Dissecting differential binding in the forward and reverse reaction of Escherichia coli maltodextrin phosphorylase using 2-deoxyglucosyl substrates.
Substrate analogs are used in combination with site-directed mutagenesis to probe specific interactions between substrate and enzyme in the forward and reverse direction of the Escherichia coli maltodextrin phosphorylase reaction. In the phosphorolysis (degradation) mode, removal of the 2-OH group of the terminal glucose of the polysaccharide results in a 30-fold reduction of Km while similar changes were of no influence when the same polysaccharide was used for priming the synthesis. Mutation of active site residues Glu637 or Tyr538 does not change apparent affinity of substrates during degradation. In the synthesis mode, 2-deoxyglucose-1-P as substrate causes a 2-fold reduction of the wild-type kcat/Km while for the Y538F mutant a approximately 7-fold reduction is observed. In contrast, the mutation of Glu637 to Asp causes a 10-fold increase in kcat/Km. Therefore, different binding sites for the terminal glucose residue of the oligosaccharide and glucose-1-P exist. Glu637 and Tyr538 are part of the glucose-1-P binding site and do not interact with the terminal glucose residue. A 2-fold increase in rate was observed in both directions using the 2-deoxy derivatives. This confirms the role of intrinsic electronic effects in stabilizing the transition state. Uncompetitive substrate inhibition at high concentrations of maltoheptaose in the phosphorolysis direction is explained by inhibitory binding of the sugar in the synthesis mode.
Becker
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Theodor-Boveri-Institut für Biowissenschaften (Biozentrum), Physiologische Chemie I, Am Hubland, Würzburg, Germany.
Palm
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D
Schinzel
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R
D000595:Amino Acid Sequence; D001665:Binding Sites; D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D003847:Deoxyglucose / Q000031:analogs & derivatives* / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology*; D005936:Glucans / Q000494:pharmacology; D005964:Glucosyltransferases / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D005971:Glutamates; D018698:Glutamic Acid; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009844:Oligosaccharides / Q000138:chemical synthesis / Q000378:metabolism; D011135:Polysaccharides, Bacterial / Q000096:biosynthesis; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity; D014443:Tyrosine
D005936:Glucans; D005971:Glutamates; D009844:Oligosaccharides; D011135:Polysaccharides, Bacterial; D011994:Recombinant Proteins; C031146:maltoheptaose; D018698:Glutamic Acid; D014443:Tyrosine; D003847:Deoxyglucose; D005964:Glucosyltransferases; C033938:maltodextrin phosphorylase