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PubMed1995

Evidence for the extended helical nature of polysaccharide epitopes. The 2.8 A resolution structure and thermodynamics of ligand binding of an antigen binding fragment specific for alpha-(2-->8)-polysialic acid.

The antigen binding fragment from an IgG2a kappa murine monoclonal antibody with specificity for alpha-(2-->8)-linked sialic acid polymers has been prepared and crystallized in the absence of hapten. Crystals were grown by vapor diffusion equilibrium with 16-18% polyethylene glycol 4000 solutions. The structure was solved by molecular replacement methods and refined to a conventional R factor of 0.164 for data to 2.8 A. The binding site is observed to display a shape and distribution of charges that is complementary to that of the predicted conformation of the oligosaccharide epitope. A thermodynamic description of ligand binding has been compiled for oligosaccharides ranging in length from 9 to 41 residues, and the data for the largest ligand has been used in a novel way to estimate the size of the antigen binding site. A model of antigen binding is presented that satisfies this thermodynamic data, as well as a previously reported requirement of conformational specificity of the oligosaccharide. X-ray crystallographic and thermodynamic evidence are consistent with a binding site that accommodates at least eight sialic acid residues.

Antibodies, Monoclonal↗
PubMed2000

Factors affecting the thermodynamic stability of small asymmetric internal loops in RNA.

Optical melting experiments were used to determine the thermodynamic parameters for oligoribonucleotides containing small asymmetric internal loops. The results show a broad range of thermodynamic stabilities, which depend on loop size, asymmetry, sequence, closing base pairs, and length of helix stems. Imino proton NMR experiments provide evidence for possible hydrogen bonding in GA and UU mismatches in some asymmetric loops. The stabilizing effects of GA, GG, and UU mismatches on the thermodynamic stability of internal loops vary depending on the size and asymmetry of the loop. The dependence of loop stability on Watson-Crick closing base pairs may be explained by an account of hydrogen bonds. Models are presented for approximating the free energy increments of 2 x 3 and 1 x 3 internal loops.

Adenine↗
PubMed1995

Thermodynamic parameters to predict stability of RNA/DNA hybrid duplexes.

The thermodynamic parameters (delta H degree, delta S degree, and delta G degree 37) for 16 nearest-neighbor sets and one initiation factor are presented here in order to predict stability of RNA/DNA hybrid duplexes. To determine the nearest-neighbor parameters, thermodynamics for 68 different hybrid sequences (136 single-stranded oligonucleotides) with 5-13 nucleotide length including several duplexes with identical nearest-neighbors were measured by UV melting procedure. These sequences were selected to have many different combinations of nearest-neighbor pairs, and so that the number of the 16 nearest-neighbor sequences in the oligomers were as equal as possible. The structures of the hybrids were also investigated by measuring circular dichroism spectra. Comparing delta G degree 37 values of the hybrids with DNA/DNA and RNA/RNA parameters reported previously (Breslauer, K.J., Frank, R., Blöcker, H., & Marky, L.A. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 3746-3750; Freier, S.M., Kierzek, R., Jaeger, J.A., Sugimoto, N., Caruthers, M.H., Neilson, T., & Turner, D.H. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 9373-9377), RNA/RNA double helix is the most stable of the three kinds of helixes with the same nearest-neighbor sequences. Which is more stable between DNA/DNA and RNA/DNA hybrid duplexes depends on its sequence. Calculated thermodynamic values of hybrid formation with the present parameters reproduce the experimental values within reasonable errors.

Base Sequence↗
PubMed2000

Multistate equilibrium unfolding of Escherichia coli dihydrofolate reductase: thermodynamic and spectroscopic description of the native, intermediate, and unfolded ensembles.

The thermodynamic and spectroscopic properties of a cysteine-free variant of Escherichia coli dihydrofolate reductase (AS-DHFR) were investigated using the combined effects of urea and temperature as denaturing agents. Circular dichroism (CD), absorption, and fluorescence spectra were recorded during temperature-induced unfolding at different urea concentrations and during urea-induced unfolding at different temperatures. The first three vectors obtained by singular-value decomposition of each set of unfolding spectra were incorporated into a global analysis of a unique thermodynamic model. Although individual unfolding profiles can be described as a two-state process, a simultaneous fit of 99 vectors requires a three-state model as the minimal scheme to describe the unfolding reaction along both perturbation axes. The model, which involves native (N), intermediate (I), and unfolded (U) states, predicts a maximum apparent stability, DeltaG degrees (NU), of 6 kcal mol(-)(1) at 15 degrees C, an apparent m(NU) value of 2 kcal mol(-)(1) M(-)(1), and an apparent heat capacity change, DeltaC(p)()(-NU), of 2.5 kcal mol(-)(1) K(-)(1). The intermediate species has a maximum stability of approximately 2 kcal mol(-)(1) and a compactness closer to that of the native than to that of the unfolded state. The population of the intermediate is maximal ( approximately 70%) around 50 degrees C and falls below the limits of detection of > or =2 M urea or at temperatures of <35 or >65 degrees C. The fluorescence properties of the equilibrium intermediate resemble those of a transient intermediate detected during refolding from the urea-denatured state, suggesting that a tryptophan-containing hydrophobic cluster in the adenosine-binding domain plays a key role in both the equilibrium and kinetic reactions. The CD spectroscopic properties of the native state reveal the presence of two principal isoforms that differ in ligand binding affinities and in the packing of the adenosine-binding domain. The relative populations of these species change slightly with temperature and do not depend on the urea concentration, implying that the two native isoforms are well-structured and compact. Global analysis of data from multiple spectroscopic probes and several methods of unfolding is a powerful tool for revealing structural and thermodynamic properties of partially and fully folded forms of DHFR.

Circular Dichroism↗
PubMed2000

Thermodynamic and structural studies of cavity formation in proteins suggest that loss of packing interactions rather than the hydrophobic effect dominates the observed energetics.

The hydrophobic effect is widely believed to be an important determinant of protein stability. However, it is difficult to obtain unambiguous experimental estimates of the contribution of the hydrophobic driving force to the overall free energy of folding. Thermodynamic and structural studies of large to small substitutions in proteins are the most direct method of measuring this contribution. We have substituted the buried residue Phe8 in RNase S with alanine, methionine, and norleucine. Binding thermodynamics and structures were characterized by titration calorimetry and crystallography, respectively. The crystal structures of the RNase S F8A, F8M, and F8Nle mutants indicate that the protein tolerates the changes without any main chain adjustments. The correlation of structural and thermodynamic parameters associated with large to small substitutions was analyzed for nine mutants of RNase S as well as 32 additional cavity-containing mutants of T4 lysozyme, human lysozyme, and barnase. Such substitutions were typically found to result in negligible changes in DeltaC(p)() and positive values of both DeltaDeltaH degrees and DeltaDeltaS of folding. Enthalpic effects were dominant, and the sign of DeltaDeltaS is the opposite of that expected from the hydrophobic effect. Values of DeltaDeltaG degrees and DeltaDeltaH degrees correlated better with changes in packing parameters such as residue depth or occluded surface than with the change in accessible surface area upon folding. These results suggest that the loss of packing interactions rather than the hydrophobic effect is a dominant contributor to the observed energetics for large to small substitutions. Hence, estimates of the magnitude of the hydrophobic driving force derived from earlier mutational studies are likely to be significantly in excess of the actual value.

Amino Acid Sequence↗
PubMed1993

Influence of two substrate analogues on thermodynamic properties of medium-chain acyl-CoA dehydrogenase.

The objective of this work was to identify the key structural functionalities of substrate or product that modulate the thermodynamic properties of medium-chain acyl-CoA dehydrogenase (MCAD). In order to achieve this, two classes of substrate analogues, acetyl-CoA and thioether-CoAs, were complexed with MCAD and their effects on the redox properties of MCAD were measured. A pH dependence study of the redox potential of uncomplexed MCAD allowed us to compare redox properties between complexed and uncomplexed MCAD and to calculate the dissociation constants of the analogues to the three redox states of MCAD. The results from this work indicate that these analogues are not influencing the thermodynamic behavior of MCAD in the same way as natural substrate. Thus, we propose that the following two key structural features of the binding ligand are necessary for mimicking the thermodynamic effects natural substrate has on MCAD: a thioester carbonyl on carbon 1 and a fatty acyl-CoA chain length around 8 carbon units. Furthermore, with the advent of structural knowledge, insights into the interactions of these structural features with MCAD and their influence on MCAD's highly regulated dehydrogenation mechanism are discussed.

Acetyl Coenzyme A↗
PubMed1992

H NMR investigation of the influence of interacting sites on the dynamics and thermodynamics of substrate and ligand binding to horseradish peroxidase.

The influence of substrate benzhydroxamic acid (BHA) and iron ligand (cyanide) on the thermodynamics and dynamics of each of the two binding sites of horseradish peroxidase (HRP) isozyme C has been investigated by 1H NMR spectroscopy. A combination of line-width analysis and saturation transfer spectroscopy has allowed the direct determination of the off-rate of substrate and ligand in the absence or presence of the other. These off-rates, together with available dissociation constants obtained by optical spectroscopy (Schonbaum, 1973), provide estimates for kon. The dissociation constant for cyanide binding to the BHA.HRP complex was also directly determined by NMR. In all cases the 1H NMR determined dynamic and thermodynamic data agree well with those values available in the literature. BHA binding leads to a 200-fold decrease in CN- affinity that arises from a factor greater than 10 decrease in koff(CN-) and greater than 2 x 10(3) decrease in kon(CN-). While a portion of the decrease in kon(CN-) can be rationalized by water coordination of the iron in the BHA.HRP complex, the additional decrease in kon(CN-) and that in koff(CN-) indicates that BHA in the binding pocket blocks the CN- ligation channel and serves as a "gate" to CN- exchange. This view is supported by observing a factor greater than 4 decrease in distal His labile proton exchange with bulk water in HRP-CN upon BHA binding. The ternary complex BHA.HRP-CN is shown to be heterogeneous. While the thermodynamics of BHA and CN- binding appear similar in the two ternary complexes, the BHA on- and off-rates for the two complexes differ by a factor of approximately 10. The two heterogeneous forms interconvert at 25 degrees C at approximately 2 x 10(2) s-1, precluding the determination of any difference in the CN- binding rates by saturation transfer. The greater lability of one of the two ternary complexes is attributed to an alternate orientation of some distal residue that blocks the substrate binding channel in one of the forms. Transferred nuclear Overhauser effects from the heme to BHA in the ternary complex reveal that the BHA substrate is in contact not only with the heme pyrrole D substituents but also with the distal His 42, indicating that the polar side chain of BHA extends well into the distal heme pocket.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗
PubMed1994

The thermodynamics of formation of a three-strand, DNA three-way junction complex.

Isothermal titration calorimetry (ITC) is used to study the thermodynamics of assembly of the three DNA oligonucleotides S1 (5'-GCCTGCCACCGC), S2 (5'-GCGGTGCGTCCG), and S3AA (5'-CGGACGAAGCAGGC) to form a three-way junction (TWJ) complex consisting of three double-helical arms radiating from a junction region having two unpaired adenosines in one strand (S3AA). The thermodynamics of assembly were measured for three different orders of addition of the component oligonucleotides at four temperatures between 10 and 25 degrees C. At each temperature studied, the overall values of delta H, delta S degrees, and delta G degrees for assembly of the complex from the component single strands were found to be independent of the order of addition. The enthalpy of binding, delta H, was found to be linearly dependent on temperature. From the temperature dependence of delta H, the change in heat capacity delta Cp, for the overall assembly of three strands to form the junction complex was calculated and found to be -1.6 kcal mol-1K-1. This work represents the first attempt to evaluate the thermodynamics of DNA TWJ formation by ITC.

Base Sequence↗
PubMed2001

Thermodynamic and kinetic effects of N3'-->P5' phosphoramidate modification on pyrimidine motif triplex DNA formation.

I have investigated the thermodynamic and kinetic effects of N3'-->P5' phosphoramidate (PN) backbone modification of triplex-forming oligonucleotide (TFO) on the pyrimidine motif triplex formation between a 23-bp target duplex and a 15-mer TFO using electrophoretic mobility shift assay, UV melting, isothermal titration calorimetry, and interaction analysis system. The thermodynamic and kinetic analyses have clearly indicated that the PN modification of TFO not only significantly increased the thermal stability of the pyrimidine motif triplex at neutral pH but also increased the binding constant of the pyrimidine motif triplex formation at room temperature and neutral pH by nearly 2 orders of magnitude. The consideration of the observed thermodynamic parameters has suggested that the more rigidity of the PN TFO in the free state relative to the unmodified TFO may enable the significant increase in the binding constant of the pyrimidine motif triplex formation at neutral pH. Kinetic data have also demonstrated that the observed PN modification-mediated promotion of pyrimidine motif triplex formation at neutral pH resulted from the considerable decrease in the dissociation rate constant rather than the increase in the association rate constant. This information will present an effective approach for designing chemically modified TFO with higher binding affinity in the triplex formation under physiological conditions, which may eventually lead to progress in therapeutic applications of the antigene strategy in vivo.

Amides↗
PubMed1994

Product binding modulates the thermodynamic properties of a Megasphaera elsdenii short-chain acyl-CoA dehydrogenase active-site mutant.

Previous work has shown that the redox properties of Megasphaera elsdenii short-chain acyl-CoA dehydrogenase (SCAD) are specifically modulated upon the binding of the substrate/product couple, allowing the reaction to proceed thermodynamically [Stankovich, M.T., & Soltysik, S. (1987) Biochemistry 26, 2627-2632]. The focus of this study on the Glu367Gln SCAD mutant protein is to gain an understanding of this phenomenon. The active-site mutant Glu367Gln SCAD inactivates the reductive and oxidative pathways and allows the effects of substrate (butyryl-CoA) and product (crotonyl-CoA) binding on the redox properties of the Glu367Gln SCAD mutant protein to be determined separately. Red anionic semiquinone was found to be thermodynamically stabilized in coulometric/potentiometric reductions of both butyryl-CoA- and crotonyl-CoA-complexed Glu367Gln SCAD. Reduction potential measurements showed that butyryl-CoA binding has little effect on the reduction potential of Glu367Gln SCAD. Crotonyl-CoA complexation, however, shifted the reduction potential of the Glu367Gln SCAD mutant protein by 30 mV in the positive direction. This modulation is similar to the 60-mV positive shift observed in native M. elsdenii SCAD upon complexation with the substrate/product couple [Stankovich, M.T., & Soltysik, S. (1987) Biochemistry 26, 2627-2632]. Thus, product binding and not substrate binding, thermodynamically regulates M. elsdenii SCAD. We propose that this observation is best explained by assuming that the product resembles an intermediate in the catalytic mechanism that is responsible for facilitating isopotential electron transfer from the substrate to the enzyme.

Acyl Coenzyme A↗
PubMed1994

Kinetics and thermodynamics of the molecular mechanism of the reductive half-reaction of xanthine oxidase.

The kinetics and thermodynamics of the reductive half-reaction of xanthine oxidase with xanthine as substrate have been investigated by stopped-flow kinetic measurements. The temperature dependence of the steady-state and transient kinetics of the reductive half-reaction reveals the existence of at least three molecular intermediates during this half-reaction. All the microscopic rate constants and the thermodynamic activation parameters of the elementary steps of the reductive half-reaction have been determined for the first time. The microscopic rate constants and the thermodynamic activation parameters of the individual steps show wide variations in their magnitudes. The present work provides the most detailed and incisive description of the reaction of xanthine oxidase with its physiological substrate xanthine.

Animals↗
PubMed1991

Human platelet factor 4 subunit association/dissociation thermodynamics and kinetics.

Platelet factor 4 (PF4) monomers (7800 daltons) form dimers and tetramers in varying molar ratios under certain solution conditions [Mayo, K. H., & Chen, M. J. (1989) Biochemistry 28, 9469]. The presence of a simplified aromatic region (one Tyr and two His) and resolved monomer, dimer, and tetramer Y60 3,5 ring proton resonances makes study of PF4 aggregate association/dissociation thermodynamics and kinetics possible. PF4 protein subunit association/dissociation equilibrium thermodynamic parameters have been derived by 1H NMR (500MHz) resonance line-fitting analysis of steady-state Y60 3,5 ring proton resonance monomer-dimer-tetramer populations as a function of temperature from 10 to 40 degrees C. Below 10 degrees C and above 40 degrees C, resonance broadening and overlap severely impaired analysis. Enthalpic and entropic contributions to dimer association Gibb's free energy [-5.1 kcal/mol (30 degrees C)] are +2.5 +/- 1 kcal/mol and +26 +/- 7 eu, respectively, and for tetramer association Gibb's free energy [-5.7 kcal/mol (30 degrees C)], they are -7.5 +/- 1 kcal/mol and -7 +/- 3 eu, respectively. These thermodynamic parameters are consistent with low dielectric medium electrostatic/hydrophobic interactions governing dimer formation and hydrogen bonding governing tetramer formation. Association/dissociation kinetic parameters, i.e., steady-state jump rates, have been derived from exchange-induced line-width increases and from 1H NMR (500 MHz) saturation-transfer and spin-lattice (Tl) relaxation experiments. From dissociation jump rates and equilibrium constants, association rate constants were estimated. For dimer and tetramer equilibria at 30 degrees C, unimolecular dissociation rate constants are 35 +/- 10 s-1 for dimer dissociation and 6 +/- 2 s-1 for tetramer dissociation.(ABSTRACT TRUNCATED AT 250 WORDS)

Humans↗
PubMed1987

Thermodynamics of follitropin binding to solubilized calf testis receptor.

Thermodynamic parameters of follitropin binding to solubilized testicular receptors were measured in order to assess the forces involved in the binding reaction. Reversibility of follitropin binding to solubilized receptor decreased only 20% over the temperature range 4-24 degrees C, whereas earlier studies indicated reversibility of binding to membrane-bound receptor decreased by more than 40% over the same range [Anderson, T. T., Curatolo, L. M., & Reichert, L. E., Jr. (1983) Mol. Cell. Endocrinol. 33, 37-52]. Thermodynamic analysis of follitropin binding to solubilized receptors showed that the hydrophobic effect was important in the binding reaction. The mean values, at 25 degrees C, for delta H and delta S were -31.8 kcal/mol and -66.0 cal mol-1 K-1, respectively, and delta Cp was -3.0 kcal mol-1 K-1. This is an unusually large heat capacity for protein-protein association reactions, indicating an enhanced role for the hydrophobic effect with the solubilized (compared to membrane-bound) receptor. Since glycerol was necessary to stabilize the solubilized receptor, we determined whether glycerol affected the thermodynamic parameters measured for the binding reaction. Control experiments, performed with membrane-bound receptor in the presence or absence of glycerol, indicated that delta Cp actually decreased upon addition of glycerol (-0.8 kcal mol-1 K-1 in the presence of glycerol compared to -2.3 kcal mol-1 K-1 in the absence of glycerol). Thus, the large negative delta Cp observed for the soluble receptor was a result of its removal from the membrane and was not due to the presence of glycerol.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1988

Unfolding free energy changes determined by the linear extrapolation method. 2. Incorporation of delta G degrees N-U values in a thermodynamic cycle.

The linear extrapolation method was used to evaluate the unfolding free energy changes (delta G degrees N-U) for phenylmethanesulfonyl chymotrypsin (PMS-Ct) at pH 6.0. The nonlinear least-squares fits of difference spectral data using urea and guanidinium chloride as denaturants gave identical values for delta G degrees N-U and delta epsilon degrees U, the latter being extinction coefficient differences between native and unfolded forms of the protein in the limit of zero concentration of denaturant. The independence of these parameters from the nature of solvent suggests strongly that they are characteristic properties of the protein alone. The delta G degrees N-U data at pH 6.0 and 4.0, which differ by more than 100-fold in stability of the protein, were incorporated into a thermodynamic cycle involving free energy changes for titration of native and unfolded PMS-Ct from pH 4.0 to 6.0. The purpose of the cycle was to test whether delta G degrees N-U obtained by use of the linear extrapolation method exhibits the characteristics required of a thermodynamic function of state. Within error, the thermodynamic cycle was found to accommodate the delta G degrees N-U quantities obtained at pH 4.0 and 6.0 for PMS-Ct.

Calorimetry↗
PubMed1981

Determination of protein--ligand equilibria by difference spectroscopy. Hemerythrin--ligand thermodynamic studies.

A difference spectrophotometric method for the rapid determination of equilibrium constants for protein--ligand interactions has been developed. The method requires no knowledge of the extinction coefficient of either reactants or products. Furthermore the method allows rapid determination of the temperature dependence of a reaction and thus leads to rapid determination of thermodynamic parameters. The method has been tested by following the interactions of ligands with hemerythrin, the nonheme iron, oxygen storage protein isolated from Phasocolopsis gouldii. The reactions were studied at various temperatures and ionic strengths, and standard thermodynamic parameters were determined. The standard thermodynamic parameters for the conversion of metaquohemerythrin to methydroxyhemerythrin were found to be delta H degrees = 5.8 +/- 1.3 kcal mol-1 and delta S degrees = -11.5 +/- 1.5 cal mol-1 deg-1. For the reaction of metaquohemerythrin with thiocyanate ion to produce metthiocyanatohemerythrin delta H degrees = --13.0 +/- 1.6 kcal mol-1 and delta S degrees - --25.3 +/- 5.5 cal mol-1 deg-1. For the reaction of thiocyanate ion with methydroxy-hemerythrin delta H degrees = --6.6 +/- 0.8 kcal mol-1 and delta S degrees = --38.3 +/- 4.0 cal mol-1 deg-1. Perchlorate ion decreases the affinity of metaquohemerhythrin for thiocyanate ion. This is reflected in both the entropy and enthalpy being more unfavorable for the reaction in the presence of perchlorate ion.

Animals↗
PubMed1981

Self-association of insulin and the role of hydrophobic bonding: a thermodynamic model of insulin dimerization.

The association constant for insulin dimerization calculated from concentration-dependent circular dichroic (CD) spectra of insulin, K12 = 7.5 X 10(5) M-1, is used along with other association constants (K24, K46, and K26) in an attempt to decipher the complex association behavior in solution and in crystal of this protein hormone. The free-energy change associated with dimerization, -RT ln K12, is -8.01 kcal mol-1, a value which is used to test a semiquantitative thermodynamic model of the process based in part on the X-ray crystallographic data of insulin. By delineation of the hydrophobic core on the surface of insulin, which is implicated in the dimer formation, the free energy of association, delta G degrees assoc, is estimated as -8.27 kcal mol-1 by using the thermodynamic parameters of Némethy & Scheraga [Némethy, G., & Scheraga, H. A. (1962) J Phys. Chem. 66, 1773-1789] and as -10.27 kcal mol-1 by using the values of Nozaki & Tanford [Nozaki, Y., & Tanford, C. (1971) J. Biol. Chem. 246, 2211-2217]. The role of hydrophobic bonding in the dimerization of insulin is discussed, and the calculated values of free energy of association are compared with the experimental value. The importance of this thermodynamic model is delineated in regard to both hormone-hormone and hormone-receptor interactions.

Chemical Phenomena↗
PubMed2001

Thermodynamic linked-function analysis of Mg(2+)-activated yeast pyruvate kinase.

Yeast pyruvate kinase (YPK) is regulated by intermediates of the glycolytic pathway [e.g., phosphoenolpyruvate (PEP), fructose 1,6-bisphosphate (FBP), and citrate] and by the ATP charge of the cell. Recent kinetic and thermodynamic data with Mn(2+)-activated YPK show that Mn(2+) mediates the allosteric communication between the substrate, PEP, and the allosteric effector, FBP [Mesecar, A., and Nowak, T. (1997) Biochemistry 36, 6792, 6803]. These results indicate that divalent cations modulate multiligand interactions, and hence cooperativity with YPK. The nature of multiligand interactions on YPK was investigated in the presence of the physiological divalent activator Mg(2+). The binding interactions of PEP, Mg(2+), and FBP were monitored by fluorescence spectroscopy. The binding data were subject to thermodynamic linked-function analysis to determine the magnitudes of the multiligand interactions governing the allosteric activation of YPK. The two ligand coupling free energies between PEP and Mg(2+), PEP and FBP, and FBP and Mg(2+) are 0.88, -0.38, and -0.75 kcal/mol, respectively. The two-ligand coupling free energies between PEP and Mn(2+) and FBP and Mn(2+) are more negative than those with Mg(2+) as the cation. This indicates that the interactions between the divalent cation and PEP with YPK are different for Mg(2+) and Mn(2+) and that the interaction is not simply electrostatic in nature, as originally hypothesized. The magnitude of the heterotropic interaction between the metal and FBP is similar with Mg(2+) and Mn(2+). The simultaneous binding of Mg(2+), PEP, and FBP to YPK is favored by 3.21 kcal/mol compared to independent binding. This complex is destabilized by 3.30 kcal/mol relative to the analogous YPK-Mn(2+)-PEP-FDP complex. Interpretation of K(d) values when cooperative binding occurs must be done with care as these are not simple thermodynamic constants. These data demonstrate that the divalent metal, which activates phosphoryl transfer in YPK, plays a key role in modulating the various multiligand interactions that define the overall allosteric properties of the enzyme.

Adenosine Triphosphate↗
PubMed2001

Thermodynamics of electron transfer in oxygenic photosynthetic reaction centers: volume change, enthalpy, and entropy of electron-transfer reactions in the intact cells of the cyanobacterium Synechocystis PCC 6803.

The volume and enthalpy changes for charge transfer in the 0.1-10 micros time window in photosynthetic reaction centers of the intact cells of Synechocystis PCC 6803 were determined using pulsed, time-resolved photoacoustics. This required invention of a method to correct for the cell artifact at the temperature of maximum density of water caused by the heterogeneous system. Cells grown under either white or red light had different PS I/PS II molar ratios, approximately 3 and approximately 1.7, respectively, but invariable action spectra and effective antenna sizes of the photosystems. In both cultures, the photoacoustic measurements revealed that their thermodynamic parameters differed strongly in the spectral regions of predominant excitation of PS I (680 nm) and PS II (625 nm). On correcting for contribution of the two photosystems at these wavelengths, the volume change was determined to be -27 +/- 3 and -2 +/- 3 A3 for PS I and PS II, respectively. The energy storage on the approximately 1 micros time scale was estimated to be 80 +/- 15% and 45 +/- 10% per trap in PS I and PS II, respectively. These correspond to enthalpies of -0.33 +/- 0.2 and -1 +/- 0.2 eV for the assumed formation of ion radical pairs P700+F(AB-) and Y(Z*)P680Q(A-), respectively. Taking the free energy of the above reactions as the differences of their redox potentials in situ, apparent entropy changes were estimated to be +0.4 +/- 0.2 and -0.2 +/- 0.2 eV for PS I and PS II, respectively. These values are similar to that obtained in vitro for the purified reaction center complexes on the microsecond time scale [Hou et al. (2001) Biochemistry 40, 7109-7116, 7117-7125]. The constancy of these thermodynamic values over a 2-fold change of the ratio of PS I/PS II is support for this method of in vivo analysis. Our pulsed PA method can correct the "cell" or heterogeneous artifact and thus opens a new route for studying the thermodynamics of electron transfer in vivo.

Calorimetry↗