Maintenance of the monomeric structure of glucokinase under reacting conditions

Cárdenas, Marí­a Luz; Rabajille, Eliana; Niemeyer, Hermann M.

doi:10.1016/0003-9861(78)90261-8

Cited by 50 publications

(22 citation statements)

References 32 publications

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“…The unique non-Michaelis-Menten kinetics observed in the steady state velocity is intriguing because the enzyme functions exclusively as a monomer (7, 8). Based upon the transient state glucose binding curves presented herein, we propose a minimal kinetic model in which the enzyme samples more than one conformationally distinct state, both in the absence and presence of glucose.…”

Section: Discussionmentioning

confidence: 99%

Global Fit Analysis of Glucose Binding Curves Reveals a Minimal Model for Kinetic Cooperativity in Human Glucokinase

Larion

Miller

2010

Biochemistry

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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 pre-existing 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 do not support the glucose binding component of the mnemonic mechanism, the simplified LIST mechanism or a mechanism that includes three unliganded enzyme species. Instead, our experimental data are consistent with a LIST kinetic model that includes two additional 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.

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Section: Discussionmentioning

confidence: 99%

Global Fit Analysis of Glucose Binding Curves Reveals a Minimal Model for Kinetic Cooperativity in Human Glucokinase

Larion

Miller

2010

Biochemistry

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“…Notably, sigmoidal kinetics is only observed with variable glucose concentrations; the reaction rate adheres to Michaelis-Menten kinetics with respect to the concentration of MgATP 2− [58]. The positive cooperativity observed as a function of glucose concentration is intriguing since glucokinase functions exclusively as a monomer [59]. Much of the early work on mammalian glucokinase was centered on establishing the validity of the sigmoidal kinetic response and investigating the oligomeric state of the enzyme.…”

Section: Functional Attributes Of Mammalian Glucokinasementioning

confidence: 99%

Homotropic allosteric regulation in monomeric mammalian glucokinase

Larion

Miller

2012

Archives of Biochemistry and Biophysics

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Glucokinase catalyzes the ATP-dependent phosphorylation of glucose, a chemical transformation that represents the rate-limiting step of glycolytic metabolism in the liver and pancreas. Glucokinase is a central regulator of glucose homeostasis as evidenced by its association with two disease states, maturity onset diabetes of the young (MODY) and persistent hyperinsulinemia of infancy (PHHI). Mammalian glucokinase is subject to homotropic allosteric regulation by glucose — the steady-state velocity of glucose 6-phosphate production is not hyperbolic, but instead displays a sigmoidal response to increasing glucose concentrations. The positive cooperativity displayed by glucokinase is intriguing since the enzyme functions as a monomer under physiological conditions and contains only a single binding site for glucose. Despite the existence of several models of kinetic cooperativity in monomeric enzymes, a consensus has yet to be reached regarding the mechanism of allosteric regulation in glucokinase. Experimental evidence collected over the last 45 years by a number of investigators supports a link between cooperativity and slow conformational reorganizations of the glucokinase scaffold. In this review, we summarize advances in our understanding of glucokinase allosteric regulation resulting from recent X-ray crystallographic, pre-equilibrium kinetic and high-resolution nuclear magnetic resonance investigations. We conclude with a brief discussion of unanswered questions regarding the mechanistic basis of kinetic cooperativity in mammalian glucokinase.

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“…This is an important property of glucokinase because it sensitizes this "glucose-sensing" enzyme to its substrate. Because glucokinase is a monomeric enzyme whether in the absence or presence of its substrates (39), classical models of cooperat i v i t y, such as the symmetrical model of Monod et al (40) or the sequential model of Koshland et al (41) are not applicable. Random nonequilibrium addition of substrates can give rise, under some conditions, to cooperativity for one of the substrates (42), but this explanation can be excluded on the basis that there is no significant inhibition by ATP (43).…”

Section: Binding Of Nag and Mh To Different Conformations Of G L U C mentioning

confidence: 99%

Study of the regulatory properties of glucokinase by site-directed mutagenesis: conversion of glucokinase to an enzyme with high affinity for glucose.

2000

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To identify the amino acids involved in the specific regulatory properties of glucokinase, and particularly its low affinity for glucose, mutants of the human islet enzyme have been prepared, in which glucokinase-specific residues have been replaced. Two mutations increased the affinity for glucose by twofold (K296M) and sixfold (Y214A), the latter also decreasing the Hill c o e fficient from 1.75 to 1.2 with minimal change in the a ffinity for AT P. Combining these two mutations with N166R resulted in a 50-fold decrease in the half-saturating substrate concentration (S 0 . 5 ) value, which became then comparable to the K m of hexokinase II. The location of N166, Y214, and K296 in the three-dimensional structure of glucokinase suggests that these mutations act by favoring closure of the catalytic cleft. As a rule, mutations changed the affinity for glucose and for the competitive inhibitor mannoheptulose (MH) in parallel, whereas they barely affected the affinity for N-acetylglucosamine (NAG). These and other results suggest that NAG and MH bind to the same site but to d i fferent conformations of glucokinase. A small reduction in the affinity for the regulatory protein was observed with mutations of residues on the smaller domain and in the hinge region, confirming the bipartite nature of the binding site for the regulatory protein. The K296M mutant was found to have a threefold decreased affinity for palmitoyl CoA; this effect was additive to that previously observed for the E279Q mutant, indicating that the binding site for long-chain acyl CoAs is located on the upper face of the larger domain. D i a b e t e s 4 9 :1 9 5-201, 2000

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Maintenance of the monomeric structure of glucokinase under reacting conditions

Cited by 50 publications

References 32 publications

Global Fit Analysis of Glucose Binding Curves Reveals a Minimal Model for Kinetic Cooperativity in Human Glucokinase

Global Fit Analysis of Glucose Binding Curves Reveals a Minimal Model for Kinetic Cooperativity in Human Glucokinase

Homotropic allosteric regulation in monomeric mammalian glucokinase

Study of the regulatory properties of glucokinase by site-directed mutagenesis: conversion of glucokinase to an enzyme with high affinity for glucose.

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