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

被引:18
作者
Zhang, YN [1 ]
Malinovskii, VA [1 ]
Fiedler, TJ [1 ]
Brew, K [1 ]
机构
[1] Univ Miami, Sch Med, Dept Biochem & Mol Biol, Miami, FL 33101 USA
关键词
beta 1,4 galactosyltransferase; expression in E. coli; mutagenesis; reaction mechanism;
D O I
10.1093/glycob/9.8.815
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
The truncated catalytic domain of bovine beta 1,4 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 beta 4GT-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 beta 4GT-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.
引用
收藏
页码:815 / 822
页数:8
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