acetyllactosamine Synthase; D000117:Acetylglucosamine