Structural and kinetic studies of induced fit in xylulose kinase from Escherichia coli

J Mol Biol. 2007 Jan 19;365(3):783-98. doi: 10.1016/j.jmb.2006.10.068. Epub 2006 Oct 25.

Abstract

The primary metabolic route for D-xylose, the second most abundant sugar in nature, is via the pentose phosphate pathway after a two-step or three-step conversion to xylulose-5-phosphate. Xylulose kinase (XK; EC 2.7.1.17) phosphorylates D-xylulose, the last step in this conversion. The apo and D-xylulose-bound crystal structures of Escherichia coli XK have been determined and show a dimer composed of two domains separated by an open cleft. XK dimerization was observed directly by a cryo-EM reconstruction at 36 A resolution. Kinetic studies reveal that XK has a weak substrate-independent MgATP-hydrolyzing activity, and phosphorylates several sugars and polyols with low catalytic efficiency. Binding of pentulose and MgATP to form the reactive ternary complex is strongly synergistic. Although the steady-state kinetic mechanism of XK is formally random, a path is preferred in which D-xylulose binds before MgATP. Modelling of MgATP binding to XK and the accompanying conformational change suggests that sugar binding is accompanied by a dramatic hinge-bending movement that enhances interactions with MgATP, explaining the observed synergism. A catalytic mechanism is proposed and supported by relevant site-directed mutants.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adenosine Triphosphatases / chemistry
  • Amino Acid Sequence
  • Carbohydrates / chemistry
  • Catalysis
  • Conserved Sequence
  • Cryoelectron Microscopy
  • Crystallography, X-Ray
  • Enzyme Inhibitors / pharmacology
  • Escherichia coli / enzymology*
  • Glycerol Kinase / chemistry
  • Kinetics
  • Models, Molecular
  • Molecular Sequence Data
  • Phosphotransferases (Alcohol Group Acceptor) / antagonists & inhibitors
  • Phosphotransferases (Alcohol Group Acceptor) / chemistry*
  • Phosphotransferases (Alcohol Group Acceptor) / metabolism*
  • Phosphotransferases (Alcohol Group Acceptor) / ultrastructure
  • Protein Structure, Quaternary
  • Protein Structure, Secondary
  • Sequence Alignment
  • Substrate Specificity

Substances

  • Carbohydrates
  • Enzyme Inhibitors
  • Phosphotransferases (Alcohol Group Acceptor)
  • xylulokinase
  • Glycerol Kinase
  • Adenosine Triphosphatases

Associated data

  • PDB/1ITM
  • PDB/2NLX