MUTATIONAL ANALYSIS OF THE OLIGOSACCHARIDE RECOGNITION SITE AT THE ACTIVE SITE OF ESCHERICHIA COLI MALTODEXTRIN PHOSPHORYLASE

A mutagenesis approach was applied to identify specific amino acid residues that are tentatively involved in binding of the oligosaccharide substrate at the active site of Escherichia coli maltodextrin phosphorylase. From ten residues located within a proposed channel connecting the enzyme surface with the active site, nine displayed significant effects on the reaction with oligosaccharide substrates when exchanged by mutagenesis. While several mutant enzymes (N258A/D259A/N260A, N307A, E350A, and Y578F) exhibited moderate decreases in apparent binding (about 4−17-fold), two mutations, H536L and E67A, weakenend apparent binding of oligosaccharide substrates by 2 orders of magnitude. Two further mutant enzymes (T346G and H310A) displayed a 10-fold increase in the apparent Km of the oligosaccharide in the degradation reaction, while binding in the synthesis direction seemed less affected, indicating partially differential binding modes of oligosaccharides in synthesis and degradation. Quite uniquely, the H31...