Analysis of the cooperativity of human beta-cell glucokinase through the stimulatory effect of glucose on fructose phosphorylation.
Using overexpressed Escherichia coli sorbitol-6-phosphate dehydrogenase to monitor fructose 6-phosphate formation, we found that the stimulation of fructose phosphorylation by glucose was reduced in two human beta-cell glucokinase mutants with a low Hill coefficient or when the activity of wild type glucokinase was decreased by replacing ATP with poorer nucleotide substrates. Mutation of two other residues, neighboring glucose-binding residues in the catalytic site, also reduced the affinity for glucose as a stimulator of fructose phosphorylation. Among a series of glucose analogs, only 3, all substrates of glucokinase, stimulated fructose phosphorylation; other analogs were either inactive or inhibited glucokinase. Glucose increased the apparent affinity for inhibitors that are glucose analogs but not for the glucokinase regulatory protein or palmitoyl-CoA. These data indicate that the stimulatory effect of glucose on fructose phosphorylation reflects the positive cooperativity for glucose and is mediated by binding of glucose to the catalytic site. They support models involving the existence of two slowly interconverting conformations of glucokinase that differ through their affinity for glucose and for glucose analogs. We show by computer simulation that such a model can account for the kinetic properties of glucokinase, including the differential ability of mannoheptulose and N-acetylglucosamine to suppress cooperativity (Agius, L., and Stubbs, M. (2000) Biochem. J. 346, 413-421).
Moukil
M A
MA
Laboratoire de Chimie Physiologique, Christian de Duve Institute of Cellular Pathology and Université Catholique de Louvain, B-1200 Brussels, Belgium.
Van Schaftingen
E
E
D020134:Catalytic Domain; D005632:Fructose / Q000378:metabolism*; D005941:Glucokinase / Q000378:metabolism*; D005947:Glucose / Q000378:metabolism*; D006801:Humans; D007515:Islets of Langerhans / Q000201:enzymology*; D010766:Phosphorylation; D013379:Substrate Specificity
D005632:Fructose; D005941:Glucokinase; D005947:Glucose