FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2023332924> ?p ?o ?g. }

• W2023332924 endingPage "22757" @default.
• W2023332924 startingPage "22747" @default.
• W2023332924 abstract "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. For each of these enzymes, the minor effects of changing viscosity indicate that kcat/Km is governed by the central chemical events in the enzyme-substrate complex rather than by enzyme-substrate encounter. Individual Arrhenius plots, obtained at intervals between pH 4.8 and 11.0, yielded ΔH‡ and TΔS‡ for the nonenzymatic methanolysis of ATP2−, ATP3−, and ATP4− in the absence of Mg2+. The addition of Mg2+ led to partly compensating changes in ΔH‡ and TΔS‡, accelerating the nonenzymatic methanolysis of ATP 11-fold at pH 7 and 25 °C. The rate enhancements produced by yeast hexokinase, homoserine kinase, and N-acetylgalactosamine kinase (obtained by comparison of their kcat/Km values in the presence of saturating phosphoryl acceptor with the second order rate constant for methanolysis of MgATP) ranged between 1012- and 1014-fold. Their nominal affinities for the altered substrates in the transition state were 2.1 × 10−16m for N-acetylgalactosamine kinase, 7.4 × 10−17m for homoserine kinase, and 6.4 × 10−18m for hexokinase. Compared with nonenzymatic phosphoryl transfer, all three kinases were found to produce major reductions in the entropy of activation, in accord with the likelihood that substrate juxtaposition and desolvation play prominent roles in their catalytic action. 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. For each of these enzymes, the minor effects of changing viscosity indicate that kcat/Km is governed by the central chemical events in the enzyme-substrate complex rather than by enzyme-substrate encounter. Individual Arrhenius plots, obtained at intervals between pH 4.8 and 11.0, yielded ΔH‡ and TΔS‡ for the nonenzymatic methanolysis of ATP2−, ATP3−, and ATP4− in the absence of Mg2+. The addition of Mg2+ led to partly compensating changes in ΔH‡ and TΔS‡, accelerating the nonenzymatic methanolysis of ATP 11-fold at pH 7 and 25 °C. The rate enhancements produced by yeast hexokinase, homoserine kinase, and N-acetylgalactosamine kinase (obtained by comparison of their kcat/Km values in the presence of saturating phosphoryl acceptor with the second order rate constant for methanolysis of MgATP) ranged between 1012- and 1014-fold. Their nominal affinities for the altered substrates in the transition state were 2.1 × 10−16m for N-acetylgalactosamine kinase, 7.4 × 10−17m for homoserine kinase, and 6.4 × 10−18m for hexokinase. Compared with nonenzymatic phosphoryl transfer, all three kinases were found to produce major reductions in the entropy of activation, in accord with the likelihood that substrate juxtaposition and desolvation play prominent roles in their catalytic action. 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 (1Wolfenden R. Snider M. Ridgway C. Miller B. J. Am. Chem. Soc. 1999; 121: 7419-7420Crossref Scopus (107) Google Scholar). 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 (1Wolfenden R. Snider M. Ridgway C. Miller B. J. Am. Chem. Soc. 1999; 121: 7419-7420Crossref Scopus (107) Google Scholar). 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 (2Sievers A. Beringer M. Rodnina M.V. Wolfenden R. Proc. Natl. Acad. Sci. U.S.A. 2004; 101: 7897-7901Crossref PubMed Scopus (244) Google Scholar, 3Schroeder G.K. Wolfenden R. Biochemistry. 2007; 46: 4037-4044Crossref PubMed Scopus (54) Google Scholar). The peptidyl transferase center of the ribosome was found to produce a 3 × 107-fold enhancement of the rate of peptidyl ester aminolysis entirely by rendering TΔS‡ more favorable, whereas ΔH‡ was actually found to become less favorable on the enzyme than in solution (3Schroeder G.K. Wolfenden R. Biochemistry. 2007; 46: 4037-4044Crossref PubMed Scopus (54) Google Scholar). 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 (4Trobro S. Aqvist J. Biochemistry. 2006; 45: 7049-7056Crossref PubMed Scopus (79) Google Scholar, 5Sharma P.K. Xiang Y. Kato M. Warshel A. Biochemistry. 2005; 44: 11307-11314Crossref PubMed Scopus (106) Google Scholar). 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 (6Welch M. Chastang J. Yarus M. Biochemistry. 1995; 34: 385-390Crossref PubMed Scopus (73) Google Scholar). 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 ribosome seems unlikely to be typical of two-substrate enzymes in general. The present work was undertaken with the limited objective of determining the magnitude and thermodynamic basis of the rate enhancements that are produced by several more conventional protein enzymes that catalyze reactions involving more than one substrate. The three enzymes examined here, yeast hexokinase, N-acetylgalactosamine kinase, and homoserine kinase, all bind the phosphoryl acceptor and MgATP in random order (7Danenberg K.D. Cleland W.W. Biochemistry. 1975; 14: 28-39Crossref PubMed Scopus (109) Google Scholar, 8Pastuszak I. Drake R. Elbein A.D. J. Biol. Chem. 1996; 271: 20776-20782Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar, 9Shames S.L. Wedler F.C. Arch. Biochem. Biophys. 1984; 235: 359-370Crossref PubMed Scopus (31) Google Scholar), and phosphoryl transfer is pH-insensitive over the pH range between ∼7.0 and 8.5 (8Pastuszak I. Drake R. Elbein A.D. J. Biol. Chem. 1996; 271: 20776-20782Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar, 10Viola R.E. Cleland W.W. Biochemistry. 1978; 17: 4111-4117Crossref PubMed Scopus (52) Google Scholar, 11Daugherty M. Vonstein V. Overbeek R. Osterman A. J. Bacteriol. 2001; 183: 292-300Crossref PubMed Scopus (75) Google Scholar). These enzymes are stable and active over the temperature range between 0 and 50 °C, and product inhibition is negligible under the conditions of these experiments. These three enzymes were chosen because they have different active site architectures that have led to different hypotheses about their mechanisms of action. The active site of yeast hexokinase is equipped with an aspartate residue that may act as a general base that deprotonates the acceptor alcohol (12Cleland W.W. Hengge A.C. FASEB J. 1995; 9: 1585-1594Crossref PubMed Scopus (110) Google Scholar). Many kinases are equipped with such a residue (13Jones J.P. Weiss P.M. Cleland W.W. Biochemistry. 1991; 30: 3634-3639Crossref PubMed Scopus (62) Google Scholar), although the extent of its involvement in the rate-determining step is unclear (14Matte A. Tari L.W. Delbaere L.T. Structure. 1998; 6: 413-419Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar). In contrast, the crystal structures of N-acetylgalactosamine kinase (GalNAcK) 2The abbreviations used are: GalNAcKN-acetylgalactosamine kinaseITCisothermal titration calorimetryMePmethyl phosphateHSKhomoserine kinase. 2The abbreviations used are: GalNAcKN-acetylgalactosamine kinaseITCisothermal titration calorimetryMePmethyl phosphateHSKhomoserine kinase. and homoserine kinase (HSK) suggest the absence of an active site residue that might act as a general base. Thoden and Holden (15Thoden J.B. Holden H.M. J. Biol. Chem. 2005; 280: 32784-32791Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar) have suggested that GalNAcK catalyzes phosphoryl transfer by juxtaposition of its two substrates, with a dissociative transition state like that of the spontaneous reaction. In the case of HSK, Krishna et al. (16Krishna S.S. Zhou T. Daugherty M. Osterman A. Zhang H. Biochemistry. 2001; 40: 10810-10818Crossref PubMed Scopus (71) Google Scholar) have proposed that the enzyme juxtaposes the substrates and stabilizes the non-bridging oxygen atoms of ATP in the transition state by interaction with backbone NH groups and by the positive end of a helix dipole. That proposal would seem to imply an associative transition state in which the non-bridging oxygen atoms carry more negative charge than they carry in the ground state. In a dissociative transition state, the non-bridging oxygen atoms would be expected to bear less negative charge than they do in the ground state, so that pairing them with positively charged groups would actually be expected to be anti-catalytic. N-acetylgalactosamine kinase isothermal titration calorimetry methyl phosphate homoserine kinase. N-acetylgalactosamine kinase isothermal titration calorimetry methyl phosphate homoserine kinase. For comparison with these enzyme-catalyzed reactions of MgATP, we sought to determine the rate of a similar reaction in the absence of a catalyst. Earlier work has shown that at elevated temperatures, the γ-phosphorus atom of MgATP undergoes spontaneous attack by water in the absence of enzyme (17Tetas M. Lowenstein J.M. Biochemistry. 1963; 2: 350-357Crossref PubMed Scopus (117) Google Scholar), that normal alcohols compete effectively with water in water-alcohol mixtures (18Admiraal S.J. Herschlag D. Chem. Biol. 1995; 2: 729-739Abstract Full Text PDF PubMed Scopus (197) Google Scholar), 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 (18Admiraal S.J. Herschlag D. Chem. Biol. 1995; 2: 729-739Abstract Full Text PDF PubMed Scopus (197) Google Scholar). 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. We, therefore, chose to examine the thermodynamics of activation for the methanolysis of ATP in the presence and absence of Mg2+ at various pH values using proton NMR to follow the course of reactions conducted in sealed tubes at elevated temperatures. Unless otherwise noted, reagents were obtained from Sigma. The β-O-methyl ester of ADP (ADP-O-CH3) and the α-O-methyl ester of AMP (AMP-O-CH3) were prepared by condensation of ADP or AMP with methanol in the presence of 0.1 m dicyclohexylcarbodiimide in methanol as described by Darzykiewicz et al. (19Darzynkiewicz E. Ekiel I. Tahara S.M. Seliger L.S. Shatkin A.J. Biochemistry. 1985; 24: 1701-1707Crossref Scopus (75) Google Scholar). In a typical experiment, ADP or ATP (0.025 m), methanol (2 m), and anionic buffers (0.1 m, pH 4.8–9.0) were sealed in quartz tubes under vacuum and incubated in convection ovens (Barnstead/Thermolyne Corp., model 47900) at temperatures ranging between 50 and 110 °C (±1.5 °C as indicated by an American Society for Testing and Materials thermometer) for varying periods of time in buffers (0.1 m) consisting of potassium acetate (pH 4.8–6.0), methyl phosphonate (pH 7.0–8.0), or sodium carbonate (pH 9.0–11.0). Separate experiments involving the addition of potassium chloride showed that at the buffer concentrations used, reactions were insensitive to changes in ionic strength (less than 3% variation with ionic strength varying from 0.1 to 1.0). After reaction, samples were prepared for analysis by evaporation to dryness and dilution with D2O containing sodium carbonate buffer (0.1 m, pD 10.0), with dioxane (1.8 × 10−4m) added as an internal integration standard (8 H, δ = 3.7 ppm). The integrated intensities of the proton signals arising from methyl phosphate and from the C8 adenine protons of ATP, ADP, and AMP (Fig. 1) were then measured using a Varian 600 MHz spectrometer. Data were acquired for a minimum of four transients using a standard water suppression pulse sequence and processed using SpinWorks (20Marat K. SpinWorks. University of Manitoba, Winnipeg, Manitoba, Canada1999–2007Google Scholar). In reactions conducted in the presence of MgCl2 and MnCl2, the concentration of metal ions (0.025 m) exceeded the value of Kd by at least 2 orders of magnitude (21Smith R.M. Alberty R.A. J. Am. Chem. Soc. 1956; 78: 2376-2380Crossref Scopus (163) Google Scholar). In experiments with added Mn2+, samples were prepared for NMR analysis by first removing Mn2+ ions by stirring with Chelex 100 beads (25% by volume) for 30 min followed by filtration to remove the beads. That treatment was repeated three times, and the filtrate was evaporated to dryness and taken up in D2O for 1H NMR as described above. Homoserine kinase from Methanococcus jannaschii and human N-acetylgalactosamine kinase were a gift from Drs. Hazel Holden and James Thoden of the University of Wisconsin at Madison. Enzyme assays were performed using isothermal titration calorimetry (VP-ITC, Microcal, Northampton, MA) as described by Todd and Gomez (22Todd M.J. Gomez J. Anal. Biochem. 2001; 296: 179-187Crossref PubMed Scopus (274) Google Scholar). To determine kcat/Km using ATP as the variable substrate, the enzyme (∼1 × 10−8m active sites) and saturating (0.01 m) substrate (see below) were first prepared in HEPES buffer (0.1 m, pH 8.0) with magnesium chloride (5 × 10−3m) and degassed for 10 min to prevent bubble formation during the experiment. After thermal equilibration and a 200-s time lag to establish a base line, the second substrate, ATP (0.0075 ml), was injected into the reaction mixture (1.45 ml) to initiate the reaction. The final concentration of ATP (1 × 10−5m) was well below its Km value for each of the enzymes examined (1.0 × 10−4m for homoserine kinase (Table 1), 6.3 × 10−5m for hexokinase (23Sem D.S. Cleland W.W. Biochemistry. 1991; 30: 4978-4984Crossref PubMed Google Scholar), and 8.3 × 10−5m for N-acetylgalactosamine kinase (Table 1)). In experiments of this kind, the enzyme reaction releases an amount of heat directly proportional to the amount of substrate turned over. The instrument output (Fig. 2A) reflects the amount of energy required to offset the heat generated by the reaction, and the lightly shaded area under the instrument response curve (Fig. 2A) represents the amount of heat generated in the turnover of 1 × 10−5m ATP. The integrated area between substrate injection and time t (dark gray area) divided by the total area in the well represents the percentage of reaction that has occurred at time t, P(t)=[SL]∫0rdQdtdt∫0∞dQdtdt(Eq. 1) TABLE 1kcat and Km values for GalNAcK, HSK, and hexokinase measured calorimetrically compared with those reported in the literatureEnzymeKm,ATPKm,acceptorkcatITCLiteratureITCLiteratureITCLiteraturemmmms−1s−1GalNAcK9.3 × 10−56.3 × 10−5 (8Pastuszak I. Drake R. Elbein A.D. J. Biol. Chem. 1996; 271: 20776-20782Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar)1.2 × 10−41.4 × 10−4 (8Pastuszak I. Drake R. Elbein A.D. J. Biol. Chem. 1996; 271: 20776-20782Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar)2.5n.d.HSK1.1 × 10−41.0 × 10−4 (11Daugherty M. Vonstein V. Overbeek R. Osterman A. J. Bacteriol. 2001; 183: 292-300Crossref PubMed Scopus (75) Google Scholar)8.7 × 10−51.9 × 10−4 (11Daugherty M. Vonstein V. Overbeek R. Osterman A. J. Bacteriol. 2001; 183: 292-300Crossref PubMed Scopus (75) Google Scholar)4557 (11Daugherty M. Vonstein V. Overbeek R. Osterman A. J. Bacteriol. 2001; 183: 292-300Crossref PubMed Scopus (75) Google Scholar)hexokinasen.d.1.2 × 10−4 (23Sem D.S. Cleland W.W. Biochemistry. 1991; 30: 4978-4984Crossref PubMed Google Scholar)n.d.7.2 × 10−5 (22Todd M.J. Gomez J. Anal. Biochem. 2001; 296: 179-187Crossref PubMed Scopus (274) Google Scholar)n.d.270 (22Todd M.J. Gomez J. Anal. Biochem. 2001; 296: 179-187Crossref PubMed Scopus (274) Google Scholar) Open table in a new tab A continuous reaction curve was generated from the integral of the calorimeter output (Fig. 2B). Typically, conditions were arranged so that reactions ran to completion in ∼2000 s. The first 10% of the curve (excluding ∼30 s allowed for mixing immediately after injection) was used to calculate the rate constant. In a plot of the product formed as a function of time (Fig. 1B), that portion of the curve is approximately linear, and its slope is proportional to the second order rate constant, kcat/Km, kcatKm=slope[EA][ATP](Eq. 2) where [EA] is the concentration of the enzyme saturated with the phosphoryl acceptor. To verify the accuracy of this method, the results obtained calorimetrically at 25 °C were compared with those obtained using a coupled assay to detect ADP generation by the decrease in UV absorbance at 340 nm, arising from the oxidation of NADH in the presence of pyruvate kinase and lactate dehydrogenase (24Kornberg A. Pricer Jr., W.E. J. Biol. Chem. 1951; 193: 481-495Abstract Full Text PDF PubMed Google Scholar) at 25 °C. The results obtained by these methods were in close agreement with each other, and the kcat and Km values determined by ITC were in close agreement with values from the literature (Table 1). Relative viscosities were adjusted over the range between 1.0 and 6.5 by adding trehalose, polyethylene glycol (average molecular weight = 300), or sucrose. Viscosities of buffered solutions were measured using a Cannon-Fiske kinetic viscometer at 25 °C. Rate constants (kcat and kcat/Km) were determined using both the ITC method and the coupled enzyme assay described above. Effects of viscosity on kcat/Km were determined for each enzyme in the presence of saturating phosphoryl-accepting substrate (0.01 m) and subsaturating ATP (1 × 10−5m). Effects of viscosity on kcat were determined with both ATP and the phosphoryl acceptor at saturating concentrations (0.01 m). In the presence of methanol the major reaction product was methyl phosphate (MeP), formed by transfer of the γ-phosphoryl group to methanol in a reaction analogous to the reactions catalyzed by kinases that phosphorylate hydroxyl groups. In principle, other products could also be formed by the reaction of ATP with methanol. Thus, methanol attack could occur at the β-position in a reaction analogous to that catalyzed by GTP diphosphokinase and thiamine diphosphokinase, or cleavage could occur between the α- and β-phosphoryl groups to yield ADP-O-CH3 (by methanol attack at the β-phosphoryl group) or AMP-O-CH3 (by attack at the α-phosphoryl group). Although no enzymes appear to catalyze reactions of the first type, many enzymes catalyze reactions of the second type, including DNA polymerases, aminoacyl-tRNA synthetases, and acyl-CoA synthetases. Finally, in a reaction analogous to that catalyzed by S-adenosyl methionine synthetase, ATP could undergo attack at the γ-phosphoryl group to yield 5′-methyl adenosine and inorganic triphosphate. In fact, a small amount of ADP-O-CH3, equal to ∼1.5% of the amount of MeP formed, was observed in the 1H NMR spectrum of the product mixture arising from ATP methanolysis at pH 7.0 (doublet; δ = 3.55). Its identity was confirmed by the addition of authentic ADP-O-CH3. At low pH, but not at pH 7, a small amount of adenosine was also formed (∼1% the amount of MeP). No other products were observed by 31P or 1H NMR at pH 4.8 or 9.0 or in the presence of 0.025 m Mg2+ at pH 7.0. The sensitivity of the instrument was such that formation of methyl pyrophosphate or AMP-O-CH3 would have been observable if either of those reactions had occurred at rates equivalent to >0.1% of the rate of formation of MeP. In aqueous methanol, ATP hydrolysis and methanolysis occur concurrently, and ADP formed during the reaction can then undergo a second hydrolysis or methanolysis reaction (Fig. 3) (the amount of AMP obtained by hydrolysis is equal to the difference between total amounts of AMP and MeP formed). To calculate the rate of ADP methanolysis, the observed rate of ADP decomposition (k3 + k4), k3+k4=In([ADP]t[ADP]0)t(Eq. 3) was divided into methanolysis and hydrolysis terms based on the relative amounts of MeP and AMP formed, k3k4=[AMP]−[MeP][MeP](Eq. 4) These equations were combined to evaluate k4, k3+k4=k4[([AMP]−[MeP][MeP])+1](Eq. 5) ATP methanolysis is more complicated to analyze because MeP derived from ADP also contributes to the amount of product observed. To estimate the value of k2, the disappearance of ATP was monitored to determine the composite rate constant (k1 + k2). The amount of “transient ADP” that decomposes in a second phosphorylation reaction is equal to the amount of AMP formed. Based on transient ADP and the ratio of k3 to k4 determined previously, the amount of MeP derived from ADP was estimated and subtracted from the total amount of MeP formed. The corrected result, representing the amount of MeP derived from ATP, was used to determine k2. The resulting rate constants were found to vary in proportion to the methanol concentration (data not shown). Arrhenius plots obtained for ADP and ATP methanolysis at pH 5.5 and 7.0 were found to be virtually superimposable (Fig. 4, top and middle panels). But at pH 9.0, where ADP and ATP are completely unprotonated, ATP4− reacted approximately twice as rapidly as ADP3− over the temperature range from 50 to 110 °C (Fig. 4, lower panel). That difference was also noted by Admiraal and Herschlag (18Admiraal S.J. Herschlag D. Chem. Biol. 1995; 2: 729-739Abstract Full Text PDF PubMed Scopus (197) Google Scholar) in earlier experiments at high pH. To obtain information about the methanolysis of ATP2−, ATP3−, and ATP4− in the absence of Mg2+, Arrhenius plots with at least six data points were gathered over the temperature range from 50 to 110 °C (R2 ≥ 0.975) at each of seven pH values between 4.8 and 9.0. Apparent pKa values of 6.5 and 4.3 were determined by titration at 25 °C under the conditions of the experiment. Comparable pKa values were reported earlier at this temperature and ionic strength (25Alberty R.A. Smith R.M. Bock R.M. J. Biol. Chem. 1951; 193: 425-434Abstract Full Text PDF PubMed Google Scholar). The methanolysis of ATP showed a gradual decrease in rate with increasing pH (Fig. 5). Similar behavior was described earlier by Tetas and Lowenstein (17Tetas M. Lowenstein J.M. Biochemistry. 1963; 2: 350-357Crossref PubMed Scopus (117) Google Scholar) for ATP hydrolysis. Interestingly, ΔH‡ and TΔS‡ differed for each species of ATP, leading to an unusual pH profile with a maximum in the enthalpic barrier and a minimum in the entropic barrier at pH 6.0 (Fig. 5). The complexity of this behavior presumably arises from the very different heats of ionization of amines and phosphoric acid derivatives, and the fact that the ionization of ATP at pH 4.2 involves the loss of a proton from N1 of the adenine ring, whereas the ionization of ATP at pH 6.5 involves deprotonation of the γ-phosphoryl group (see “Discussion” for further information about the ionization of ATP). At pH 6.0–9.0, the contributions of ATP3− and ATP4− dominate the observed activation parameters, and at pH 5.5 and 4.8 the contribution of ATP2− to the observed activation parameters becomes evident (shaded portion of Fig. 5). Rate constants and activation parameters for ATP3− methanolysis (Table 2) were estimated from the observed rates at pH 6.0–8.0, the measured rate constant for ATP4− methanolysis at pH 9.0, and the relative abundance of the ionized species of ATP3− derived from a titration curve. For example, at pH 8.0, 92.5% of ATP is present as ATP4−, and 7.5% of ATP is present as ATP3−. The rate of methanolysis of ATP4−, measured at pH 9.0, was found to be 7.1 × 10−11m−1 s−1. The observed rate constant for methanolysis at pH 8.0, 1.6 × 10−10m−1 s−1, is the sum of the rate constants for ATP4− methanolysis and ATP3− methanolysis, with both values adjusted to reflect the relative abundance of the reacting species: (0.925)(7.1×10−11)+(0.075)(kATP3−)=1.6×10−10M−1S−1(Eq. 6) In the same manner, the rate constant for ATP3− methanolysis was calculated for experiments carried out at pH 6.0, 7.0, 7.5, and 8.0. The average of the four values obtained for the rate constant for ATP3− methanolysis was 1.3 × 10−9m−1 s−1, with a ΔG‡ value of 29.5 kcal/mol. No single value deviated from that average by more than 0.4 kcal/mol.TABLE 2Rate constants and thermodynamics of activation for the methanolysis (boldface type) and hydrolysis (lightface type) of different species of ATPΔG‡ΔH‡TΔS‡k25cMethanolysis rate constants are expressed in s−1m−1. Hydrolysis rate constants are expressed in s−1.t½ (25 °C)dMethanolysis t½ values are for 1 m methanol.kcal/molkcal/molkcal/molATP4−30.3aMethanolysis reaction values are in boldface rows.25.0−5.33.4 × 10−10 s−1m−167 years m28.9bHydrolysis reaction values are in lightface rows.27.9−1.01.5 × 10−9 s−16.3 yearsATP3−29.526.8−2.71.3 × 10−9 s−1m−117 years m28.027.6−0.41.7 × 10−8 s−1500 daysMg·ATP2−28.822.8−6.03.9 × 10−9 s−1m−15.6 years m27.525.6−1.93.8 × 10−8 s−1210 daysMn·ATP2−28.122.1−6.01.4 × 10−8 s−1m−11.6 years m26.825.4−1.41.3 × 10−7 s−163 daysMg·ADP−30.324.5−5.73.4 × 10−10 s−1m−164 years m28.725.8−2.85.3 × 10−9 s−14.1 yearsa Methanolysis reaction values are in boldface rows.b Hydrolysis reaction values are in lightface rows.c Methanolysis rate constants are expressed in s−1m−1. Hydrolysis rate constants are expressed in s−1.d Methanolysis t½ values are for 1 m methanol. Open table in a new tab The value of ΔH‡ was estimated from the slopes of the Arrhenius plots obtained at pH 6.0, 7.0, 7.5, and 8.0. The average value for ΔH‡ obtained from the four experiments was 26.8 kcal/mol. No single value deviated from that average by more than 0.8 kcal/mol. Because excessive amounts of divalent cation lead to aggregation of MgATP complexes (26Glonek T. Int. J. Biochem. 1992; 24: 1533-1559Crossref PubMed Scopus (26) Google Scholar), Mg2+ was maintained in these experiments at a concentration equivalent to that of the nucleotide. ITC experiments (not shown) indicated dissociation constants of 1.7 × 10−5m for MgATP and 1.47 × 10−4m for MgADP at 25 °C, in reasonable agreement with values from the literature (21Smith R.M. Alberty R.A. J. Am. Chem. Soc. 1956; 78: 2376-2380Crossref Scopus (163) Google Scholar). At 25 °C, with both components present at a concentration of 0.025 m, 97% of the ATP and 93% of the ADP are present as their Mg2+ complexes, and their Kd values have been shown to decrease with increasing temperature (27Wang P. Oscarson J.L. Izatt R.M. Watt G.D. Larsen C.D. J. Solution Chem. 1995; 24: 989-1012Crossref Scopus (16) Google Scholar). Titrations of MgATP or MgADP indicated that at pH 7.0 the phosphoryl groups are completely unprotonated (MgATP2− and MgADP−). Whereas ADP and ATP are similarly reactive in the absence of Mg2+ (as described above), these nucleotides were found to exhibit differing reactivities in the presence of Mg2+ (Table 2). Fig. 6 shows Arrhenius plots for the methanolysis of MgATP, the methanolysis of MgADP, and the methanolysis of ATP in the absence of Mg2+ at pH 7.0. Data are shown in pairs to facilitate visual comparison. At all temperatures examined Mg2+ enhanced the rate of ATP methanolysis. And when the Arrhenius plots were extrapolated to room temperature, it became evident that Mg2+ accelerates ATP methanolysis by ∼1 order of magnitude at 25 °C (Fig. 6A), a larger factor than had been observed in earlier experiments at higher temperatures (17Tetas M. Lowenstein J.M. Biochemistry. 1963; 2: 350-357Crossref PubMed Scopus (117) Google Scholar, 18Admiraal S.J. Herschlag D. Chem. Biol. 1995; 2: 729-739Abstract Full Text PDF PubMed Scopus (197) Google Scholar). But ADP methanolysis behaved somewhat differently in that Mg2+ actually inhibited ADP me" @default.
• W2023332924 created "2016-06-24" @default.
• W2023332924 creator A5018640288 @default.
• W2023332924 creator A5055076632 @default.
• W2023332924 date "2009-08-01" @default.
• W2023332924 modified "2023-10-17" @default.
• W2023332924 title "The Intrinsic Reactivity of ATP and the Catalytic Proficiencies of Kinases Acting on Glucose, N-Acetylgalactosamine, and Homoserine" @default.
• W2023332924 cites W109567242 @default.
• W2023332924 cites W1501596101 @default.
• W2023332924 cites W1533460149 @default.
• W2023332924 cites W1579399439 @default.
• W2023332924 cites W188344449 @default.
• W2023332924 cites W1953904050 @default.
• W2023332924 cites W1964141556 @default.
• W2023332924 cites W1964598711 @default.
• W2023332924 cites W1966578342 @default.
• W2023332924 cites W1969158313 @default.
• W2023332924 cites W1970583752 @default.
• W2023332924 cites W1972136966 @default.
• W2023332924 cites W1975308721 @default.
• W2023332924 cites W1979285357 @default.
• W2023332924 cites W1987350479 @default.
• W2023332924 cites W1991954334 @default.
• W2023332924 cites W1993889668 @default.
• W2023332924 cites W1996613940 @default.
• W2023332924 cites W1998010694 @default.
• W2023332924 cites W2002836712 @default.
• W2023332924 cites W2010000945 @default.
• W2023332924 cites W2019564793 @default.
• W2023332924 cites W2027233056 @default.
• W2023332924 cites W2029396826 @default.
• W2023332924 cites W2031456024 @default.
• W2023332924 cites W2033929454 @default.
• W2023332924 cites W2036735263 @default.
• W2023332924 cites W2041435911 @default.
• W2023332924 cites W2044654702 @default.
• W2023332924 cites W2046251264 @default.
• W2023332924 cites W2064750009 @default.
• W2023332924 cites W2072407456 @default.
• W2023332924 cites W2072660673 @default.
• W2023332924 cites W2074367710 @default.
• W2023332924 cites W2076687655 @default.
• W2023332924 cites W2081109219 @default.
• W2023332924 cites W2081609898 @default.
• W2023332924 cites W2086337577 @default.
• W2023332924 cites W2090347337 @default.
• W2023332924 cites W2094719712 @default.
• W2023332924 cites W2095470273 @default.
• W2023332924 cites W2118527243 @default.
• W2023332924 cites W2312789057 @default.
• W2023332924 cites W2313720416 @default.
• W2023332924 cites W2316706024 @default.
• W2023332924 cites W2331377560 @default.
• W2023332924 cites W2951635181 @default.
• W2023332924 doi "https://doi.org/10.1074/jbc.m109.017806" @default.
• W2023332924 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/2755683" @default.
• W2023332924 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/19531469" @default.
• W2023332924 hasPublicationYear "2009" @default.
• W2023332924 type Work @default.
• W2023332924 sameAs 2023332924 @default.
• W2023332924 citedByCount "57" @default.
• W2023332924 countsByYear W20233329242012 @default.
• W2023332924 countsByYear W20233329242013 @default.
• W2023332924 countsByYear W20233329242014 @default.
• W2023332924 countsByYear W20233329242015 @default.
• W2023332924 countsByYear W20233329242016 @default.
• W2023332924 countsByYear W20233329242017 @default.
• W2023332924 countsByYear W20233329242018 @default.
• W2023332924 countsByYear W20233329242019 @default.
• W2023332924 countsByYear W20233329242020 @default.
• W2023332924 countsByYear W20233329242021 @default.
• W2023332924 countsByYear W20233329242022 @default.
• W2023332924 countsByYear W20233329242023 @default.
• W2023332924 crossrefType "journal-article" @default.
• W2023332924 hasAuthorship W2023332924A5018640288 @default.
• W2023332924 hasAuthorship W2023332924A5055076632 @default.
• W2023332924 hasBestOaLocation W20233329241 @default.
• W2023332924 hasConcept C104317684 @default.
• W2023332924 hasConcept C118687296 @default.
• W2023332924 hasConcept C142724271 @default.
• W2023332924 hasConcept C161790260 @default.
• W2023332924 hasConcept C184235292 @default.
• W2023332924 hasConcept C185592680 @default.
• W2023332924 hasConcept C204787440 @default.
• W2023332924 hasConcept C2776910235 @default.
• W2023332924 hasConcept C2779840497 @default.
• W2023332924 hasConcept C55493867 @default.
• W2023332924 hasConcept C60987743 @default.
• W2023332924 hasConcept C71240020 @default.
• W2023332924 hasConcept C71924100 @default.
• W2023332924 hasConceptScore W2023332924C104317684 @default.
• W2023332924 hasConceptScore W2023332924C118687296 @default.
• W2023332924 hasConceptScore W2023332924C142724271 @default.
• W2023332924 hasConceptScore W2023332924C161790260 @default.
• W2023332924 hasConceptScore W2023332924C184235292 @default.
• W2023332924 hasConceptScore W2023332924C185592680 @default.
• W2023332924 hasConceptScore W2023332924C204787440 @default.
• W2023332924 hasConceptScore W2023332924C2776910235 @default.

• next