Mechanistic origin of the sigmoidal rate behaviour of glucokinase

Pettersson, Gösta

doi:10.1042/bj2330347

Cited by 32 publications

(24 citation statements)

References 12 publications

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“…We cannot completely exclude an alternative mechanism in which the substrates bind in random order, and the flux through the oxygen binding first pathway versus the cholesterol binding first pathway shifts upon changing oxygen concentration (46,47). This mechanism requires that substrate binding not be at equilibrium, that is, steps subsequent to ternary complex formation are fast relative to substrate binding.…”

Section: Discussionmentioning

confidence: 99%

The Binding and Release of Oxygen and Hydrogen Peroxide Are Directed by a Hydrophobic Tunnel in Cholesterol Oxidase

et al. 2008

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The usage by enzymes of specific binding pathways for gaseous substrates or products is debated. The crystal structure of the redox enzyme cholesterol oxidase, solved at sub-Ångstrom resolution, revealed a hydrophobic tunnel that may serve as a binding pathway for oxygen and hydrogen peroxide. This tunnel is formed by a cascade of conformational rearrangements and connects the active site with the exterior surface of the protein. To understand the relationship between this tunnel and gas binding and release, three mutant enzymes were constructed to block the tunnel or its putative gate. Mutation of the proposed gating residue Asn485 to Asp or the tunnel residues Phe359 or Gly347 to Trp or Asn, reduces the catalytic efficiency of oxidation. The K mO2 increases from 300 ± 35 μM for the wild-type enzyme to 617 ± 15 μM for the F359W mutant. The k cat for the F359W mutant catalyzed reaction decreases 13-fold relative to the wild-type catalyzed reaction. The N485D and G347N mutants could not be saturated with oxygen. Hydride transfer from the sterol to the flavin prosthetic group is no longer rate limiting for these tunnel mutants. The steady-state kinetics of both wild-type and tunnel-mutant enzymes are consistent with formation of a ternary complex of steroid and oxygen during catalysis. Furthermore, kinetic cooperativity with respect to molecular oxygen is observed with the tunnel mutants, but not with the wild-type enzyme. A rate-limiting conformational change for binding and release of oxygen and hydrogen peroxide, respectively are consistent with the cooperative kinetics. In the atomic resolution structure of F359W, the indole ring of the tryptophan completely fills the tunnel and is only observed in a single conformation. The size of the indole is proposed to limit conformational rearrangement of residue 359 that leads to tunnel opening in the wild-type enzyme. Overall, these results substantiate the functional importance of the tunnel for substrate binding and product release.

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Section: Discussionmentioning

confidence: 99%

The Binding and Release of Oxygen and Hydrogen Peroxide Are Directed by a Hydrophobic Tunnel in Cholesterol Oxidase

et al. 2008

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“…This model was first delineated by Ferdinand [7] and further developed by Pettersson [30] in an attempt to reconcile the sigmoidal kinetic response of mammalian liver glucokinase. It requires that the enzymatic reaction be capable of proceeding through a random order mechanism.…”

Section: Mechanisms Of Monomeric Kinetic Cooperativitymentioning

confidence: 99%

Cooperativity in monomeric enzymes with single ligand-binding sites

Porter

Miller

2012

Bioorganic Chemistry

100

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Cooperativity is widespread in biology. It empowers a variety of regulatory mechanisms and impacts both the kinetic and thermodynamic properties of macromolecular systems. Traditionally, cooperativity is viewed as requiring the participation of multiple, spatially distinct binding sites that communicate via ligand-induced structural rearrangements; however, cooperativity requires neither multiple ligand binding events nor multimeric assemblies. An underappreciated manifestation of cooperativity has been observed in the non-Michaelis-Menten kinetic response of certain monomeric enzymes that possess only a single ligand-binding site. In this review, we present an overview of kinetic cooperativity in monomeric enzymes. We discuss the primary mechanisms postulated to give rise to monomeric cooperativity and highlight modern experimental methods that could offer new insights into the nature of this phenomenon. We conclude with an updated list of single subunit enzymes that are suspected of displaying cooperativity, and a discussion of the biological significance of this unique kinetic response.

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“…One such mechanism postulates that random addition of substrates, which could create multiple pathways for substrate binding, could produce cooperativity if formation of one of these binary complexes is slower than k cat [83]. It is well established that the glucokinase mechanism is ordered.…”

Section: Models Of Kinetic Cooperativity In Monomeric Enzymesmentioning

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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Mechanistic origin of the sigmoidal rate behaviour of glucokinase

Cited by 32 publications

References 12 publications

The Binding and Release of Oxygen and Hydrogen Peroxide Are Directed by a Hydrophobic Tunnel in Cholesterol Oxidase

The Binding and Release of Oxygen and Hydrogen Peroxide Are Directed by a Hydrophobic Tunnel in Cholesterol Oxidase

Cooperativity in monomeric enzymes with single ligand-binding sites

Homotropic allosteric regulation in monomeric mammalian glucokinase

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