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

K. K. Arora, P. Shenbagamurthi, M. Fanciulli, P. L. Pedersen

Research output: Contribution to journalArticlepeer-review

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 μM peptide interacts with 20 μM 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.

Original languageEnglish (US)
Pages (from-to)5324-5328
Number of pages5
JournalJournal of Biological Chemistry
Volume265
Issue number9
StatePublished - 1990

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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