Global fit analysis of glucose binding curves reveals a minimal model for kinetic cooperativity in human glucokinase.
Human pancreatic glucokinase is a monomeric enzyme that displays kinetic cooperativity, a feature that facilitates enzyme-mediated regulation of blood glucose levels in the body. Two theoretical models have been proposed to describe the non-Michaelis-Menten behavior of human glucokinase. The mnemonic mechanism postulates the existence of one thermodynamically favored enzyme conformation in the absence of glucose, whereas the ligand-induced slow transition model (LIST) requires a preexisting equilibrium between two enzyme species that interconvert with a rate constant slower than turnover. To investigate whether either of these mechanisms is sufficient to describe glucokinase cooperativity, a transient-state kinetic analysis of glucose binding to the enzyme was undertaken. A complex, time-dependent change in enzyme intrinsic fluorescence was observed upon exposure to glucose, which is best described by an analytical solution comprised of the sum of four exponential terms. Transient-state glucose binding experiments conducted in the presence of increasing glycerol concentrations demonstrate that three of the observed rate constants decrease with increasing viscosity. Global fit analyses of experimental glucose binding curves are consistent with a kinetic model that is an extension of the LIST mechanism with a total of four glucose-bound binary complexes. The kinetic model presented herein suggests that glucokinase samples multiple conformations in the absence of ligand and that this conformational heterogeneity persists even after the enzyme associates with glucose.
Larion
Mioara
M
Department of Chemistry and Biochemistry, The Florida State University, Tallahassee, FL 32306-4390, USA.
Miller
Brian G
BG
D001786:Blood Glucose / Q000737:chemistry* / Q000378:metabolism; D005941:Glucokinase / Q000737:chemistry* / Q000378:metabolism; D005990:Glycerol / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008956:Models, Chemical; D010179:Pancreas / Q000201:enzymology*; D014783:Viscosity
D001786:Blood Glucose; D005941:Glucokinase; D005990:Glycerol