Kinetics of the monomer-dimer reaction of yeast hexokinase PI

Hoggett, J. G.; Kellett, George L.

doi:10.1042/bj2870567

Cited by 19 publications

(24 citation statements)

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“…In the case of ScHxk1, the same crystal form was analyzed (PDB entry, 3B8A) (29), whereas a new crystal form was obtained for ScHxk2 (PDB entry, 1IG8) (30). Remarkably, in all of these structures the enzymes are present in the monomeric state, even though ScHxk1 and ScHxk2 are known to form dimers (31). In contrast, dimeric arrangements have been observed in crystal forms BI (32) and BII (33) of ScHxk2; however, these dimers are asymmetric and consist of two subunits related by screw axes of 156°(BI) and 180°(BII) rotation and 13.8 Å (BI) and 3.6 Å (BII) translation.…”

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Crystal Structure of Hexokinase KlHxk1 of Kluyveromyces lactis

Kuettner

Kettner

Keim

et al. 2010

Journal of Biological Chemistry

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Crystal structures of the unique hexokinase KlHxk1 of the yeast Kluyveromyces lactis were determined using eight independent crystal forms. In five crystal forms, a symmetrical ringshaped homodimer was observed, corresponding to the physiological dimer existing in solution as shown by small-angle x-ray scattering. The dimer has a head-to-tail arrangement such that the small domain of one subunit interacts with the large domain of the other subunit. Dimer formation requires favorable interactions of the 15 N-terminal amino acids that are part of the large domain with amino acids of the small domain of the opposite subunit, respectively. The head-to-tail arrangement involving both domains of the two KlHxk1 subunits is appropriate to explain the reduced activity of the homodimer as compared with the monomeric enzyme and the influence of substrates and products on dimer formation and dissociation. In particular, the structure of the symmetrical KlHxk1 dimer serves to explain why phosphorylation of conserved residue Ser-15 may cause electrostatic repulsions with nearby negatively charged residues of the adjacent subunit, thereby inducing a dissociation of the homologous dimeric hexokinases KlHxk1 and ScHxk2. The enzymes of the hexokinase family catalyze the intracellular trapping and the initiation of metabolism of glucose, fructose, and mannose. In addition to their role in glycolysis, an increasing number of yeast, plant, and mammalian hexokinases have been found to represent multifunctional proteins that are implicated in glucose sensing and signaling (1-4), whereas their glycolytic sugar substrate plays a dual role as a carbon source and hormone-like regulator (4, 5). The molecular basis underlying the involvement of hexokinases in the transcriptional control of glucose metabolism and in glucose homeostasis is their ability to interact with mitochondria and to reversibly translocate to nuclei (3, 6 -9).In the Crabtree-positive yeast Saccharomyces cerevisiae, glucose abundance is accompanied by the translocation of the cytosolic hexokinase isoenzyme 2 (ScHxk2) 4 and the transcriptional repressor Mig1 (ScMig1) into the nucleus, where both proteins participate in the formation of a hetero-oligomeric complex that suppresses the transcription of ScMig1 target genes like SUC2 encoding invertase (9). The mechanism of glucose signaling in glucose-repressible strains of the Crabtree-negative yeast Kluyveromyces lactis, used increasingly as a model organism in comparative functional genomics (10 -12), is largely unknown; however, the unique hexokinase KlHxk1 encoded by the RAG5 gene, the expression of glucose transporters, and the capacity for glucose transport seem to be involved (13)(14)(15).Contrary to the situation in bakers' yeast, glucose and fructose limitation causes the translocation of the mammalian hexokinase isoenzyme IV (also referred to as "glucokinase" or hexokinase D) to the nucleus of the liver parenchymal cell where it binds to its regulatory protein, GKRP, and retranslocates when glucose is abundantly av...

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confidence: 99%

Crystal Structure of Hexokinase KlHxk1 of Kluyveromyces lactis

Kuettner

Kettner

Keim

et al. 2010

Journal of Biological Chemistry

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“…It has been suggested that hysteresis in yeast hexokinase activity could be due to isomerization or dissociation of the molecule [41]. The slow isomerizations observed here, like the slow association-dissociation reactions observed previously [34], would be able to generate hysteretic effects.…”

Section: Quantitative Analysismentioning

confidence: 55%

“…Under these conditions, three relaxation effects were observed which, as discussed above, cannot be attributed to the association-dissociation reaction of hexokinase; this reaction is known to intervene at much higher pH and ionic strength [34]. The present results are interpreted within the framework of the concerted MWC model which is conceptually more straightforward and involves fewer independent parameters than either the sequential model or the more general model of Eigen [27].…”

Section: Quantitative Analysismentioning

confidence: 89%

“…Relaxations attributable to the monomer-dimer reaction of hexokinase have been shown to intervene only at higher pH and ionic strength, and these relaxations are slow and depend strongly on enzyme concentration [34].…”

Section: Kinetic Measurementsmentioning

confidence: 99%

“…Fluorescence temperature-jump measurements were carried out on apparatus described fully elsewhere [33,34]. Temperature- …”

Section: Kinetic Measurementsmentioning

confidence: 99%

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Kinetics of the cooperative binding of glucose to dimeric yeast hexokinase P-I

Hoggett

Kellett

1995

Biochemical Journal

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Kinetic studies of the cooperative binding of glucose to yeast hexokinase P-I at pH 6.5 have been carried out using the fluorescence temperature-jump technique. Three relaxation effects were observed: a fast low-amplitude effect which could only be resolved at low glucose concentrations (tau 1(-1) = 500-800 s-1), an intermediate effect (tau 2) which showed a linear dependence of reciprocal relaxation time on concentration, and a slow effect (tau 3) which showed a curved dependence on glucose concentration, increasing from approximately 28 s-1 at low concentrations to 250 s-1 at high levels. The findings are interpreted in terms of the concerted Monod-Wyman-Changeux mechanism, the two faster relaxations being assigned to binding to the R and T states, and the slow relaxation to isomerization between the states. Quantitative fitting of the kinetic data to the mechanism has been carried out using independent estimates of the equilibrium parameters of the model; these have been derived from equilibrium dialysis data and by determining the enhancement of the intrinsic ATPase activity of the enzyme by the non-phosphorylatable sugar lyxose, which switches the conformation of the enzyme to the active R state.

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Respiratory inhibitors affect incorporation of glucose into Saccharomyces cerevisiae cells, but not the activity of glucose transport

Walsh

Smits

Dam

1994

Yeast

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Incubation of starved galactose-grown S. cerevisiae cells with cyanide reduced glucose uptake as measured over a 5-s period. The Vmax for glucose uptake was decreased by over a factor of two but the apparent affinity for glucose doubled. When measured in the sub-second time scale, however, there was no significant inhibition of glucose uptake, by cyanide, up to 200-ms, clearly demonstrating that, in cyanide treated cells, glucose uptake was not linear for the first 5-s. After a 200-ms exposure of untreated cells to radio-labelled glucose, less than 10% of the intracellular label resided in soluble uncharged compounds. In cyanide-treated cells up to 43% of the labelled compounds were uncharged, with a concurrent reduction of intracellular label residing in anionic compounds. The results suggest that, in the presence of 10 mM cyanide when respiration is inhibited, a reduction in the cellular ATP concentration causes a reduction in hexose-kinase activity which results in an accumulation of internal free glucose, which in turn causes a reduction in net glucose transport.

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Kinetics of the monomer-dimer reaction of yeast hexokinase PI

Cited by 19 publications

References 27 publications

Crystal Structure of Hexokinase KlHxk1 of Kluyveromyces lactis

Crystal Structure of Hexokinase KlHxk1 of Kluyveromyces lactis

Kinetics of the cooperative binding of glucose to dimeric yeast hexokinase P-I

Respiratory inhibitors affect incorporation of glucose into Saccharomyces cerevisiae cells, but not the activity of glucose transport

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