Fluorescence-quenching study of glucose binding by yeast hexokinase isoenzymes

Feldman, Isaac; Kramp, Douglas C.

doi:10.1021/bi00601a029

Cited by 30 publications

(16 citation statements)

References 26 publications

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“…1, we calculate a binding constant, K b , of 3.1±0.2 mM −1 for glucose association with yeast hexokinase PII at 25°C and pH 8.7. Our binding constant is in reasonable agreement with the previously reported values of 5.6 mM −1 [13] and 3.2 mM −1 [25]. Using Δ G b =− RT ln K b , we determine a binding free energy, Δ G b , of −4.8±0.5 kcal/mol.…”

Section: Resultssupporting

confidence: 90%

Volumetric and spectroscopic characterizations of glucose–hexokinase association

Filfil

Chalikian

2003

FEBS Letters

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The binding of D-glucose to hexokinase PII at 25 ‡C and pH 8.7 has been investigated by a combination of ultrasonic velocimetry, high precision densimetry, and £uorescence spectroscopy. The binding of glucose to the enzyme results in signi¢cant dehydration of the two interacting molecules, while the intrinsic coe⁄cient of adiabatic compressibility of hexokinase slightly decreases. Glucose^hexokinase association is an entropy-driven process. The favorable change in entropy results from compensation between two large contributions. The binding-induced increase in hydrational entropy slightly prevails over the decrease in the con¢gurational entropy of the enzyme. Taken together, our results emphasize the crucial role of water in modulating the energetics of protein recognition.

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Section: Resultssupporting

confidence: 90%

Volumetric and spectroscopic characterizations of glucose–hexokinase association

Filfil

Chalikian

2003

FEBS Letters

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“…Yeast HKes exist in solution as dimers and monomers. The dissociation of dimers into monomers is promoted by glucose binding and an increase in pH and/or ionic strength 15–17…”

Section: Introductionmentioning

confidence: 99%

Crystal structure of yeast hexokinase PI in complex with glucose: A classical “induced fit” example revised

Kuser

Cupri

Bleicher³

et al. 2008

Proteins

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Hexokinase is the first enzyme in the glycolytic pathway that catalyzes the transfer of a phosphoryl group from ATP to glucose to form glucose-6-phosphate and ADP. Two yeast hexokinase isozymes are known, namely PI and PII. Here we redetermined the crystal structure of yeast hexokinase PI from Saccharomyces cerevisiae as a complex with its substrate, glucose, and refined it at 2.95 A resolution. Comparison of the holo-PI yeast hexokinase and apo-hexokinase structures shows in detail the rigid body domain closure and specific loop movements as glucose binds and sheds more light on structural basis of the "induced fit" mechanism of reaction in the HK enzymatic action. We also performed statistical coupling analysis of the hexokinase family, which reveals two co-evolved continuous clusters of amino acid residues and shows that the evolutionary coupled amino acid residues are mostly confined to the active site and the hinge region, further supporting the importance of these parts of the protein for the enzymatic catalysis.

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“…The affinity of the monomer was found to be independent of ionic strength over the range of KCl concentrations 0-1.Omol dm-3 at low and high pH; the large (up to about 8-fold at neutrality) increases in the affinity of the enzyme observed with increases in ionic strength under these conditions, where the enzyme exists as a mixture of monomers and dimers, were fully accounted for by increases in the dissociation of dimer to monomer. Subsequently, Feldman & Kramp (1978) have criticized these conclusions, claiming that at pH 8.3 the affinity of glucose for the monomer is only about 1.6-to 2.0fold greater than that for the dimer and attributing most of the dependence of glucose binding to the enzyme on ionic strength to a 4-fold decrease in the intrinsic dissociation constant of glucose from the monomer with increase in ionic strength.…”

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confidence: 99%

The binding of glucose to yeast hexokinase monomers is independent of ionic strength

Mayes¹,

Hoggett²,

Kellett³

1982

Biochemical Journal

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Hoggett & Kellett [Eur. J. Biochem. 66, 65-77 (1976)] have reported that the binding of glucose to the monomer of hexokinase PII isoenzyme is independent of ionic strength, in contrast to the subsequent claim of Feldman & Kramp [Biochemistry 17, 1541-1547 (1978)] that the binding is strongly dependent on ionic strength. Since measurements with native hexokinase P forms are complicated by the fact that the enzyme exists in a monomer-dimer association-dissociation equilibrium, we have now studied the binding of glucose to the proteolytically-modified S forms which are monomeric. At pH 8.5, the affinity of glucose for both SI and SII monomers is independent of salt concentration over the range of KCl concentrations 0-1.0 mol . dm-3 and is in good agreement with that of the corresponding P forms in both low and high salt. These observations confirm that the binding of glucose to hexokinase P monomers is independent of ionic strength and that the affinity of glucose for the hexokinase PII monomer is about an order of magnitude greater than that for the dimer.

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Fluorescence-quenching study of glucose binding by yeast hexokinase isoenzymes

Cited by 30 publications

References 26 publications

Volumetric and spectroscopic characterizations of glucose–hexokinase association

Volumetric and spectroscopic characterizations of glucose–hexokinase association

Crystal structure of yeast hexokinase PI in complex with glucose: A classical “induced fit” example revised

The binding of glucose to yeast hexokinase monomers is independent of ionic strength

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