The Effect of Bivalent Metal Ions on the Hydrolysis of Adenosine Di- and Triphosphate<sup>*</sup>

Tetas, Montserrat; Lowenstein, John M.

doi:10.1021/bi00902a030

Cited by 139 publications

(44 citation statements)

References 29 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…And when the Arrhenius plots were extrapolated to room temperature, it became evident that Mg 2ϩ accelerates ATP methanolysis by ϳ1 order of magnitude at 25°C (Fig. 6A), a larger factor than had been observed in earlier experiments at higher temperatures (17,18). But ADP methanolysis behaved somewhat differently in that Mg 2ϩ actually inhibited ADP methanolysis at high temperatures, whereas its effect was found to be almost negligible at 25°C (Fig.…”

Section: The Catalytic Proficiencies Of Kinasesmentioning

confidence: 66%

“…Effect of the Divalent Cation-In earlier comparisons of the rates of uncatalyzed solvolysis of ATP and MgATP, carried out at a single elevated temperature (17,18), the presence of Mg 2ϩ was shown to result in an approximate doubling of the rate. We confirmed those results, observing a 3.4-fold rate enhancement for ATP hydrolysis or methanolysis by Mg 2ϩ at 80°C (equivalent to a decrease of 0.7 kcal/mol in ⌬G ‡ at 80°C).…”

Section: ϫmentioning

confidence: 99%

“…Earlier work has shown that at elevated temperatures, the ␥-phosphorus atom of MgATP undergoes spontaneous attack by water in the absence of enzyme (17), that normal alcohols compete effectively with water in water-alcohol mixtures (18), and that in the transition state for spontaneous alcoholysis and hydrolysis, the bond to the leaving group appears to be almost fully broken, whereas the bond to the attacking nucleophile has only begun to form (18). Because Admiraal and Herschlag have shown that the rate of reaction is insensitive to the nature of the nucleophilic alcohol, a simple alcohol such as methanol would be expected to serve as a reasonable model for the reaction catalyzed by any kinase that mediates direct phosphoryl transfer from MgATP to an alcoholic acceptor.…”

mentioning

confidence: 99%

See 2 more Smart Citations

The Intrinsic Reactivity of ATP and the Catalytic Proficiencies of Kinases Acting on Glucose, N-Acetylgalactosamine, and Homoserine

Stockbridge

Wolfenden

2009

Journal of Biological Chemistry

View full text Add to dashboard Cite

To evaluate the rate enhancements produced by representative kinases and their thermodynamic basis, rate constants were determined as a function of changing temperature for 1) the spontaneous methanolysis of ATP and 2) reactions catalyzed by kinases to which different mechanisms of action have been ascribed. A common property shared by enzymes catalyzing biological reactions that involve a single substrate and hydrolytic and hydration reactions in which the effective concentration of water (the second substrate) cannot be elevated much above its concentration in living tissue is their ability to lower the reaction heat of activation. The rates of these enzymatic reactions are less sensitive to temperature than are the rates of the uncatalyzed reactions, so that the rate enhancements and transition state affinities generated by enzymes of this kind increase with decreasing temperature. Entropic effects tend to be minor, with an average change in the entropy of activation that approaches zero (1). That behavior seems understandable in view of the absence of a second substrate or the fact that the second substrate (water) is present in abundance. Thus, approximation effects seem relatively unlikely to play a major role in catalysis by enzymes of those types. Instead, enthalpic effects play a prominent role in catalysis, consistent with the formation in the transition state of new electrostatic and H-bonds between the enzyme and substrate, for which much evidence exists in the structures of enzymes crystallized with transition state analogue inhibitors (1).The effects of enzymes on the enthalpies and entropies of activation of two-substrate reactions remain largely unexplored. The thermodynamics of activation (for an enzyme reaction and for the corresponding uncatalyzed reaction) were examined recently for an unusual two-substrate enzyme (2, 3). The peptidyl transferase center of the ribosome was found to produce a 3 ϫ 10 7 -fold enhancement of the rate of peptidyl ester aminolysis entirely by rendering T⌬S ‡ more favorable, whereas ⌬H ‡ was actually found to become less favorable on the enzyme than in solution (3). Juxtaposition, that is, the gathering of two or more substrates at an enzyme active site in a configuration appropriate for reaction, appears to be partly responsible for that rate enhancement, and desolvation of the substrates may also contribute to the observed rate enhancement (4, 5). Thus, the ribosome might be said to function as a predominantly "physical" catalyst, bringing the substrates near each other in a water-poor environment rather than as a predominantly "chemical" catalyst that participates in the reaction as a general acid or a general base. Consistent with that interpretation is the finding that ribosomal peptidyl transfer is very strongly inhibited by a bisubstrate analogue devised by Yarus and co-workers (6).Because of its unusual composition (consisting entirely of RNA) and the relatively small rate enhancement that it produces, the behavior of the peptidyltransferase center of the r...

show abstract

Section: The Catalytic Proficiencies Of Kinasesmentioning

confidence: 66%

Section: ϫmentioning

confidence: 99%

mentioning

confidence: 99%

See 1 more Smart Citation

The Intrinsic Reactivity of ATP and the Catalytic Proficiencies of Kinases Acting on Glucose, N-Acetylgalactosamine, and Homoserine

Stockbridge

Wolfenden

2009

Journal of Biological Chemistry

View full text Add to dashboard Cite

show abstract

“…The situation is further complicated by the influence of reaction conditions on metabolite/coenzyme stability. For example, ATP stability is greatly affected by pH and metal ions [11], and NADH stability is very pH sensitive [12].…”

Section: D)mentioning

confidence: 99%

[24] Assaying activity and assessing thermostability of hyperthermophilic enzymes

Daniel¹,

Danson²

2001

Methods in Enzymology

View full text Add to dashboard Cite

There is now a wide variety of intra-and extra-cellular enzymes available from organisms growing above 75°C, and having sufficient stability to allow assay well above this temperature. For some of these enzymes, to assay below even 95°C will involve measurement below the optimal growth temperature for the organism. The purpose of this chapter is to cover practical aspects of enzyme assay procedures that are specific to high temperatures. Since by far the commonest routine assessment of enzyme stability is activity loss, and because it is always unwise to measure enzyme activity without being confident of its stability during the assay, we include an outline of procedures for measuring enzyme activity loss/stability at high temperatures.There are a number of useful reviews of the effects of temperature upon enzyme activity [1], and these apply as much to reactions at 100°C as at 37°C. However, enzymes stable at 100°C have a number of advantages as research subjects. For example, they can be used to investigate enzymes that are particularly unstable when isolated from mesophiles, to slow reactions without the use of cryosolvents, and to probe the effect of temperature on enzyme and protein behavior over a very wide temperature range. They can also be used to study enzyme behavior under conditions that would denature most enzymes, since enzymes resistant to heat are also, at room temperature, resistant to organic solvents, 2 chaotropic agents, and proteolysis. Clearly, such stable enzymes also have a variety of applications in biotechnology, where protein stability may be an important practical and economic factor.Enzyme activity increases with temperature, usually by a factor of 1.4 -2.0 per 10°C, depending upon the Arrhenius activation energy. As the temperature is raised, at some point the enzyme will begin to denature during the assay. The extent will depend upon the stability of the enzyme and the temperature, but also on the duration of the assay, and on factors such as the buffer composition and the degree of stabilisation of the enzyme by substrate/cofactor, etc. This combination of activity acceleration and increasing denaturation with temperature has unfortunately led to the production of activity versus temperature graphs that exhibit a so-called "temperature-optimum" peak for the enzyme. The position of this peak can of course be shifted by tens of degrees by varying the assay duration, and these graphs give little biochemical information above the temperature at which denaturation becomes significant during the assay. As a tool to aid in the development of enzyme technology processes, with the assay duration linked to the process time, these graphs may be useful in comparing enzymes. Their use should otherwise be avoided, as should the term "temperatureoptimum" derived from them.However, there is recent evidence [2] that some enzymes may have genuine temperature optima. That is, at some point in the temperature profile, an enzyme may actually become less active as the temperature is raised, but this is...

show abstract

“…However, Mg 2+ itself has little effect on non-enzymatic rates 1 , and reports of the rate acceleration attributable to the divalent metal differ markedly for different enzymes. Empirical valence bond simulations of two nuclease mechanisms 2 suggest that the metal contributes > 10 orders of magnitude in rate enhancement to phosphodiester bond hydrolysis catalyzed by enzymes using one 3 and two 4 Mg 2+ ions.…”

Section: Introductionmentioning

confidence: 99%

Mg²⁺-Free Bacillus stearothermophilus Tryptophanyl-tRNA Synthetase Retains a Major Fraction of the Overall Rate Enhancement for Tryptophan Activation

Weinreb

Carter

2008

J. Am. Chem. Soc.

View full text Add to dashboard Cite

Few experimental data are available for rates of enzymatic phosphoryl-transfer reactions in the absence of the divalent metal ions associated with such reactions. Such data are of interest for amino acid activation by class Ic aminoacyl-tRNA synthetases, for which there is substantial evidence that binding energy of ATP may account for a major fraction of the overall rate enhancement, and it is crucial to know if these effects themselves depend on the divalent metal ion. We describe a nested, non-linear model for the sum of metal-free and metal-catalyzed activities and its use in determining metal-free enzyme activity jointly with transition-state metal binding affinity, by fitting observed values obtained from Mg 2+ -depleted assays with increasing [EDTA] at known [Mg 2+ ] total . Tryptophan activation by B. stearothermophilus tryptophanyl-tRNA synthetase falls asymptotically to a plateau value five orders of magnitude below that observed for the Mg 2+ -supplemented enzyme at EDTA concentrations that reduce the free metal concentration to <1 pmolar. The fitted regression model parameters yield a relative rate acceleration of 9.3 × 10 4 attributable to the catalytic effect of Mg 2+ and an enhanced (K E ‡ = 1.15 × 10 −7 M) transition-state binding of Mg 2+ . Factorial analysis indicates that 80% of the reduction in free energy of activation effected by TrpRS arises from proteinligand interactions.

show abstract

The Effect of Bivalent Metal Ions on the Hydrolysis of Adenosine Di- and Triphosphate^*

Cited by 139 publications

References 29 publications

The Intrinsic Reactivity of ATP and the Catalytic Proficiencies of Kinases Acting on Glucose, N-Acetylgalactosamine, and Homoserine

The Intrinsic Reactivity of ATP and the Catalytic Proficiencies of Kinases Acting on Glucose, N-Acetylgalactosamine, and Homoserine

[24] Assaying activity and assessing thermostability of hyperthermophilic enzymes

Mg²⁺-Free Bacillus stearothermophilus Tryptophanyl-tRNA Synthetase Retains a Major Fraction of the Overall Rate Enhancement for Tryptophan Activation

Contact Info

Product

Resources

About

The Effect of Bivalent Metal Ions on the Hydrolysis of Adenosine Di- and Triphosphate*

Cited by 139 publications

References 29 publications

The Intrinsic Reactivity of ATP and the Catalytic Proficiencies of Kinases Acting on Glucose, N-Acetylgalactosamine, and Homoserine

The Intrinsic Reactivity of ATP and the Catalytic Proficiencies of Kinases Acting on Glucose, N-Acetylgalactosamine, and Homoserine

[24] Assaying activity and assessing thermostability of hyperthermophilic enzymes

Mg2+-Free Bacillus stearothermophilus Tryptophanyl-tRNA Synthetase Retains a Major Fraction of the Overall Rate Enhancement for Tryptophan Activation

Contact Info

Product

Resources

About

The Effect of Bivalent Metal Ions on the Hydrolysis of Adenosine Di- and Triphosphate^*

Mg²⁺-Free Bacillus stearothermophilus Tryptophanyl-tRNA Synthetase Retains a Major Fraction of the Overall Rate Enhancement for Tryptophan Activation