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, weakened 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 H310A mutant enzyme exhibits a more than 100-fold-lowered Ki for gluconolactone, indicating the existence of an inhibitor binding site similar to that expected for a carbonium ion-like transition state. This is further confirmed by the finding that glucose, which does not inhibit wild-type enzyme, became an inhibitor of the H310A mutant enzyme (Ki = 20 mM). Since mutation of D308 did reduce kcat about 10-100-fold while Km values remained unchanged, a participation of this residue in transition state binding is probable. The insight into substrate recognition derived from this mutagenesis study corroborates a binding model where maltopentaose fits into the phosphorylase b structure in a distorted form.
Drueckes
P
P
Theodor-Boveri-Institut für Biowissenschaften, Universität Würzburg, Germany.
Boeck
B
B
Palm
D
D
Schinzel
R
R
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D004252:DNA Mutational Analysis; D004795:Enzyme Stability; D005964:Glucosyltransferases / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D006358:Hot Temperature; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D009844:Oligosaccharides / Q000378:metabolism*; D017354:Point Mutation; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity
D009838:Oligodeoxyribonucleotides; D009844:Oligosaccharides; D011994:Recombinant Proteins; D005964:Glucosyltransferases; C033938:maltodextrin phosphorylase