title: Demonstration of a slow conformational change in liver glucokinase by fluorescence spectroscopy.
abstract: A slow interconversion between two enzyme forms of rat liver glucokinase has been previously inferred from kinetic assay studies. Two different conformations of the enzyme-substrate complex have now been directly demonstrated by an intrinsic fluorescence enhancement in glucokinase upon addition of glucose. The transition between these two conformations upon glucose addition is measurably slow in the presence of glycerol, with a glucose-dependent half-life of 0.5 to more than 10 min. In the presence of 5% glycerol, the forward and reverse isomerization rate constants are 2.6 x 10(-2) s-1 and 8.5 x 10(-4) s-1, respectively. Correspondingly, the overall equilibrium dissociation constant (0.13 mM) is more than 30-fold lower than the first binding step, i.e. the affinity for glucose is greatly increased. This result was also verified by equilibrium titration of the enzyme with glucose. A similar slow transition was analyzed in the presence of 30% glycerol and observed without glycerol. The dilution of stock glucokinase to promote glucose dissociation from the enzyme showed an exponential fluorescence decay, exactly the reverse phenomenon of glucose addition. The deduced rate constant for the reverse reaction coincided with that calculated from the association results. The conformational change is specific for glucose and responsible for the generation of the kinetic cooperativity of this monomeric enzyme, thus playing a regulatory role in the uptake of glucose in liver.
authors.affiliation: Department of Biochemistry, Case Western Reserve University, Cleveland, Ohio 44106.
authors.forename: S X
authors.initials: SX
authors.lastname: Lin
authors.forename: K E
authors.initials: KE
authors.lastname: Neet
mesh_terms: D000818:Animals; D005941:Glucokinase / Q000378:metabolism*; D005947:Glucose / Q000378:metabolism; D007536:Isomerism; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008433:Mathematics; D008962:Models, Theoretical; D011487:Protein Conformation; D051381:Rats; D013050:Spectrometry, Fluorescence
chemical_list: D005941:Glucokinase; D005947:Glucose