Magnesium ion requirements for yeast enolase activity

Faller, Larry D.; Baroudy, Bahige M.; Johnson, Alan M.; Ewall, Ralph X.

doi:10.1021/bi00636a023

Cited by 98 publications

(82 citation statements)

References 23 publications

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“…The use of two Mg 2ϩ ions to bind PEP is consistent with biochemical evidence that suggests several molar equivalents of Mg 2ϩ bind to PEP in the active site (11). Such an arrangement would be similar to that seen in the active site of enolase (8,22).…”

Section: Resultssupporting

confidence: 77%

The Structural Determination of Phosphosulfolactate Synthase from Methanococcus jannaschii at 1.7-Å Resolution

Wise

Graham

White

et al. 2003

Journal of Biological Chemistry

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Members of the enolase mechanistically diverse superfamily catalyze a wide variety of chemical reactions that are related by a common mechanistic feature, the abstraction of a proton adjacent to a carboxylate group. Recent investigations into the function and mechanism of the phosphosulfolactate synthase encoded by the ComA gene in Methanococcus jannaschii have suggested that ComA, which catalyzes the stereospecific Michael addition of sulfite to phosphoenolpyruvate to form phosphosulfolactate, may be a member of the enolase superfamily. The ComA-catalyzed reaction, the first step in the coenzyme M biosynthetic pathway, likely proceeds via a Mg 2؉ ion-stabilized enolate intermediate in a manner similar to that observed for members of the enolase superfamily. ComA, however, has no significant sequence similarity to any known enolase. Here we report the x-ray crystal structure of ComA to 1.7-Å resolution. The overall fold for ComA is an (␣/␤) 8 barrel that assembles with two other ComA molecules to form a trimer in which three active sites are created at the subunit interfaces. From the positions of two ordered sulfate ions in the active site, a model for the binding of phosphoenolpyruvate and sulfite is proposed. Despite its mechanistic similarity to the enolase superfamily, the overall structure and active site architecture of ComA are unlike any member of the enolase superfamily, which suggests that ComA is not a member of the enolase superfamily but instead acquired an enolase-type mechanism through convergent evolution.

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Section: Resultssupporting

confidence: 77%

The Structural Determination of Phosphosulfolactate Synthase from Methanococcus jannaschii at 1.7-Å Resolution

Wise

Graham

White

et al. 2003

Journal of Biological Chemistry

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“…This suggests that subunit association and binding of two moles of 'conformational' metal are not influenced by substrate binding, the latter being consistent with recent findings [5,21,22].…”

Section: Kinetics Of Ansyl Fluorescence Changessupporting

confidence: 92%

Stopped‐Flow Studies of Changes in Fluorescence of 8‐Anilino‐1‐naphthalene Sulfonic Acid Caused by Magnesium and Salt Binding to Yeast Enolase

Brewer

1976

European Journal of Biochemistry

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Stopped-flow studies of magnesium and salt (potassium chloride and acetate) effects on yeast enolase were carried out by following 8-anilino-1 -naphthalenesulfonic acid fluorescence changes. The fluorescence changes appear to be largely caused by subunit association and dissociation, though there is evidence in some reactions for large changes in fluorescence occurring within the dead time of the stopped-flow measurements. These data are combined with measurements of initial enzyme activity after incubation in various solvents with or without magnesium to obtain subunit association and dissociation rates. From these, it is concluded that magnesium and the salts act by directly changing the affinities of the subunits for each other, apparently by producing a rapid change in protein conformation.Yeast enolase is a dimeric protein of identical subunits [l -31 which requires certain divalent cations, particularly magnesium, for activity [4]. The mechanism of activation by magnesium is complex: two moles of metal are bound with different dissociation constants in the absence of substrate, and two more in the presence of substrate [5]. Metal binding at the former two sites produces a conformational change in fully associated enzyme [5,6] and, in the case of partially dissociated enzyme, the most strongly-bound metal is also known to increase the extent of subunit association [l, 21 (i. e. decrease the dissociation equilibrium constant). In addition, high concentrations (% 1 M) of potassium acetate increase subunit association, and potassium chloride, at 1 M concentrations, promotes dissociation [l]. The effect of the anions is related to their position in the Hofmeister series of anions [7]. The two salts also appear to cause conformational changes in the enzyme [7], though the changes observed have been influenced by dissociation. It has been shown that these changes can be correlated with the fluorescence and extent of binding of the hydrophobic probe, 8-anilino-1 -naphthalene sulfonic acid (ansyl) Recently the effect of magnesium addition and removal on the enzyme has been studied using stoppedflow techniques [S]. In this study, advantage was taken of the fact that in Tris-HC1 of 0.05-0.1 M ionic strength, pH 8.0, magnesium causes a change in the absorption spectrum of the enzyme, with a maximum change of 296 nm. It was found that the metal bound to both monomeric and dimeric enzyme within the dead time of the stopped-flow. Association of monomers then occurred and after this, enzyme activity returned in a first-order reaction. That is:(neglecting the effect of the ligand). Here E,* is the active enzyme.On reaction of magnesium-enzyme with EDTA, the metal is removed within the dead time of the stopped-flow measurement. Following this, Brewer and DeSa observed a 296-nm absorbance change whose kinetics suggested a slow reaction followed by a faster one. The same kinetics were obtained whether or not subunit dissociation occurred, that is, the two reactions observed preceded any dissociation, i. e . , E,Mg = E, e E...

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“…2,14 ± 18 Two magnesium ions per subunit are required for enolase's catalytic activity. 24 The second magnesium ion has lower af®nity and binds after the substrate has bound. 25,26 Comparison of E. coli (red), yeast (yellow; 3enl) and lobster (blue; 1pdy) enolase structures.…”

Section: The Active Sitementioning

confidence: 99%

Crystal structure of the Escherichia coli RNA degradosome component enolase

Kühnel

Luisi

2001

Journal of Molecular Biology

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The crystal structure of Escherichia coli enolase (EC 4.2.1.11, phosphopyruvate hydratase), which is a component of the RNA degradosome, has been determined at 2.5 A Ê . There are four molecules in the asymmetric unit of the C2 cell, and in one of the molecules,¯exible loops close onto the active site. This closure mimics the conformation of the substratebound intermediate. A comparison of the structure of the E. coli enolase with the eukaryotic enolase structures available (lobster and yeast) indicates a high degree of conservation of the hydrophobic core and the subunit interface of this homodimeric enzyme. The dimer interface is enriched in charged residues compared with other protein homodimers, which may explain our observations from analytical ultracentrifugation that dimerisation is affected by ionic strength. The putative role of enolase in the RNA degradosome is discussed; although it was not possible to ascribe a speci®c role to it, a structural role is possible.

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Magnesium ion requirements for yeast enolase activity

Cited by 98 publications

References 23 publications

The Structural Determination of Phosphosulfolactate Synthase from Methanococcus jannaschii at 1.7-Å Resolution

The Structural Determination of Phosphosulfolactate Synthase from Methanococcus jannaschii at 1.7-Å Resolution

Stopped‐Flow Studies of Changes in Fluorescence of 8‐Anilino‐1‐naphthalene Sulfonic Acid Caused by Magnesium and Salt Binding to Yeast Enolase

Crystal structure of the Escherichia coli RNA degradosome component enolase

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