Role of a conserved acidic cluster in bovine β1,4 galactosyltransferase-1 probed by mutagenesis of a bacterially expressed recombinant enzyme

Abstract

The truncated catalytic domain of bovine β1,4 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 β4GT-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 β4GT-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 β-galactosyltransferase reactions.