Glucose phosphorylation: Site-directed mutations which impair the catalytic function of hexokinase

Krishan K. Arora, Charles R. Filburn, Peter L Pedersen

Research output: Contribution to journalArticle

Abstract

Recent studies from this and other laboratories have resulted in the cloning and sequencing of hexokinases from a variety of tissues including yeast, human kidney, rat brain, rat liver, and mouse hepatoma. Significantly, studies on the hepatoma enzyme conducted in this laboratory (Arora, K. K., Fanciulli, M., and Pedersen, P. L. (1990) J. Biol. Chem. 265, 6481-6488) resulted also in its overexpression in Escherichia coli in active form. We have now used site-directed mutagenesis for the first time in studies of hexokinase to evaluate the role of amino acid residues predicted to interact with either glucose or ATP. Four amino acid residues (Ser-603, Asp-657, Glu-708, and Glu-742) believed to interact with glucose were mutated to alanine or glycine, whereas a lysine residue (Lys-558) thought to be directly involved in binding ATP was mutated to either methionine or arginine. Of all the mutations in residues believed to interact with glucose, the Asp-657 → Ala mutation is the most profound, reducing the hexokinase activity to a level max values for Ser-603 → Ala, Glu-708 → Ala, and Glu-742 → Ala enzymes are 6, 10, and 6.5%, respectively, of the wild-type enzyme. Glu-708 and Glu-742 mutations increase the apparent Km for glucose 50- and 14-fold, respectively, while the Ser-603 → Ala mutation decreases the apparent Km for glucose 5-fold. At the putative ATP binding site, the relative Vmax for Lys-558 → Arg and Lys-558 → Met enzymes are 70 and 29%, respectively, of the wild-type enzyme with no changes in apparent Km for glucose. No changes were observed in the apparent Km for ATP with any mutation. These results support the view that all 4 residues predicted to interact with glucose from earlier x-ray studies may play a role in binding and/or catalysis. The Asp-657 and Ser-603 residues may be involved in both, while Glu-708 and Glu-742 clearly contribute to binding but are not essential for catalysis. In contrast, Lys-558 appears to be essential neither for binding nor catalysis.

Original languageEnglish (US)
Pages (from-to)5359-5362
Number of pages4
JournalJournal of Biological Chemistry
Volume266
Issue number9
StatePublished - 1991

Fingerprint

Phosphorylation
Hexokinase
Glucose
Mutation
Adenosine Triphosphate
Catalysis
Enzymes
Rats
Hepatocellular Carcinoma
Amino Acids
Mutagenesis
Cloning
Site-Directed Mutagenesis
Alanine
Methionine
Liver
Yeast
Glycine
Escherichia coli
Lysine

ASJC Scopus subject areas

  • Biochemistry

Cite this

Glucose phosphorylation : Site-directed mutations which impair the catalytic function of hexokinase. / Arora, Krishan K.; Filburn, Charles R.; Pedersen, Peter L.

In: Journal of Biological Chemistry, Vol. 266, No. 9, 1991, p. 5359-5362.

Research output: Contribution to journalArticle

Arora, Krishan K. ; Filburn, Charles R. ; Pedersen, Peter L. / Glucose phosphorylation : Site-directed mutations which impair the catalytic function of hexokinase. In: Journal of Biological Chemistry. 1991 ; Vol. 266, No. 9. pp. 5359-5362.
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