Yeast Hexokinase. I. Preparation of the Pure Enzyme<sup>*</sup>

Lazarus, Norman R.; Ramel, A.; Rustum, Youcef M.; Barnard, Eric A.

doi:10.1021/bi00876a035

Cited by 101 publications

(74 citation statements)

References 16 publications

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“…Model 11, which is based on the flexible mnemonical enzyme concept, gives rise to rate equations that are similar in form to Eqns (6) and (7), as previously shown [29]. However, in this case, the formulation of coefficients CI, p, y , 6, E is different from that given in Table 2 and can be found in Table 4 of the previous paper [29].…”

Section: Discussionmentioning

confidence: 89%

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Regulatory Behavior of Monomeric Enzymes

Buc

Navarro

et al. 1974

European Journal of Biochemistry

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The wheat-germ hexokinase L, is made up of one polypeptide chain only and exhibits one active site for glucose per enzyme molecule as shown by equilibrium dialysis experiments. However, MgATP2-cannot be bound to a significant extent on the free enzyme in the absence of the hexose.Plots of the reciprocal of the reaction rate versus the reciprocal of glucose concentration exhibit a significant downward curvature, but they are linear versus the reciprocal of MgATP' concentration. The curvature of the plots with regard to glucose is modified neither by the concentration of MgATP'; nor by that of the product MgADP-. The intercepts and the slopes of the straight lines obtained versus the reciprocal of MgATP2 -concentrations give, when replotted against the reciprocal of glucose concentration, a curve concave downwards and a straight line, respectively. The downward curvature of the reciprocal plots, obtained with regard to glucose, is strongly increased by glucose 6-phosphate.The whole set of kinetic results along with equilibrium dialysis data strongly suggest that the nonMichaelian behavior is due to a mnemonical transition of the wheat germ hexokinase L,. The free enzyme very probably exists in two conformation states. The collision of any of these enzymes forms with glucose induces the same new conformation. After the binding of MgATP'; which is thus the second substrate, and the release of MgADP, the enzyme appears in one of the initial conformations which is stabilized by glucose 6-phosphate. That ligand can thus be bound on one only of the two conformation states of the free enzyme.The view that wheat germ hexokinase L, is a mnemonical enzyme gains further support from the study of the effect of denaturing agents on the reaction kinetics. Low concentrations of urea or dodecylsulfate "desensitize" the enzyme and completely suppress its non-Michaelian behavior, which clearly appears as a consequence of a conformational transition.

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Section: Discussionmentioning

confidence: 89%

“…2). If inequality of Eqn (5) The explicit formulation of the parameters a, p, y, 6, E is to be found in Table 2.…”

Section: Discussionmentioning

confidence: 99%

Regulatory Behavior of Monomeric Enzymes

Buc

Navarro

et al. 1974

European Journal of Biochemistry

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“…Protein concentrations were measured from the absorbance a t 280 nm using (hexokinase) = 9.2 [20]. Molar concentrations were determined assuming a molecular weight of 98 000 for hexokinase [22].…”

Section: Protein Concentration and Specific Activity Measurementsmentioning

confidence: 99%

Yeast Hexokinase: Interaction with Substrates and Analogs Studied by Difference Spectrophotometry

et al. 1974

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The mechanism of the reaction catalyzed by yeast hexokinase has been investigated by means of spectrophotometric investigations. Two binding sites, one for the sugar and one for the nucleotide substrate, have been characterized.The interaction of sugar substrates with yeast hexokinase induces spectral perturbations of aromatic residues of the protein molecule. The specificity of the enzyme towards its sugar substrates is discussed.The interaction of ATP-Mg can only be visualized spectrophotometrically when the sugar substrate or inhibitor is already bound to the enzyme.Furthermore, spectrophotometric studies evidence the formation of a binary complex (sugar However, the true mechanism of the reaction and the way in which the ternary complex is formed is still a matter of controversy. properties suggest the formation of a catalytic transitory phosphoryl enzyme [ll]. To explain the glucose-6-phosphate exchange, a second hypothesis in which glucose-6-phosphate is supposed to be bound a t the ATP site and glucose at the sugar site has been postulated [13]. Finally, the binding of some pentoses like lyxose or xylose produces an induced fit of the protein which enhances the hydrolysis rate of ATP [la]. After a n initial activating effect of xylose, De La Fuente has observed that the enzyme is inactivated [15]. I n a recent paper, Colowick has shown that this inactivation is related to the phosphorylation of hexokinase [16]. From this result, he has postulated [ 171 that "catalysis involves an active phosphoryl enzyme derivative which in the presence of D-xylose undergoes rearrangement to form the inactive derivative. It is also possible that the phosphorylation of the protein occurs only as a side reaction".By studying the interaction of various substrates with yeast hexokinase by means of difference spectrophotometry we can provide some information on the mechanism of this reaction; herein, we report the data on the formation of binary and ternary complexes between hexokinase and various substrates and analogs. We have also characterized a binding site specific for nucleotide substrates and another one for sugar phosphorylated sugar.Eur. J. Bioohem. 44 (1974)

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“…In the case of the Crabtree-positive yeast Saccharomyces cerevisiae, the regulatory phenomenon of glucose repression allows the preferential uptake and utilization of glucose, which is trapped inside the cell and activated for metabolism via phosphorylation (1). The genome of S. cerevisiae encodes three enzymes that are capable of catalyzing glucose phosphorylation: the differentially regulated hexokinases ScHxk1 3 and ScHxk2 and the glucokinase ScGlk1 (2,3). ScHxk2 is the predominating hexose kinase when glucose is abundantly available (4).…”

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confidence: 99%

“…1 Both authors contributed equally to this work. 2 To whom correspondence should be addressed. -Met 16 of ScHxk2 (16), its phosphorylation is likely to play a key role in ScHxk2-dependent glucose signaling.…”

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confidence: 99%

Protein Kinase Ymr291w/Tda1 Is Essential for Glucose Signaling in Saccharomyces cerevisiae on the Level of Hexokinase Isoenzyme ScHxk2 Phosphorylation*

Kaps

Kettner

Migotti

et al. 2015

Journal of Biological Chemistry

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Background: Monomer-dimer equilibrium, substrate affinity, and subcellular localization of yeast hexokinase ScHxk2 depend on the state of phosphorylation of serine 15. Results: Serine/threonine protein kinase Ymr291w/Tda1 is essentially required for ScHxk2-S15 phosphorylation. Conclusion: Ymr291w/Tda1 is the ScHxk2-S15 kinase or an upstream regulatory enzyme. Significance: The analysis of Ymr291w/Tda1 function(s) is indispensable for understanding glucose signaling in yeast.

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Yeast Hexokinase. I. Preparation of the Pure Enzyme^*

Cited by 101 publications

References 16 publications

Regulatory Behavior of Monomeric Enzymes

Regulatory Behavior of Monomeric Enzymes

Yeast Hexokinase: Interaction with Substrates and Analogs Studied by Difference Spectrophotometry

Protein Kinase Ymr291w/Tda1 Is Essential for Glucose Signaling in Saccharomyces cerevisiae on the Level of Hexokinase Isoenzyme ScHxk2 Phosphorylation*

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Yeast Hexokinase. I. Preparation of the Pure Enzyme*

Cited by 101 publications

References 16 publications

Regulatory Behavior of Monomeric Enzymes

Regulatory Behavior of Monomeric Enzymes

Yeast Hexokinase: Interaction with Substrates and Analogs Studied by Difference Spectrophotometry

Protein Kinase Ymr291w/Tda1 Is Essential for Glucose Signaling in Saccharomyces cerevisiae on the Level of Hexokinase Isoenzyme ScHxk2 Phosphorylation*

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Yeast Hexokinase. I. Preparation of the Pure Enzyme^*