Escherichia Coli Maltodextrin Phosphorylase: Contribution of Active Site Residues glutamate-637 and tyrosine-538 to the Phosphorolytic Cleavage of Alpha-Glucans

Biochemistry. 1990 Oct 23;29(42):9956-62. doi: 10.1021/bi00494a028.


The role of Escherichia coli maltodextrin phosphorylase (EC 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.

Publication types

  • Comparative Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Bacterial Proteins / genetics
  • Bacterial Proteins / metabolism*
  • Base Sequence
  • Binding Sites
  • Escherichia coli / enzymology*
  • Escherichia coli / genetics
  • Glucans / metabolism*
  • Glucosyltransferases / genetics
  • Glucosyltransferases / metabolism*
  • Glutamates
  • Glutamic Acid
  • Kinetics
  • Molecular Sequence Data
  • Mutagenesis, Site-Directed
  • Phosphorylation
  • Protein Conformation
  • Pyridoxal Phosphate / metabolism
  • Sequence Homology, Nucleic Acid
  • Tyrosine


  • Bacterial Proteins
  • Glucans
  • Glutamates
  • Glutamic Acid
  • Tyrosine
  • Pyridoxal Phosphate
  • Glucosyltransferases
  • maltodextrin phosphorylase