Glucose phosphorylation. Interaction of a 50-amino acid peptide of yeast hexokinase with trinitrophenyl ATP

J Biol Chem. 1990 Mar 25;265(9):5324-8.

Abstract

A 50-amino acid peptide predicted by chemical modification studies of yeast hexokinase to contain an ATP-binding site has been synthesized and purified. The peptide, which includes residues from glutamate 78 at the NH2-terminal end to leucine 127 at the COOH-terminal, resides within the smaller of the two lobes found in the three-dimensional structure of yeast hexokinase. It is this region which has been reported recently to exhibit significant sequence homology with hexokinase types I and IV of higher eukaryotic cells and sequence homology with the active site of protein kinases. Similar to native yeast hexokinase, the 50-amino acid peptide interacts strongly with the fluorescent analog TNP-ATP [2',(3')-O-(2,4,6-trinitrophenyl)-adenosine-5'-triphosphate]. A 5-fold enhancement is observed when 8 microM peptide interacts with 20 microM TNP-ATP. The stoichiometry of binding is very close to 1 mol of TNP-ATP/mol peptide. Also, similar to native yeast hexokinase, the fluorescent enhancement observed upon TNP-ATP binding to the synthetic peptide is greater than that observed upon TNP-ADP binding. Finally, TNP-AMP exhibits a much lower fluorescent enhancement in the presence of hexokinase or the synthetic peptide. The additional findings that ATP can readily prevent TNP-ATP binding and that TNP-ATP can substitute for ATP as a weak substrate for hexokinase in the phosphorylation of glucose indicate that the synthetic peptide described here comprises part of the catalytic site.

Publication types

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

MeSH terms

  • Adenosine Triphosphate / analogs & derivatives*
  • Adenosine Triphosphate / metabolism
  • Amino Acid Sequence
  • Animals
  • Brain / enzymology
  • Computer Graphics
  • Glucose / metabolism
  • Hexokinase / genetics
  • Hexokinase / metabolism*
  • Humans
  • Kidney / enzymology
  • Kinetics
  • Models, Molecular
  • Molecular Sequence Data
  • Peptide Fragments / metabolism
  • Phosphorylation
  • Protein Conformation
  • Protein Kinases / genetics
  • Rats
  • Sequence Homology, Nucleic Acid

Substances

  • 2',3'-O-(2,4,6-trinitrophenyl)adenosine 5'-triphosphate
  • Peptide Fragments
  • Adenosine Triphosphate
  • Protein Kinases
  • Hexokinase
  • Glucose