Yeast hexokinase in solution exhibits a large conformational change upon binding glucose or glucose 6-phosphate

McDonald, Robert; Steitz, Thomas A.; Engelman, Donald M.

doi:10.1021/bi00569a017

Cited by 141 publications

(64 citation statements)

References 17 publications

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“…From SAXS data, a radius of gyration of 31.3 Å was obtained for ScHxk2 (34), which closely corresponds to the calculated radius of gyration of 31.5 Å of the dimer in crystal form BII (33). However, the latter dimer is asymmetric with a rotation angle of 156°and a translation of 13.8 Å.…”

Section: Discussionsupporting

confidence: 75%

“…Their calculated radii of gyration are 40.2 Å (BI) and 31.3 Å (BII). Only the radius of the latter crystal dimer is in agreement with the radius of gyration of 30 -31.3 Å determined from small-angle x-ray scattering of dimeric ScHxk2 in solution (34). It remains unclear whether the asymmetric dimer in crystal form BII corresponds to the dimer observed in solution, as oligomers usually exhibit a closed symmetry.…”

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confidence: 57%

“…However, the latter dimer is asymmetric with a rotation angle of 156°and a translation of 13.8 Å. Another asymmetric dimer of ScHxk2 observed in crystal form BI (32), exhibiting a calculated radius of gyration of 40.2 Å, was considered unphysiological (34). The close similarity of the radii of gyration of the symmetric KlHxk1 dimer (32.0 Å) and the asymmetric ScHxk2 dimer (31.3 Å) suggests that both hexokinases might form similar ring-shaped dimeric structures in solution.…”

Section: Discussionmentioning

confidence: 99%

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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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Section: Discussionsupporting

confidence: 75%

mentioning

confidence: 57%

Section: Discussionmentioning

confidence: 99%

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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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“…The conformation change in the E . glucose complex has been described previously, for example by McDonald et al [15] and by Wilkinson and Rose [20]. There has also been a pH-dependent conformation change described for free enzyme by Jentoft et al [I31 but this is a rapid process (5 x lo4 s-l) at pH 8.…”

Section: Discussionmentioning

confidence: 80%

“…This is unlikely, however, since these two isoenzymes have been shown to be very similar in both their kinetic behavior 113,141 and the crystal structure of the E . glucose complex [15].…”

Section: Discussionmentioning

confidence: 99%

Solvent Isotope Effects on the Reaction Catalyzed by Yeast Hexokinase

Taylor

Cook

Cleland

1983

European Journal of Biochemistry

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The pH dependence of the maximum velocity (v) for the phosphorylation of glucose, the V/%,ucose and the V/KMgATp have been obtained in H2O and 2H20. In H20, V decreases below a pK of 5.8, V/Kglucose decreases below a pK of 6.1 and V / K M~A T~ decreases below a pK of 6.7. In 2H20, complex behavior is observed for these parameters as a function of pD. The ratios of the parameters in HzO and 2Hz0 above their respective pK values give solvent deuterium isotope effects of about 1.5 -1.7 for all three parameters. When 1,5-anhydromannitol is used as an alternative substrate, an isotope effect different than unity is obtained only for V/K~,5-anhydromannito, which gives a value of about 0.7. Both the complex pH profiles and the relative magnitude of the isotope effects are interpreted in terms of a pH-dependent change in the E . glucose complex.Hexokinase catalyzes the transfer of a phosphoryl group from ATP and other nucleoside triphosphates to the primary alcohol in the 6-position of a number of hexoses. X-ray crystallographic studies of yeast hexokinase and its binary E . glucose complex have shown that glucose binds in the chair conformation (an axial hydroxyl group on C-1) and that an aspartic acid residue is positioned properly to facilitate phosphorylation at C-6 [l]. An acid-base catalytic role for a carboxylic acid group has been confirmed by the kinetic studies of Viola and Cleland [2].The steady-state kinetic mechanism for yeast hexokinase is consistent with random addition of glucose and MgATP, but with a marked preference for the addition of glucose prior to MgATP [3,4]. Glucose is released from the E . glucose . MgATP complex much slower than V/El [5], whereas MgATP dissociates rapidly from both binary and ternary complexes. In the direction of MgADP phosphorylation, glucose 6-phosphate dissociates from the ternary E . glucose-6-P . MgADP complex slowly (although it is not as sticky as glucose), but dissociates from the E . glucose-6-P complex about 30-times faster than the V/E, for phosphorylation of glucose [6]. The principal rate-determining step for the overall reaction in the direction of glucose phosphorylation is the release of MgADP (or a concomitant conformation change) [7l.Additional information concerning the location of ratedetermining steps along the reaction path can be obtained by the use of solvent deuterium isotope effects which detect slow steps that include perturbation of protons. This study reports the solvent deuterium isotope effect for the yeast hexokinase reaction as a function of pH. MATERIALS AND METHODS MaterialsHexokinase (type C-302) and other enzymes were from Sigma. 1,5-Anhydro-~-mannitol was prepared by treatment of D-mannitol with HCl [8].Other chemicals were purchased from commercial supplies and were of the highest purity available. Kinetic MeasurementsThe phosphorylation of glucose by hexokinase was monitored by the use of coupling the production of MgADP to the pyruvate kinase and lactate dehydrogenase reactions so that the disappearance of adsorbance at 340 nm could ...

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References

1988

Advances in Chemical Physics

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Yeast hexokinase in solution exhibits a large conformational change upon binding glucose or glucose 6-phosphate

Cited by 141 publications

References 17 publications

Crystal Structure of Hexokinase KlHxk1 of Kluyveromyces lactis

Crystal Structure of Hexokinase KlHxk1 of Kluyveromyces lactis

Solvent Isotope Effects on the Reaction Catalyzed by Yeast Hexokinase

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