Role of a conserved acidic cluster in bovine beta1,4 galactosyltransferase-1 probed by mutagenesis of a bacterially expressed recombinant enzyme.
The truncated catalytic domain of bovine beta1,4 galactosyltransferase-1 was expressed as inclusion bodies in E.coli and folded to generate 10-15 mg of active enzyme per liter of bacterial culture after extraction and purification under denaturing conditions. Mutations were introduced to investigate the roles of Trp312, Asp318, and Asp320, components of a highly conserved region of sequence in all known beta4GT-1 homologues that includes a cluster of acidic residues. Near and far UV CD spectra of the mutants indicate that the substitutions did not perturb the secondary and tertiary structure of beta4GT-1, and steady state kinetic studies indicate only minor effects on the response to an essential metal cofactor. However substitutions for the two aspartyl residues result in a reduction in catalytic efficiency of a magnitude that suggests they are important for catalysis. It seems possible that this anionic center may act in stabilizing a carbocation formed from the galactose component of the donor substrate in the transition state, reflecting a common reaction mechanism for beta-galactosyltransferase reactions.
Zhang
Y
Y
Department of Biochemistry and Molecular Biology, University of Miami School of Medicine, Miami, FL 33101, USA.
Malinovskii
V A
VA
Fiedler
T J
TJ
Brew
K
K
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001483:Base Sequence; D002417:Cattle; D002942:Circular Dichroism; D017124:Conserved Sequence; D017931:DNA Primers; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D005700:Galactosyltransferases / Q000737:chemistry* / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017510:Protein Folding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid
D017931:DNA Primers; D011994:Recombinant Proteins; D005700:Galactosyltransferases; C119701:beta-1,4-galactosyltransferase I