Escherichia coli maltodextrin phosphorylase: contribution of active site residues glutamate-637 and tyrosine-538 to the phosphorolytic cleavage of alpha-glucans.
The role of Escherichia coli maltodextrin phosphorylase (EC 2.4.1.1) active site residues Glu637 and Tyr538 which line the sugar-phosphate contact region of the enzyme was investigated by site-directed mutagenesis. Substitution of Glu637 by an Asp or Gln residue reduced kcat to approximately 0.2% of wild-type activity, while the Km values were affected to a minor extent. This indicated participation of Glu637 in transition-state binding rather than in ground-state binding. 31P NMR analysis of the ionization state of enzyme-bound pyridoxal phosphate suggested that Glu637 is also involved in modulation of the protonation state of the coenzyme phosphate observed during catalysis. Despite loss of proposed hydrogen-bonded substrate contacts, the Tyr538Phe mutant enzyme retained more than 10% activity; the apparent affinity of all substrates was slightly decreased. Mutations at either site affected the error rate of the enzyme (ratio of hydrolysis/phosphorolysis). Besides a role in substrate binding, the hydrogen-bond network of Tyr538 supports the exclusion of water from the active site.
Schinzel
R
R
Department of Physiological Chemistry, University of Würzburg, Federal Republic of Germany.
Palm
D
D
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D005936:Glucans / Q000378:metabolism*; D005964:Glucosyltransferases / Q000235:genetics / Q000378:metabolism*; D005971:Glutamates; D018698:Glutamic Acid; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D011487:Protein Conformation; D011732:Pyridoxal Phosphate / Q000378:metabolism; D012689:Sequence Homology, Nucleic Acid; D014443:Tyrosine
D001426:Bacterial Proteins; D005936:Glucans; D005971:Glutamates; D018698:Glutamic Acid; D014443:Tyrosine; D011732:Pyridoxal Phosphate; D005964:Glucosyltransferases; C033938:maltodextrin phosphorylase