Identification of fructose 6-phosphate- and fructose 1-phosphate-binding residues in the regulatory protein of glucokinase.
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.
Veiga-da-Cunha
Maria
M
Laboratory of Physiological Chemistry, University of Louvain and Christian de Duve Institute of Cellular Pathology, B-1200 Brussels, Belgium. viegadacunha@bchm.ulc.ac.be
Van Schaftingen
Emile
E
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D003001:Cloning, Molecular; D003545:Cysteine / Q000737:chemistry; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D005636:Fructosephosphates / Q000378:metabolism*; D005941:Glucokinase / Q000737:chemistry* / Q000378:metabolism; D005945:Glutamine-Fructose-6-Phosphate Transaminase (Isomerizing) / Q000737:chemistry / Q000378:metabolism; D005998:Glycine / Q000737:chemistry; D006801:Humans; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D008969:Molecular Sequence Data; D016296:Mutagenesis; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D020125:Mutation, Missense; D011485:Protein Binding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013012:Sorbitol / Q000494:pharmacology; D013379:Substrate Specificity; D014981:Xenopus
D005636:Fructosephosphates; D011994:Recombinant Proteins; C032284:fructose-1-phosphate; D013012:Sorbitol; C027618:fructose-6-phosphate; D005945:Glutamine-Fructose-6-Phosphate Transaminase (Isomerizing); D005941:Glucokinase; D003545:Cysteine; D005998:Glycine