Phosphoenolpyruvate- and ATP-dependent dihydroxyacetone kinases: covalent substrate-binding and kinetic mechanism

Biochemistry. 2004 Oct 19;43(41):13037-45. doi: 10.1021/bi048575m.


Dihydroxyacetone (Dha) kinases are a sequence-conserved family of enzymes, which utilize two different phosphoryldonors, ATP in animals, plants, and some bacteria, and a multiphosphoprotein of the phosphoenolpyruvate carbohydrate phosphotransferase system (PTS) in most bacteria. Here, we compare the PTS-dependent kinase of Escherichia coli and the ATP-dependent kinase of Citrobacter freundii. They display 30% sequence identity. The binding constants of the E. coli kinase for eleven short-chain carbonyl compounds were determined by acetone precipitation of the enzyme-substrate complexes. They are 3.4 microM for Dha, 780 microM for Dha-phosphate (DhaP), 50 microM for D,L-glyceraldehyde (GA), and 90 microM for D,L-glyceraldehyde-3-phosphate. The k(cat) for Dha of the PTS-dependent kinase is 290 min(-1), and that of the ATP-dependent kinase is 1050 min(-1). The Km for Dha of both kinases is <6 microM. The X-ray structures of the enzyme-GA and the enzyme-DhaP complex show that substrates as well as products are bound in hemiaminal linkage to an active-site histidine. Quantum-mechanical calculations offer no indication for activation of the reacting hydroxyl group by the formation of the hemiaminal. However, the formation of the hemiaminal bond allows selection for short-chain carbonyl compounds and discrimination against structurally similar polyols. The Dha kinase remains fully active in the presence of 2 M glycerol, and phosphorylates trace impurities of carbonyl compounds present in glycerol.

Publication types

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

MeSH terms

  • Acetone / analogs & derivatives*
  • Acetone / chemistry
  • Adenosine Triphosphate / chemistry*
  • Amines / chemistry
  • Binding, Competitive
  • Citrobacter freundii / enzymology*
  • Crystallography, X-Ray
  • Dihydroxyacetone Phosphate / chemistry
  • Enzyme Inhibitors / chemistry
  • Escherichia coli Proteins / chemistry*
  • Glyceraldehyde / chemistry
  • Kinetics
  • Phosphoenolpyruvate / chemistry*
  • Phosphotransferases (Alcohol Group Acceptor) / chemistry*
  • Protein Binding
  • Quantum Theory
  • Substrate Specificity


  • Amines
  • Enzyme Inhibitors
  • Escherichia coli Proteins
  • Acetone
  • Glyceraldehyde
  • Dihydroxyacetone Phosphate
  • chloroacetone
  • Phosphoenolpyruvate
  • acetol
  • Adenosine Triphosphate
  • Phosphotransferases (Alcohol Group Acceptor)
  • glycerone kinase

Associated data

  • PDB/1UOD
  • PDB/1UOE