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

Krishan K. Arora, P. Shenbagamurthi, Maurizio Fanciulli, Peter L. Pedersen

Research output: Contribution to journalArticle

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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
Pages (from-to)5324-5328
Number of pages5
JournalJournal of Biological Chemistry
Volume265
Issue number9
Publication statusPublished - 1990

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Phosphorylation
Hexokinase
Yeast
Adenosine
Adenosine Triphosphate
Yeasts
Amino Acids
Glucose
Peptides
Sequence Homology
Catalytic Domain
Chemical modification
Leucine
Stoichiometry
Protein Kinases
triphosphoric acid
Glutamic Acid
Eukaryotic Cells
Binding Sites
Substrates

ASJC Scopus subject areas

  • Biochemistry

Cite this

Glucose phosphorylation : Interaction of a 50-amino acid peptide of yeast hexokinase with trinitrophenyl ATP. / Arora, Krishan K.; Shenbagamurthi, P.; Fanciulli, Maurizio; Pedersen, Peter L.

In: Journal of Biological Chemistry, Vol. 265, No. 9, 1990, p. 5324-5328.

Research output: Contribution to journalArticle

Arora, Krishan K. ; Shenbagamurthi, P. ; Fanciulli, Maurizio ; Pedersen, Peter L. / Glucose phosphorylation : Interaction of a 50-amino acid peptide of yeast hexokinase with trinitrophenyl ATP. In: Journal of Biological Chemistry. 1990 ; Vol. 265, No. 9. pp. 5324-5328.
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