Sequential Interchange of Four Amino Acids from Blood Group B to Blood Group A Glycosyltransferase Boosts Catalytic Activity and Progressively Modifies Substrate Recognition in Human Recombinant Enzymes
Journal of Biological Chemistry(1997)
摘要
The human blood group A and B glycosyltransferase enzymes are highly homologous and the alteration of four critical amino acid residues (Arg-176 --> Gly, Gly-235 --> Ser, Leu-266 --> Met, and Gly-268 - Ala) is sufficient to change the enzyme specificity from a blood group A to a blood group B glycosyltransferase, To carry out a systematic study, a synthetic gene strategy was employed to obtain their genes and to allow facile mutagenesis. Soluble forms of a recombinant glycosyltransferase A and a set of hybrid glycosyltransferase A and B mutants were expressed in Escherichia coli in high yields, which allowed them to be kinetically characterized extensively for the first time, A functional hybrid A/B mutant enzyme was able to catalyze both A and B reactions, with the k(cat) being 5-fold higher for the A donor, Surprisingly, even a single amino acid replacement in glycosyltransferase A with the corresponding residue from glycosyltransferase B (Arg-176 --> Gly) produced enzymes with glycosyltransferase A activity only, but with very large (11-fold) increases in the k(cat) and increased specificity, The increases observed in k(cat) are among the largest obtained for a single amino acid change and are advantageous for the preparative scale synthesis of blood group antigens.
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关键词
enzyme,amino acid,catalytic activity,blood groups
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