Engineering the active center of the 6-phospho-beta-galactosidase from Lactococcus lactis

Several amino acids in the active center of the 6-phospho-beta-galactosidas e from Lactococcus lactis were replaced by the corresponding residues in ho mologous enzymes of glycosidase family 1 with different specificities, Thre e mutants, W429A, K435V/Y437F and 5428D/K435VTY437F, were constructed. W429 A was found to have an improved specificity for glucosides compared with th e wild-type, consistent with the theory that the amino acid at this positio n is relevant for the distinction between galactosides and glucosides, The k(cat)/K-m for o-nitrophenyl-beta-D-glucose-6-phosphate is 8-fold higher th an for o-nitrophenyl-beta-D-galactose-6-phosphate which is the preferred su bstrate of the wild-type enzyme. This suggests that new hydrogen bonds are formed in the mutant between the active site residues, presumably Gln19 or Trp421 and the C-4 hydroxyl group. The two other mutants with the exchanges in the phosphate-binding loop were tested for their ability to bind phosph orylated substrates, The triple mutant is inactive, The double mutant has a dramatically decreased ability to bind o-nitrophenyl-beta-D-galactose-6-ph osphate whereas the interaction with o-nitrophenyl-beta-D-galactose is bare ly altered. This result shows that the 6-phospho-beta-galactosidase and the related cyanogenic beta-glucosidase from Trifolium repens have different r ecognition mechanisms for substrates although the structures of the active sites are highly conserved.