SEQUENTIAL INTERCHANGE OF 4 AMINO-ACIDS FROM BLOOD-GROUP-B TO BLOOD-GROUP-A GLYCOSYLTRANSFERASE BOOSTS CATALYTIC ACTIVITY AND PROGRESSIVELYMODIFIES SUBSTRATE RECOGNITION IN HUMAN RECOMBINANT ENZYMES

The human blood group A and B glycosyltransferase enzymes are highly h omologous 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 allo w facile mutagenesis. Soluble forms of a recombinant glycosyltransfera se A and a set of hybrid glycosyltransferase A and B mutants were expr essed in Escherichia coli in high yields, which allowed them to be kin etically characterized extensively for the first time, A functional hy brid A/B mutant enzyme was able to catalyze both A and B reactions, wi th the k(cat) being 5-fold higher for the A donor, Surprisingly, even a single amino acid replacement in glycosyltransferase A with the corr esponding residue from glycosyltransferase B (Arg-176 --> Gly) produce d enzymes with glycosyltransferase A activity only, but with very larg e (11-fold) increases in the k(cat) and increased specificity, The inc reases observed in k(cat) are among the largest obtained for a single amino acid change and are advantageous for the preparative scale synth esis of blood group antigens.