Identification of fructose 6-phosphate- and fructose 1-phosphate-binding residues in the regulatory protein of glucokinase.
(2002) Journal of Biological Chemistry — Vol. 277, n° 10, p. 8466-8473 (2002)
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- Abstract
- Glucokinase is inhibited in the liver by a regulatory protein (GKRP) whose effects are increased by Fru-6-P and suppressed by Fru-1-P. To identify the binding site of these phosphate esters, we took advantage of the homology of GKRP to the isomerase domain of GlmS (glucosamine-6-phosphate synthase) and created 12 different mutants of rat GKRP. Mutations of three residues predicted to bind to Fru-6-P resulted in proteins that were approximately 5-fold (S110A) and 50-fold (S179A and K514A) less potent as inhibitors of glucokinase and had an at least 100-fold reduced affinity for the effectors. Mutation of another residue of the putative binding site (T109A) resulted in a 10-fold decrease in the inhibitory power and an inversion of the effect of sorbitol-6-P, a Fru-6-P analog. The replacement of Gly(107), a residue close to the binding site, by cysteine (as in GlmS and Xenopus GKRP) resulted in a protein that had 20 times more affinity for Fru-6-P and 30 times less affinity for Fru-1-P. These results are consistent with GKRP having one single binding site for phosphate esters. They also show that a missense mutation of GKRP can lead to a gain of function.
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Veiga da Cunha, M., & Van Schaftingen, E. (2002). Identification of fructose 6-phosphate- and fructose 1-phosphate-binding residues in the regulatory protein of glucokinase. Journal of Biological Chemistry, 277(10), 8466-8473. https://doi.org/10.1074/jbc.M105984200 (Original work published 2002)