Thermodynamics of the hydrolysis of adenosine 5'-triphosphate to adenosine 5'-diphosphate

The enthalpy of hydrolysis of the enzyme-catalyzed (heavy meromyosin) conversion of adenosine 5'-triphosphate (ATP) to adenosine 5'-diphosphate (ADP) and inorganic phosphate has been investigated using heat-conduction microcalorimetry. Enthalpies of reaction were measured as a function of ionic strength (0.05-0.66 mol kg-1), pH (6.4-8.8), and temperature (25-37 degrees C) in Tris/HCl buffer. The measured enthalpies were adjusted for the effects of proton ionization and metal ion binding, protonation and interaction with the Tris buffer, and ionic strength effects to obtain a value of delta H0 = -20.5 +/- 0.4 kJ mol-1 at 25 degrees C for the process, ATP4-(aq) + H2O(l) = ADP3-(aq) + HPO2-4(aq) + H+(aq) where aq is aqueous and l is liquid. Heat measurements carried out at different temperatures lead to a value of delta C0p = -237 +/- 30 J mol-1 K-1 for the above process.     

Thermodynamics of the conversion of aqueous L-aspartic acid to fumaric acid and ammonia

The thermodynamics of the conversion of aqueous L-aspartic acid to fumaric acid and ammonia have been investigated using both heat conduction microcalorimetry and high-pressure liquid chromatography. The reaction was carried out in aqueous phosphate buffer over the pH range 7.25-7.43, the temperature range 13-43 degrees C, and at ionic strengths varying from 0.066 to 0.366 mol kg(-1). The following values have been found for the conversion of aqueous L-aspartateH- to fumarate2- and NH4+ at 25 degrees C and at zero ionic strength: K = (1.48 +/- 0.10) x 10(-3), DeltaG degrees = 16.15 +/- 0.16 kJ mol(-1), DeltaH degrees = 24.5 +/- 1.0 kJ mol(-1), and DeltaC(p) degrees = -147 +/- 100 J mol(-1) K(-1). Calculations have also been performed which give values of the apparent equilibrium constant for the conversion of L-aspartic acid to fumaric acid and ammonia as a function of temperature, pH and ionic strength.     

Thermodynamics of hydrolysis of sugar phosphates

Thermodynamics of the enzyme-catalyzed (alkaline phosphatase, EC 3.1.3.1) hydrolysis of glucose 6-phosphate, mannose 6-phosphate, fructose 6-phosphate, ribose 5-phosphate, and ribulose 5-phosphate have been investigated using microcalorimetry and, for the hydrolysis of fructose 6-phosphate, chemical equilibrium measurements. Results of these measurements for the processes sugar phosphate2- (aqueous) + H2O (liquid) = sugar (aqueous) + HPO2++-(4) (aqueous) at 25 degrees C follow: delta Ho = 0.91 +/- 0.35 kJ.mol-1 and delta Cop = -48 +/- 18 J.mol-1.K-1 for glucose 6-phosphate; delta Ho = 1.40 +/- 0.31 kJ.mol-1 and delta Cop = -46 +/- 11 J.mol-1.dK-1 for mannose 6-phosphate; delta Go = -13.70 +/- 0.28 kJ.mol-1, delta Ho = -7.61 +/- 0.68 kJ.mol-1, and delta Cop = -28 +/- 42 J.mol-1.K-1 for fructose 6-phosphate; delta Ho = -5.69 +/- 0.52 kJ.mol-1 and delta Cop = -63 +/- 37 J.mol-1.K-1 for ribose 5-phosphate; and delta Ho = -12.43 +/- 0.45 kJ.mol-1 and delta Cop = -84 +/- 30 J.mol-1.K-1 for the hydrolysis of ribulose 5-phosphate. The standard state is the hypothetical ideal solution of unit molality. Estimates are made for the equilibrium constants for the hydrolysis of ribose and ribulose 5-phosphates. The effects of pH, magnesium ion concentration, and ionic strength on the thermodynamics of these reactions are considered.     

An investigation of the equilibria between aqueous ribose, ribulose, and arabinose

The thermodynamics of the equilibria between aqueous ribose, ribulose, and arabinose were investigated using high-pressure liquid chromatography and microcalorimetry. The reactions were carried out in aqueous phosphate buffer over the pH range 6.8-7.4 and over the temperature range 313.15-343.75 K using solubilized glucose isomerase with either Mg(NO3)2 or MgSO4 as cofactors. The equilibrium constants (K) and the standard state Gibbs energy (delta G degrees) and enthalpy (delta H degrees) changes at 298.15 K for the three equilibria investigated were found to be: ribose(aq) = ribulose(aq) K = 0.317, delta G degrees = 2.85 +/- 0.14 kJ mol-1, delta H degrees = 11.0 +/- 1.5 kJ mol-1; ribose(aq) = arabinose(aq) K = 4.00, delta G degrees = -3.44 +/- 0.30 kJ mol-1, delta H degrees = -9.8 +/- 3.0 kJ mol-1; ribulose(aq) = arabinose(aq) K = 12.6, delta G degrees = -6.29 +/- 0.34 kJ mol-1, delta H degrees = -20.75 +/- 3.4 kJ mol-1. Information on rates of the above reactions was also obtained. The temperature dependencies of the equilibrium constants are conveniently expressed as R in K = -delta G degrees 298.15/298.15 + delta H degrees 298.15[(1/298.15)-(1/T)] where R is the gas constant (8.31441 J mol-1 K-1) and T the thermodynamic temperature.     

Thermodynamics of the conversion of penicillin G to phenylacetic acid and 6-aminopenicillanic acid

The thermodynamics of the enzymatic conversion (penicillin acylase) of aqueous penicillin G to phenylacetic acid and 6-aminopenicillanic acid have been studied using both high-pressure liquid-chromatography and microcalorimetry. The reaction was carried out in aqueous phosphate buffer over the pH range 6.0-7.6, at ionic strengths from 0.10 to 0.40 mol kg-1, and at temperatures from 292 to 322 K. The data have been analyzed using a chemical equilibrium model with an extended Debye-Hückel expression for the activity coefficients. For the reference reaction, penicillin G- (aq) + H2O(l) = phenylacetic acid-(aq) + 6-aminopenicillanic acid-(aq) + H+ (aq), the following parameters have been obtained: K = (7.35 +/- 1.5) X 10(-8) mol kg-1, delta G0 = 40.7 +/- 0.5 kJ mol-1, delta H0 = 29.7 +/- 0.6 kJ mol-1, and delta C0p = -240 +/- 50 J mol-1 K-1 at 298.15 K and at the thermochemical standard state. The extent of reaction for the overall conversion is highly dependent upon the pH.     

Effect of temperature on the creatine kinase equilibrium.

The effect of temperature on the apparent equilibrium constant of creatine kinase (ATP:creatine N-phosphotransferase (EC 2.7.3.2)) was determined. At equilibrium the apparent K' for the biochemical reaction was defined as [formula: see text] The symbol sigma denotes the sum of all the ionic and metal complex species of the reactant components in M. The K' at pH 7.0, 1.0 mM free Mg2+, and ionic strength of 0.25 M at experimental conditions was 177 +/- 7.0, 217 +/- 11, 255 +/- 10, and 307 +/- 13 (n = 8) at 38, 25, 15, and 5 degrees C, respectively. The standard apparent enthalpy or heat of the reaction at the specified conditions (delta H' degree) was calculated from a van't Hoff plot of log10K' versus 1/T, and found to be -11.93 kJ mol-1 (-2852 cal mol-1) in the direction of ATP formation. The corresponding standard apparent entropy of the reaction (delta S' degree) was +4.70 J K-1 mol-1. The linear function (r2 = 0.99) between log10 K' and 1/K demonstrates that both delta H' degree and delta S' degree are independent of temperature for the creatine kinase reaction, and that delta Cp' degree, the standard apparent heat capacity of products minus reactants in their standard states, is negligible between 5 and 38 degrees C. We further show from our data that the sign and magnitude of the standard apparent Gibbs energy (delta G' degree) of the creatine kinase reaction was comprised mostly of the enthalpy of the reaction, with 11% coming from the entropy T delta S' degree term. The thermodynamic quantities for the following two reference reactions of creatine kinase were also determined. [formula: see text] The delta H degree for Reaction 2 was -16.73 kJ mol-1 (-3998 cal mol-1) and for Reaction 3 was -23.23 kJ mol-1 (-5552 cal mol-1) over the temperature range 5-38 degrees C. The corresponding delta S degree values for the reactions were +110.43 and +83.49 J K-1 mol-1, respectively. Using the delta H' degree of -11.93 kJ mol-1, and one K' value at one temperature, a second K' at a second temperature can be calculated, thus permitting bioenergetic investigations of organs and tissues using the creatine kinase equilibria over the entire physiological temperature range.     

Thermodynamics of porphyrin dimerization in aqueous solutions.

The dimerization equilibrium of deuteroporphyrin IX and of mesoporphyrin IX in aqueous solutions were studied by fluorimetric techniques over the 0.01-1 microM concentration range, where dimerization is the dominant aggregation process. Deuteroporphyrin IX was studied at several temperatures over the range 22-37 degrees C, and mesoporphyrin at 25 and 37 degrees C. The magnitudes determined for the dimerization equilibrium constants (25 degrees C, neutral pH, phosphate-buffered saline) are 2.3 X 10(6)M-1 and 5.4 X 10(6)M-1 for the deutero and meso derivatives respectively. The meso, deutero and haemato species tested show a similar temperature effect, namely dimerization decreasing with increasing temperature, indicating the involvement of a negative enthalpy change. Van''t Hoff isochore of the dimerization constants determined for deuteroporphyrin IX was linear within the temperature range of 22-37 degrees C, allowing the calculation of the thermodynamic parameters. For deuteroporphyrin dimerization, those were found to be delta G0 = -36. 4kJ X mol-1; delta H0 = -46. 0kJ X mol-1 and delta S0 = -32.2J X K-1 X mol-1 (at neutral pH, 25 degrees C, phosphate-buffered saline), showing the process to be enthalpy-driven. Similar trends have been found for porphyrin species other than those studied here. Our data fit with a hypothesis giving a major role to the solvent in driving porphyrins to aggregate in aqueous solution. The magnitudes and directions of the energetic changes fit better with the expectation of the '' solvophobic force'' theory predicting enthalpy-driven association, than with the classic hydrophobic bonding, predicting the association to be entropy-driven.     

Thermolysis of coenzymes B12 at physiological temperatures: activation parameters for cobalt-carbon bond homolysis and a quantitative analysis of the perturbation of the homolysis equilibrium by the ribonucleoside triphosphate reductase from Lactobacillus leichmannii

The kinetics of the thermolysis of 5'-deoxyadenosylcobalamin (AdoCbl, coenzyme B12) in aqueous solution, pH 7.5, have been studied in the temperature range 30-85 degrees C using AdoCbl tritiated at the adenine C2 position and the method of initial rates. Combined with a careful analysis of the distribution of adenine-containing products, the results permit the dissection of the competing rate constants for carbon-cobalt bond homolysis and heterolysis. After correction for the temperature-dependent occurrence of the much less reactive base-off species of AdoCbl, the activation parameters for homolysis of the base-on species were found to be delta H++homo,on = 33.8 +/- 0.2 kcal mol-1 and delta S++homo,on = 13.5 +/- 0.7 cal mol-1 K-1, values not significantly different from those determined by Hay and Finke (J. Am. Chem. Soc. 108 (1986) 4820), in the temperature range 85-115 degrees C. In contrast, the heterolysis of base-on AdoCbl was characterized by a much smaller enthalpy of activation (delta H++het,on = 18.5 +/- 0.2 kcal mol-1) and a negative entropy of activation (delta S++het,on = -34.0 +/- 0.7 cal mol-1 K-1) so that heterolysis, which is minor pathway at elevated temperatures, is the dominant pathway for AdoCbl decomposition at physiological temperatures. Using literature values for the rate constant for the reverse reaction, the equilibrium constant for AdoCbl homolysis at 37 degrees C was calculated to be 7.9 x 10(-18). Comparison with the equilibrium constant for this homolysis at the active site of the ribonucleoside triphosphate reductase from Lactobacillus leichmannii shows that the enzymes shifts the equilibrium constant towards homolysis products by a factor of 2.9 x 10(12) (17.7 kcal mol-1) by binding the thermolysis products with an equilibrium constant of 7.1 x 10(16) M-2, compared to the bonding constant for AdoCbl of 2.4 x 10(4) M-1.     

Comparison of the binding of Ca2+ and Mn2+ to bovine alpha-lactalbumin and equine lysozyme

The enthalpy change of the binding of Ca2+ and Mn2+ to equine lysozyme was measured at 25 degrees C and pH 7.5 by batch microcalorimetry: delta H degrees Ca2+ = -76 +/- 5 kJ mol-1, delta H degrees Mn2+ = -21 +/- 10 kJ mol-1. Binding constants, log KCa2+ = 6.5 +/- 0.2 and log KMn2+ = 4.1 +/- 0.5, were calculated from the calorimetric data. Therefore, delta S degrees Ca2+ = -131 +/- 20 JK-1 mol-1 and delta S degrees Mn2+ = 8 +/- 44 JK-1 mol-1. Removal of Ca2+ induces small but significant changes in the circular dichroism spectrum, indicating the existence of a partially unfolded apo-conformation, comparable with, but different from, the apo-conformation of bovine alpha-lactalbumin.     

Thermodynamics of the hydrolysis of sucrose

A thermodynamic investigation of the hydrolysis of sucrose to fructose and glucose has been performed using microcalorimetry and high-pressure liquid chromatography. The calorimetric measurements were carried out over the temperature range 298-316 K and in sodium acetate buffer (0.1 M, pH 5.65). Enthalpy and heat capacity changes were obtained for the hydrolysis of aqueous sucrose (process A): sucrose(aq) + H2O(liq) = glucose(aq) + fructose (aq). The determination of the equilibrium constant required the use of a thermochemical cycle calculation involving the following processes: (B) glucose 1-phosphate2-(aq) = glucose 6-phosphate2-(aq); (C) sucrose(aq) + HPO4(2-)(aq) = glucose 1-phosphate2-(aq) + fructose(aq); and (D) glucose 6-phosphate2-(aq) + H2O(liq) = glucose(aq) + HPO4(2-)(aq). The equilibrium constants determined at 298.15 K for processes B and C are 17.1 +/- 1.0 and 32.4 +/- 3.0, respectively. Equilibrium data for process D was obtained from the literature, and in conjunction with the data for processes B and C, used to calculate a value of the equilibrium constant for the hydrolysis of aqueous sucrose. Thus, for process A, delta G0 = -26.53 +/- 0.30 kJ mol-1, K0 = (4.44 +/- 0.54) x 10(4), delta H0 = -14.93 +/- 0.16 kJ mol-1, delta So = 38.9 +/- 1.2 J mol-1 K-1, and delta CoP = 57 +/- 14 J mol-1 K-1 at 298.15 K. Additional thermochemical cycles that bear upon the accuracy of these results are examined.     

The cholesterol-side-chain-cleaving cytochrome P450 spin-state equilibrium. 1. Thermodynamic analysis.

We have investigated the spin-state equilibrium of adrenal mitochondrial P450scc (cholesterol-side-chain-cleaving, CYP11A1) by absorption spectroscopy in the Soret band as a function of pH and temperature. The van't Hoff plot of the high-spin/low-spin equilibrium is not linear and is shifted towards high spin by lowering the pH. This non-linearity resolves clearly into two phases when the temperature range is extended from 37 degrees C to -20 degrees C using ethylene glycol as anti-freeze cosolvent. This enabled us to measure the enthalpy and entropy changes which are delta HA = 0.7 kJ.mol-1 and delta SA = 5J.K-1.mol-1 at low temperatures and delta HB = -42 kJ.mol-1 and delta SB = -152 J.K-1.mol-1 at high temperatures. The transition temperature, Tbreak, between both phases decreases as a function of pH. The experimental data can be fitted by a minimal reactional model comprising a temperature dependent conformational transition and two ionisation steps (one for each conformation), the pK of which is 1.5 +/- 0.5 higher in the low-temperature conformation. The deduced conformational equilibrium is affected by physiological effectors: Tbreak depends on the nature of the substrate intermediate and on the presence of the physiological electron donor, adrenodoxin.     

Acylation of subtilisin A by aryl esters: contribution to rate limitation by a physical step preceding general acid-base catalysis

The specificity ratios kc/Km = k for subtilisin A catalyzed hydrolysis of five aryl esters of N-(methoxycarbonyl)-L-Phe (McPhe) were determined at pH 7.03 and its pD equivalent. The ratios are independent of the electronic properties of the leaving group substituent. Kinetic solvent isotope effects, Dk, increase from about 0.9 to 1.3 as leaving group ability decreases from p-nitrophenolate to p-methoxyphenolate. The k of N-(methoxycarbonyl)-L-phenylalanine p-nitrophenyl ester (NPE) with native enzyme exhibits a strong temperature dependence; delta H* = 87 +/- 3 kJ mol-1 and delta S* = 148 +/- 14 J K-1 mol-1 at 25 degrees C (H2O). The Dk with this substrate is 1.36 at 13.6 degrees C, declines to 0.89 at 25 degrees C, and then increases to 1.04 at 39.4 degrees C. Above neutral pH(D), with McPhe NPE as substrate, the dependence of k is for the dissociated form of a single base of pKapp = 7.38 +/- 0.03 in H2O and 7.67 +/- 0.03 in D2O. The pKapp values are apparently those of the uncomplexed native protein. By contrast, k of 3-phenylpropanoic acid (Prop) p-nitrophenyl ester exhibits a weaker temperature dependence; delta H* = 20 kJ mol-1 and delta S* = -90 J K-1 mol-1 (H2O) at 25 degrees C. The Dk are larger than those for McPhe NPE, decreasing from 1.99 at 20.5 degrees C to 1.74 at 46.1 degrees C. These results, combined with those of previous studies, are consistent with limitation of k by at least two processes.(ABSTRACT TRUNCATED AT 250 WORDS)     

A calorimetric and equilibrium investigation of the hydrolysis of lactose

The thermodynamics of the hydrolysis of lactose to glucose and galactose have been investigated using both high pressure liquid chromatography and heat-conduction microcalorimetry. The reaction was carried out over the temperature range 282-316 K and in 0.1 M sodium acetate buffer at a pH of 5.65 using the enzyme beta-galactosidase to catalyze the reaction. For the process lactose(aq) + H2O(liq) = glucose(aq) + galactose(aq), delta G0 = -8.72 +/- 0.20 kJ.mol-1, K0 = 34 +/- 3, delta H0 = 0.44 +/- 0.11 kJ.mol-1, delta S0 = 30.7 +/- 0.8 J.mol-1.K-1, and delta Cop = 9 +/- 20 J.mol-1.K-1 at 298.15 K. The standard state is the hypothetical ideal solution of unit molality. Thermochemical cycle calculations using enthalpies of combustion and solution, entropies, solubilities, activity coefficients, and apparent molar heat capacities have also been performed. These calculations indicate large discrepancies which are attributable primarily to errors in literature data on the enthalpies of combustion and/or third law entropies of the crystalline forms of the substrates.     

Thermodynamics of hydrolysis of disaccharides. Cellobiose, gentiobiose, isomaltose, and maltose

The thermodynamics of the enzymatic hydrolysis of cellobiose, gentiobiose, isomaltose, and maltose have been studied using both high pressure liquid chromatography and microcalorimetry. The hydrolysis reactions were carried out in aqueous sodium acetate buffer at a pH of 5.65 and over the temperature range of 286 to 316 K using the enzymes beta-glucosidase, isomaltase, and maltase. The thermodynamic parameters obtained for the hydrolysis reactions, disaccharide(aq) + H2O(liq) = 2 glucose(aq), at 298.15 K are: K greater than or equal to 155, delta G0 less than or equal to -12.5 kJ mol-1, and delta H0 = -2.43 +/- 0.31 kJ mol-1 for cellobiose; K = 17.9 +/- 0.7, delta G0 = -7.15 +/- 0.10 kJ mol-1 and delta H0 = 2.26 +/- 0.48 kJ mol-1 for gentiobiose; K = 17.25 +/- 0.7, delta G0 = -7.06 +/- 0.10 kJ mol-1, and delta H0 = 5.86 +/- 0.54 kJ mol-1 for isomaltose; and K greater than or equal to 513, delta G0 less than or equal to -15.5 kJ mol-1, and delta H0 = -4.02 +/- 0.15 kJ mol-1 for maltose. The standard state is the hypothetical ideal solution of unit molality. Due to enzymatic inhibition by glucose, it was not possible to obtain reliable values for the equilibrium constants for the hydrolysis of either cellobiose or maltose. The entropy changes for the hydrolysis reactions are in the range 32 to 43 J mol-1 K-1; the heat capacity changes are approximately equal to zero J mol-1 K-1. Additional pathways for calculating thermodynamic parameters for these hydrolysis reactions are discussed.     

Kinetics of electron transfer between cytochrome c and laccase.

Rate constants have been determined for the electron-transfer reactions between reduced horse heart cytochrome c and resting Rhus vernicifera laccase as a function of pH, ionic strength, and temperature. The second-order rate constant for the oxidation of reduced cytochrome c was determined to be k = 125 M-1 s-1 at 25 degrees C in 0.2 M phosphate buffer at pH 6.0 with the activation parameters delta H++ = 16.2 kJ mol-1 and delta S++ = 28.9 J mol-1 K-1. The rate constants increased with decreasing buffer concentration, indicating that electron transfer from cytochrome c to laccase is favored by the local electrostatic interaction (ZAZB = -0.9 at pH 6 and -1.3 at pH 4.8) between the basic proteins with positive net charges. From the increase of the rate of electron transfer with decreasing pH, one of the driving forces of the reaction was suggested to be the difference in the redox potentials between the type 1 copper in laccase and the central iron in cytochrome c. Further, on addition of one hexametaphosphate anion per cytochrome c molecule, the rate of the electron transfer was increased, probably because the association of both proteins became more favorable.     

Thermodynamics of isomerization reactions involving sugar phosphates

Thermodynamics of isomerization reactions involving sugar phosphates have been studied using heat-conduction microcalorimetry. For the process glucose 6-phosphate2-(aqueous) = fructose 6-phosphate2- (aqueous), K = 0.285 +/- 0.004, delta Go = 3.11 +/- 0.04 kJ.mol-1, delta Ho = 11.7 +/- 0.2 kJ.mol-1, and delta Cop = 44 +/- 11 J.mol-1.K-1 at 298.15 K. For the process mannose 6-phosphate2- (aqueous) = fructose 6-phosphate2- (aqueous), K = 0.99 +/- 0.05, delta Go = 0.025 +/- 0.13 kJ.mol-1, delta Ho = 8.46 +/- 0.2 kJ.mol-1, and delta Cop = 38 +/- 25 J.mol-1.K-1 at 298.15 K. The standard state is the hypothetical ideal solution of unit molality. An approximate result (-14 +/- 5 kJ.mol-1) was obtained for the enthalpy of isomerization of ribulose 5-phosphate (aqueous) to ribose 5-phosphate (aqueous). The data from the literature on isomerization reactions involving sugar phosphates have been summarized, adjusted to a common reference state, and examined for trends and relationships to each other and to other thermodynamic measurements. Estimates are made for thermochemical parameters to predict the state of equilibrium of the several isomerizations considered herein.     

Thermodynamics of ribonuclease T1 denaturation.

Differential scanning calorimetry has been used to investigate the thermodynamics of denaturation of ribonuclease T1 as a function of pH over the pH range 2-10, and as a function of NaCl and MgCl2 concentration. At pH 7 in 30 mM PIPES buffer, the thermodynamic parameters are as follows: melting temperature, T1/2 = 48.9 +/- 0.1 degrees C; enthalpy change, delta H = 95.5 +/- 0.9 kcal mol-1; heat capacity change, delta Cp = 1.59 kcal mol-1 K-1; free energy change at 25 degrees C, delta G degrees (25 degrees C) = 5.6 kcal mol-1. Both T1/2 = 56.5 degrees C and delta H = 106.1 kcal mol-1 are maximal near pH 5. The conformational stability of ribonuclease T1 is increased by 3.0 kcal/mol in the presence of 0.6 M NaCl or 0.3 M MgCl2. This stabilization results mainly from the preferential binding of cations to the folded conformation of the protein. The estimates of the conformational stability of ribonuclease T1 from differential scanning calorimetry are shown to be in remarkably good agreement with estimates derived from an analysis of urea denaturation curves.     

Kinetic and thermodynamic characterization of the R17 coat protein-ribonucleic acid interaction

A filter retention assay is used to examine the kinetic and equilibrium properties of the interaction between phage R17 coat protein and its 21-nucleotide RNA binding site. The kinetics of the reaction are consistent with the equilibrium association constant and indicate a diffusion-controlled reaction. The temperature dependence of Ka gives delta H = -19 kcal/mol. This large favorable delta H is partially offset by a delta S = -30 cal mol-1 deg-1 to give a delta G = -11 kcal/mol at 2 degrees C in 0.19 M salt. The binding reaction has a pH optimum centered around pH 8.5, but pH has no effect on delta H. While the interaction is insensitive to the type of monovalent cation, the affinity decreases with the lyotropic series among monovalent anions. The ionic strength dependence of Ka reveals that ionic contacts contribute to the interaction. Most of the binding free energy, however, is a result of nonelectrostatic interactions.     

High-pressure effect on the equilibrium and kinetics of cyanide binding to chloroperoxidase

The kinetics of cyanide binding to chloroperoxidase were studied using a high-pressure stopped-flow technique at 25 degrees C and pH 4.7 in a pressure range from 1 to 1000 bar. The activation volume change for the association reaction is delta V not equal to + = -2.5 +/- 0.5 ml/mol. The total reaction volume change, determined from the pressure dependence of the equilibrium constant, is delta V degrees = -17.8 +/- 1.3 ml/mol. The effect of temperature was studied at 1 bar yielding delta H not equal to + = 29 +/- 1 kJ/mol, delta S not equal to + = -58 +/- 4 J/mol per K. Equilibrium studies give delta H degrees = -41 +/- 3 kJ/mol and delta S degrees = -59 +/- 10 J/mol per K. Possible contributions to the binding process are discussed: changes in spin state, bond formation and conformation changes in the protein. An activation volume analog of the Hammond postulate is considered.     

Thermodynamic study of yeast phosphoglycerate kinase

Enthalpies of binding of MgADP, MgATP, and 3-phosphoglycerate to yeast phosphoglycerate kinase have been determined by flow calorimetry at 9.95-32.00 degrees C. Combination of these data with published dissociation constants [Scopes, R.K. (1978) Eur. J. Biochem. 91, 119-129] yielded the following thermodynamic parameters for the binding of 3-phosphoglycerate at 25 degrees C: delta Go = -6.76 +/- 0.11 kcal mol-1, delta H = 3.74 +/- 0.08 kcal mol-1, delta So = 35.2 +/- 0.6 cal K-1 mol-1, and delta Cp = 0.12 +/- 0.32 kcal K-1 mol-1. The thermal unfolding of phosphoglycerate kinase in the absence and presence of the ligands listed above was studied by differential scanning calorimetry. The temperature of half-completion, t 1/2, of the denaturation and the denaturational enthalpy are increased by the binding of the ligands, the increase in t 1/2 being a manifestation of Le Chatelier's principle and that in enthalpy reflecting the enthalpy of dissociation of the ligand. Only one denaturational peak was observed under all conditions, and in contrast with the case of yeast hexokinase [Takahashi, K., Casey, J.L., & Sturtevant, J.M. (1981) Biochemistry 20, 4693-4697], no definitive evidence for the unfolding of more than one domain was obtained.