Selective DNA cleavage by elsamicin A and switch function of its amino sugar group.

We report guanine-specific recognition and selective cleavage of DNA by the antitumor antibiotic elsamicin A equipped with an amino sugar and compare these results with cleavage by chartarin and chartreusin antibiotics. The preferential cutting sites of DNA strand scission with elsamicin A are on the bases adjacent to the 3'-side of guanine residues such as 5'-GN sites, in particular 5'-GG sites. The present results also indicate that (1) the aglycon portion binds intercalatively to the 3'-side of guanine in host DNA, (2) the guanine 2-amino group has an important effect on selective DNA binding of elsamicin A, and (3) the amino sugar residue of elsamicin A facilitates the drug binding into the minor groove of B-DNA. In addition, we found that an acetylation of the amino group on the elsamicin A sugar portion plays an interesting switch function for the activity of elsamicin A. The biological implication of this switch has also been discussed.     

The vnd/NK-2 homeodomain: thermodynamics of reversible unfolding and DNA binding for wild-type and with residue replacements H52R and H52R/T56W in helix III

The conformational stabilities of the vnd (ventral nervous system defective)/NK-2 homeodomain [HD(wt); residues 1-80 that encompass the 60-residue homeodomain] and those harboring mutations in helix III of the DNA recognition site [HD(H52R) and HD(H52R/T56W)] have been investigated by differential scanning calorimetry (DSC) and ellipticity changes at 222 nm. Thermal unfolding reactions at pH 7.4 are reversible and repeatable in the presence of 50-500 mM NaCl with DeltaC(p) = 0.52 +/- 0.04 kcal K(-1) mol(-1). A substantial stabilization of HD(wt) is produced by 50 mM phosphate or by the addition of 100-500 mM NaCl to 50 mM Hepes, pH 7.4, buffer (from T(m) = 35.5 degrees C to T(m) 43-51 degrees C; DeltaH(vH) congruent with 47 +/- 5 kcal mol(-1)). The order of stability is HD(H52R/T56W) > HD(H52R) > HD(wt), irrespective of the anions present. Progress curves for ellipticity changes at 222 nm as a function of increasing temperature are fitted well by a two-state unfolding model, and the cooperativity of secondary structure changes is greater for mutant homeodomains than for HD(wt) and also is increased by adding 100 mM NaCl to Hepes buffer. A 33% quench of the intrinsic tryptophanyl residue fluorescence of HD(wt) by phosphate binding (K(D)' = 2.6 +/- 0.3 mM phosphate) is reversed approximately 60% by DNA binding. Thermodynamic parameters for vnd/NK-2 homeodomain proteins binding sequence-specific 18 bp DNA have been determined by isothermal titration calorimetry (10-30 degrees C). Values of DeltaC(p) are +0.25, -0.17, and -0.10 +/- 0.04 kcal K(-1) mol(-1) for HD(wt), HD(H52R), and HD(H52R/T56W) binding duplex DNA, respectively. Interactions of homeodomains with DNA are enthalpically controlled at 298 K and pH 7.4 with corresponding DeltaH values of -6.6 +/- 0.5, -10.8 +/- 0.1, and -9.0 +/- 0.6 kcal mol(-1) and DeltaG' values of -11.0 +/- 0.1, -11.0 +/- 0.1, and -11.3 +/- 0.3 kcal mol(-1) with a binding stoichiometry of 1.0 +/- 0.1. Thermodynamic parameters for DNA binding are not predicted from homeodomain structural changes that occur upon complexing to DNA and must reflect also solvent and possibly DNA rearrangements.     

DNA helicase RepA: cooperative ATPase activity and binding of nucleotides

The steady-state kinetic parameters of the ATPase activity of the homohexameric DNA helicase RepA and the binding of the fluorescent analogue epsilonADP to RepA have been studied. ssDNA stimulates RepA ATPase activity optimally at acidic pH 5.3-6.0. The sigmoidal kinetic curves in both the absence and presence of ssDNA show strong positive cooperativity for ATP hydrolysis, with oligonucleotides longer than 10mer optimal for ssDNA-stimulated ATPase activity. Fluorescence titrations show that, at 25 degrees C and in the absence of DNA, the binding of epsilonADP to RepA is biphasic with three high (K(1) = 1.54 x 10(6) M(-1)) and three low (K(2) = 4.71 x 10(4) M(-)(1)) affinity binding sites differing by 30-40-fold in binding constants. In the absence of cofactors, RepA melts cooperatively at T(m) = 65.8 +/- 0.1 degrees C and is more stable in the presence of ATPgammaS, T(m) = 68.1 +/- 0.2 degrees C (DeltaDeltaG 0.95 kcal/mol), than in the presence of ADP, T(m) = 66. 5 +/- 0.1 degrees C (DeltaDeltaG 0.29 kcal/mol), indicating that the additional phosphate group in ATPgammaS has a significant influence on RepA structure. A model is proposed in which individual subunits of RepA sequentially and cooperatively perform a multistep ATP hydrolytic cycle.     

Thermodynamic parameters of the binding of retinol to binding proteins and to membranes

Retinol (vitamin A alcohol) is a hydrophobic compound and distributes in vivo mainly between binding proteins and cellular membranes. To better clarify the nature of the interactions of retinol with these phases which have a high affinity for it, the thermodynamic parameters of these interactions were studied. The temperature-dependence profiles of the binding of retinol to bovine retinol binding protein, bovine serum albumin, unilamellar vesicles of dioleoylphosphatidylcholine, and plasma membranes from rat liver were determined. It was found that binding of retinol to retinol binding protein is characterized by a large increase in entropy (T delta S degrees = +10.32 kcal/mol) and no change in enthalpy. Binding to albumin is driven by enthalpy (delta H degrees = -8.34 kcal/mol) and is accompanied by a decrease in entropy (T delta S degrees = -2.88 kcal/mol). Partitioning of retinal into unilamellar vesicles and into plasma membranes is stabilized both by enthalpic (delta H degrees was -3.3 and -5.5 kcal/mol, respectively) and by entropic (T delta S degrees was +4.44 and +2.91 kcal/mol, respectively) components. The implications of these finding are discussed.     

Identification of key amino acid residues in the assembly of enzymes into the pyruvate dehydrogenase complex of Bacillus stearothermophilus: a kinetic and thermodynamic analysis

Structural studies have shown that electrostatic interactions play a major part in the binding of dihydrolipoyl dehydrogenase (E3) to the peripheral subunit-binding domain (PSBD) of the dihydrolipoyl acyltransferase (E2) in the assembly of the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus. The binding is characterized by a small, unfavorable enthalpy change (deltaH degrees = +2.2 kcal/mol) and a large, positive entropy change (TdeltaS degrees = +14.8 kcal/mol). The contributions of individual surface residues of the PSBD of E2 to its interaction with E3 have been assessed by alanine-scanning mutagenesis, surface plasmon resonance detection, and isothermal titration calorimetry. The mutation R135A in the PSBD gave rise to a significant decrease (120-fold) in the binding affinity; two other mutations (R139A and R156A) were associated with smaller effects. The binding of the R135A mutant to E3 was accompanied by a favorable enthalpy (deltaH degrees = -2.6 kcal/mol) and a less positive entropy change (TdeltaS degrees = +7.2 kcal/mol). The midpoint melting temperature (T(m)) of E3-PSBD complexes was determined by differential scanning calorimetry. The R135A mutation caused a significant decrease (5 degrees C) in the T(m), compared with the wild-type complex. The results reveal the importance of Arg135 of the PSBD as a key residue in the molecular recognition of E3 by E2, and as a major participant in the overall entropy-driven binding process. Further, the effects of mutagenesis on the deltaCp of subunit association illustrate the difficulties in attributing changes in heat capacity to specific classes of interactions.     

Crystal structure and ligand-binding studies of a screened peptide complexed with streptavidin.

The thermodynamic binding parameters and crystal structure for streptavidin-peptide complexes where the peptide sequences were obtained by random screening methods are reported. The affinities between streptavidin and two heptapeptides were determined by titrating calorimetric methods [Phe-Ser-His-Pro-Gln-Asn-Thr, Ka = 7944 (+/- 224) M-1, delta G degrees = -5.32 (+/- 0.01) kcal/mol, and delta H degrees = -19.34 (+/- 0.48) kcal/mol; His-Asp-His-Pro-Gln-Asn-Leu, Ka = 3542 (+/- 146) M-1, delta G degrees = -4.84 (+/- 0.03) kcal/mol, and delta H degrees = -19.00 (+/- 0.64) kcal/mol]. The crystal structure of streptavidin complexed with one of these peptides has been determined at 2.0-A resolution. The peptide (Phe-Ser-His-Pro-Gln-Asn-Thr) binds in a turn conformation with the histidine, proline, and glutamine side chains oriented inward at the biotin-binding site. A water molecule is immobilized between the histidine and glutamine side chains of the peptide and an aspartic acid side chain of the protein. Although some of the residues that participate in binding biotin also interact with the screened peptide, the peptide adopts an alternate method of utilizing binding determinants in the biotin-binding site of streptavidin.     

Thermodynamic analysis of the binding of component enzymes in the assembly of the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus

The peripheral subunit-binding domain (PSBD) of the dihydrolipoyl acetyltransferase (E2, EC 2.3.1.12) binds tightly but mutually exclusively to dihydrolipoyl dehydrogenase (E3, EC 1.8.1.4) and pyruvate decarboxylase (E1, EC 1.2.4.1) in the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus. Isothermal titration calorimetry (ITC) experiments demonstrated that the enthalpies of binding (DeltaH degrees ) of both E3 and E1 with the PSBD varied with salt concentration, temperature, pH, and buffer composition. There is little significant difference in the free energies of binding (DeltaG degrees = -12.6 kcal/mol for E3 and = -12.9 kcal/mol for E1 at pH 7.4 and 25 degrees C). However, the association with E3 was characterized by a small, unfavorable enthalpy change (DeltaH degrees = +2.2 kcal/mol) and a large, positive entropy change (TDeltaS degrees = +14.8 kcal/mol), whereas that with E1 was accompanied by a favorable enthalpy change (DeltaH degrees = -8.4 kcal/mol) and a less positive entropy change (TDeltaS degrees = +4.5 kcal/mol). Values of DeltaC(p) of -316 cal/molK and -470 cal/molK were obtained for the binding of E3 and E1, respectively. The value for E3 was not compatible with the DeltaC(p) calculated from the nonpolar surface area buried in the crystal structure of the E3-PSBD complex. In this instance, a large negative DeltaC(p) is not indicative of a classical hydrophobic interaction. In differential scanning calorimetry experiments, the midpoint melting temperature (T(m)) of E3 increased from 91 degrees C to 97.1 degrees C when it was bound to PSBD, and that of E1 increased from 65.2 degrees C to 70.0 degrees C. These high T(m) values eliminate unfolding as a major source of the anomalous DeltaC(p) effects at the temperatures (10-37 degrees C) used for the ITC experiments.     

Interaction of a designed interleukin-10 epitope mimic with an antibody studied by isothermal titration microcalorimetry

The mechanism of recognition of proteins and peptides by antibodies and the factors determining binding affinity and specificity are mediated by essentially the same features. However, additional effects of the usually unfolded and flexible solution structure of peptide ligands have to be considered. In an earlier study we designed and optimized six peptides (pepI to pepVI) mimicking the discontinuous binding site of interleukin-10 for the anti-interleukin-10 monoclonal antibody (mab) CB/RS/1. Three of them were selected for analysis of their solution conformation by circular dichroism measurements. The peptides differ in the content of alpha-helices and in the inducibility of helical secondary structures by trifluoroethanol. These properties, however, do not correlate with the binding affinity. PepVI, a 32-mer cyclic epitope mimic, has the highest affinity to mab CB/RS/1 identified to date. CD difference spectroscopy suggests an increase of the alpha-helix content of pepVI with complex formation. Binding of pepVI to mab CB/RS/1 is characterized by a large negative, favorable binding enthalpy and a smaller unfavorable loss of entropy (DeltaH degrees = -16.4 kcal x mol(-1), TDeltaS degrees = -6.9 kcal x mol(-1)) resulting in DeltaG degrees = -9.5 kcal x mol(-1) at 25 degrees C as determined by isothermal titration calorimetry. Binding of pepVI is enthalpically driven over the entire temperature range studied (10-35 degrees C). Complex formation is not accompanied by proton uptake or release. A negative heat capacity change DeltaC(p) of -0.354 kcal x mol(-1) x K(-1) was determined from the temperature dependence of DeltaH degrees. The selection of protein mimics with the observed thermodynamic properties is promoted by the applied identification and iterative optimization procedure.     

Binding of the recombinant proteinase inhibitor eglin c from leech Hirudo medicinalis to human leukocyte elastase, bovine alpha-chymotrypsin and subtilisin Carlsberg: thermodynamic study

The effect of pH and temperature on the apparent association equilibrium constant (Ka) for the binding of the recombinant proteinase inhibitor eglin c from leech Hirudo medicinalis to human leukocyte elastase (EC 3.4.21.37), bovine alpha-chymotrypsin (EC 3.4.21.1) and subtilisin Carlsberg (EC 3.4.21.14) has been investigated. On lowering the pH from 9.5 to 4.5, values of Ka for eglin c binding to the serine proteinases considered decrease thus reflecting the acid-pK shift of the invariant histidyl catalytic residue (His57 in human leukocyte elastase and bovine alpha-chymotrypsin, and His64 in subtilisin Carlsberg) from congruent to 6.9, in the free enzymes, to congruent to 5.1, in the enzyme:inhibitor adducts. At pH 8.0, values of the apparent thermodynamic parameters for eglin c binding are: human leukocyte elastase - Ka = 1.0 x 10(10) M-1, delta G phi = -13.4 kcal/mol, delta H phi = +1.8 kcal/mol, and delta S phi = +52 entropy units; bovine alpha-chymotrypsin -Ka = 5.0 x 10(9) M-1, delta G phi = -13.0 kcal/mol, delta H phi = +2.0 kcal/mol, and delta S phi = +51 entropy units; and subtilisin Carlsberg - Ka = 6.6 x 10(9) M-1, delta G phi = -13.1 kcal/mol, delta H phi = +2.0 kcal/mol, and delta S phi = +51 entropy units (values of Ka, delta G phi and delta S phi were obtained at 21 degrees C; values of delta H phi were temperature independent over the range explored, i.e. between 10 degrees C and 40 degrees C; 1 kcal = 4184J).(ABSTRACT TRUNCATED AT 250 WORDS)     

Catalysis by entropic effects: the action of cytidine deaminase on 5,6-dihydrocytidine

In neutral solution, 5,6-dihydrocytidine undergoes spontaneous deamination (k25 approximately 3.2 x 10(-5) s(-1)) much more rapidly than does cytidine (k25 approximately 3.0 x 10(-10) s(-1)), with a more favorable enthalpy of activation (DeltaDeltaH# = -8.7 kcal/mol) compensated by a less favorable entropy of activation (TDeltaDeltaS# = -1.8 kcal/mol at 25 degrees C). E. coli cytidine deaminase enhances the rate of deamination of 5,6-dihydrocytidine (kcat/k(non) = 4.4 x 10(5)) by enhancing the entropy of activation (DeltaDeltaH# = 0 kcal/mol; TDeltaDeltaS# = +7.6 kcal/mol, at 25 degrees C). Binding of the competitive inhibitor 3,4,5,6-tetrahydrouridine (THU), a stable analogue of 5,6-dihydrocytidine in the transition state for its deamination, is accompanied by a release of enthalpy (DeltaH = -7.1 kcal/mol, TDeltaDeltaS = +2.2 kcal/mol) that approaches the estimated enthalpy of binding of the actual substrate in the transition state for deamination of 5,6-dihydrocytidine (DeltaH = -8.1 kcal/mol, TDeltaDeltaS = +6.0 kcal/mol). Thus, the shortcomings of THU in capturing all of the binding affinity expected of an ideal transition-state analogue reflect a less favorable entropy of association. That difference may arise from the analogue's inability to displace a water molecule from the "leaving group site" at which ammonia is generated in the normal reaction. The effect on binding of removing the 4-OH group from the transition-state analogue THU, to form 3,4,5,6-tetrahydrozebularine (THZ) (DeltaDeltaH = -2.1 kcal/mol, TDeltaDeltaS = -4.4 kcal/mol), is mainly entropic, consistent with the inability of THZ to displace water from the "attacking group site". These results are consistent with earlier indications [Snider, M. J., and Wolfenden, R. (2001) Biochemistry 40, 11364] that site-bound water plays a prominent role in substrate activation and inhibitor binding by cytidine deaminase.     

Free energy contributions to direct readout of a DNA sequence

The energetic contributions of individual DNA-contacting side chains to specific DNA recognition in the human papillomavirus 16 E2C-DNA complex is small (less than 1.0 kcal mol(-1)), independent of the physical and chemical nature of the interaction, and is strictly additive. The sum of the individual contributions differs 1.0 kcal mol(-1) from the binding energy of the wild-type protein. This difference corresponds to the contribution from the deformability of the DNA, known as "indirect readout." Thus, we can dissect the energetic contribution to DNA binding into 90% direct and 10% indirect readout components. The lack of high energy interactions indicates the absence of "hot spots," such as those found in protein-protein interfaces. These results are compatible with a highly dynamic and "wet" protein-DNA interface, yet highly specific and tight, where individual interactions are constantly being formed and broken.     

Thermodynamic analysis of catalysis by the dihydroorotases from hamster and Bacillus caldolyticus, as compared with the uncatalyzed reaction

Dihydroorotase (DHOase, EC 3.5.2.3) from the extreme thermophile Bacillus caldolyticus has been subcloned, sequenced, expressed, and purified as a monomer. The catalytic properties of this thermophilic DHOase have been compared with another type I enzyme, the DHOase domain from hamster, to investigate how the thermophilic enzyme is adapted to higher temperatures. B. caldolyticus DHOase has higher Vmax and Ks values than hamster DHOase at the same temperature. The thermodynamic parameters for the binding of L-dihydroorotate were determined at 25 degrees C for hamster DHOase (deltaG = -6.9 kcal/mol, deltaH = -11.5 kcal/mol, TdeltaS = -4.6 kcal/mol) and B. caldolyticus DHOase (deltaG = -5.6 kcal/mol, deltaH = -4.2 kcal/mol, TdeltaS = +1.4 kcal/mol). The smaller enthalpy release and positive entropy for thermophilic DHOase are indicative of a weakly interacting Michaelis complex. Hamster DHOase has an enthalpy of activation of 12.3 kcal/mol, similar to the release of enthalpy upon substrate binding, rendering the kcat/Ks value almost temperature independent. B. caldolyticus DHOase shows a decrease in the enthalpy of activation from 12.2 kcal/mol at temperatures from 30 to 50 degrees C to 5.3 kcal/mol for temperatures of 50-70 degrees C. Vibrational energy at higher temperatures may facilitate the transition ES --> ES(double dagger), making kcat/Ks almost temperature independent. The pseudo-first-order rate constant for water attack on L-dihydroorotate, based on experiments at elevated temperature, is 3.2 x 10(-11) s(-1) at 25 degrees C, with deltaH(double dagger) = 24.7 kcal/mol and TdeltaS(double dagger) = -6.9 kcal/mol. Thus, hamster DHOase enhances the rate of substrate hydrolysis by a factor of 1.6 x 10(14), achieving this rate enhancement almost entirely by lowering the enthalpy of activation (delta deltaH(double dagger) = -19.5 kcal/mol). Both the rate enhancement and transition state affinity of hamster DHOase increase steeply with decreasing temperature, consistent with the development of H-bonds and electrostatic interactions in the transition state that were not present in the enzyme-substrate complex in the ground state.     

Biophysical studies of eIF4E cap-binding protein: recognition of mRNA 5' cap structure and synthetic fragments of eIF4G and 4E-BP1 proteins

mRNA 5'-cap recognition by the eukaryotic translation initiation factor eIF4E has been exhaustively characterized with the aid of a novel fluorometric, time-synchronized titration method, and X-ray crystallography. The association constant values of recombinant eIF4E for 20 different cap analogues cover six orders of magnitude; with the highest affinity observed for m(7)GTP (approximately 1.1 x 10(8) M(-1)). The affinity of the cap analogues for eIF4E correlates with their ability to inhibit in vitro translation. The association constants yield contributions of non-covalent interactions involving single structural elements of the cap to the free energy of binding, giving a reliable starting point to rational drug design. The free energy of 7-methylguanine stacking and hydrogen bonding (-4.9 kcal/mol) is separate from the energies of phosphate chain interactions (-3.0, -1.9, -0.9 kcal/mol for alpha, beta, gamma phosphates, respectively), supporting two-step mechanism of the binding. The negatively charged phosphate groups of the cap act as a molecular anchor, enabling further formation of the intermolecular contacts within the cap-binding slot. Stabilization of the stacked Trp102/m(7)G/Trp56 configuration is a precondition to form three hydrogen bonds with Glu103 and Trp102. Electrostatically steered eIF4E-cap association is accompanied by additional hydration of the complex by approximately 65 water molecules, and by ionic equilibria shift. Temperature dependence reveals the enthalpy-driven and entropy-opposed character of the m(7)GTP-eIF4E binding, which results from dominant charge-related interactions (DeltaH degrees =-17.8 kcal/mol, DeltaS degrees= -23.6 cal/mol K). For recruitment of synthetic eIF4GI, eIF4GII, and 4E-BP1 peptides to eIF4E, all the association constants were approximately 10(7) M(-1), in decreasing order: eIF4GI>4E-BP1>eIF4GII approximately 4E-BP1(P-Ser65) approximately 4E-BP1(P-Ser65/Thr70). Phosphorylation of 4E-BP1 at Ser65 and Thr70 is insufficient to prevent binding to eIF4E. Enhancement of the eIF4E affinity for cap occurs after binding to eIF4G peptides.     

Thermodynamic,kinetic, and structural basis for recognition and repair of 8-oxoguanine in DNA by Fpg protein from Escherichia coli

X-ray analysis does not provide quantitative estimates of the relative importance of the molecular contacts it reveals or of the relative contributions of specific and nonspecific interactions to the total affinity of specific DNA to enzymes. Stepwise increase of DNA ligand complexity has been used to estimate the relative contributions of virtually every nucleotide unit of 8-oxoguanine-containing DNA to its total affinity for Escherichia coli 8-oxoguanine DNA glycosylase (Fpg protein). Fpg protein can interact with up to 13 nucleotide units or base pairs of single- and double-stranded ribo- and deoxyribo-oligonucleotides of different lengths and sequences through weak additive contacts with their internucleotide phosphate groups. Bindings of both single-stranded and double-stranded oligonucleotides follow similar algorithms, with additive contributions to the free energy of binding of the structural components (phosphate, sugar, and base). Thermodynamic models are provided for both specific and nonspecific DNA sequences with Fpg protein. Fpg protein interacts nonspecifically with virtually all of the base-pair units within its DNA-binding cleft: this provides approximately 7 orders of magnitude of affinity (Delta G degrees approximately equal to -9.8 kcal/mol) for DNA. In contrast, the relative contribution of the 8-oxoguanine unit of the substrate (Delta G degrees approximately equal to -0.90 kcal/mol) together with other specific interactions is <2 orders of magnitude (Delta G degrees approximately equal to -2.8 kcal/mol). Michaelis complex formation of Fpg protein with DNA containing 8-oxoguanine cannot of itself provide the major part of the enzyme specificity, which lies in the k(cat) term; the rate is increased by 6-8 orders of magnitude on going from nonspecific to specific oligodeoxynucleotides.     

Reciprocal cooperative effects of multiple ligand binding to pyruvate kinase

The formation of multiple ligand complexes with muscle pyruvate kinase was measured in terms of dissociation constants and the standard free energies of formation were calculated. The binding of Mn2+ to the enzyme (KA = 55 +/- 5 X 10(-6) M; deltaF degrees = -5.75 +/- 0.05 kcal/mol) and to the enzyme saturated with phosphoenolpyruvate (conditional free energy) KA' = 0.8 +/- 0.4 X 10(-6) M; deltaF degrees = -8.22 +/- 0.34 kcal/mol) has been measured under identical conditions giving a free energy of coupling, delta(deltaF degrees) = -2.47 +/- 0.34 kcal/mol. Such a large negative free energy of coupling is diagnostic of a strong positively cooperative effect in ligand binding. The binding of the substrate phosphoenolpyruvate to free enzyme and the enzyme-Mn2+ complex was, by necessity, measured by different methods. The free energy of phosphoenolpyruvate binding to free enzyme (KS = 1.58 +/- 0.10 X 10(-4)M; deltaF degrees = -5.13 +/- 0.04 kcal/mol) and to the enzyme-Mn2+ complex (K3 = 0.75 +/- 0.10 X 10(-6)M; deltaF degrees = -8.26 +/- 0.07 kcal/mol) also gives a large negative free energy of coupling, delta(deltaF degrees) = -3.16 +/- 0.08 kcal/mol. Such a large negative value confirms reciprocal binding effects between the divalent cation and the substrate phosphoenolpyruvate. The binding of Mn2+ to the enzyme-ADP complex was also investigated and a free energy of coupling, delta(deltaF degrees) = -0.08 +/- 0.08 kcal/mol, was measured, indicative of little or no cooperativity in binding. The free energy of coupling with Mn2+ and pyruvate was measured as -1.52 +/- 0.14 kcal/mol, showing a significant amount of cooperativity in ligand binding but a substantially smaller effect than that observed for phosphoenolpyruvate binding. The magnitude of the coupling free energy may be related to the role of the divalent cation in the formation of the enzyme-substrate complexes. In the absence of the activating monovalent cation, the coupling free energies for phosphoenolpyruvate and pyruvate binding decrease by 40-60% and 25%, respectively, substantiating a role for the monovalent cation in the formation of enzyme-substrate complexes with phosphoenolpyruvate and with pyruvate.     

Association entropy in adsorption processes

The association of two species to form a bound complex, e.g., the binding of a ligand to a protein or the adsorption of a peptide on a lipid membrane, involves an entropy loss, reflecting the conversion of free translational and rotational degrees of freedom into bound motions. Previous theoretical estimates of the standard entropy change in bimolecular binding processes, DeltaS(o), have been derived from the root-mean-square fluctuations in protein crystals, suggesting DeltaS(o) approximately -50 e.u., i.e., TDeltaS degrees approximately -25 kT = -15 kcal/mol. In this work we focus on adsorption, rather than binding processes. We first present a simple statistical-thermodynamic scheme for calculating the adsorption entropy, including its resolution into translational and rotational contributions, using the known distance-orientation dependent binding (adsorption) potential. We then utilize this scheme to calculate the free energy of interaction and entropy of pentalysine adsorption onto a lipid membrane, obtaining TDeltaS(o) approximately -1.7 kT approximately -1.3 kcal/mol. Most of this entropy change is due to the conversion of one free translation into a bound motion, the rest arising from the confinement of two rotational degrees of freedom. The smaller entropy loss in adsorption compared to binding processes arises partly because a smaller number of degrees of freedom become restricted, but mainly due to the fact that the binding potential is much "softer."     

Thermodynamic analysis of inducer binding to the lactose repressor protein: contributions of galactosyl hydroxyl groups and beta substituents

Kinetic and equilibrium studies of the binding of modified beta-D-galactoside sugars to the lac repressor were carried out to generate thermodynamic data for protein-inducer interactions. The energetic contributions of the galactosyl hydroxyl groups to binding were assessed by using a series of methyl deoxyfluoro-beta-D-galactosides. The C-3 and C-6 hydroxyls contributed less than or equal to -2.3 and -1.7 +/- 0.3 kcal/mol to the binding free energy change, respectively, whereas the C-4 hydroxyl provided only a nominal contribution (-0.1 +/- 0.2 kcal/mol). Favorable contributions to the total binding free energy change were observed for replacement of O-methyl by S-methyl at the beta-anomeric position and for S-methyl by S-isopropyl. Negative delta H degrees values characteristic of protein-sugar complexes [Quiocho, F. A. (1986) Annu. Rev. Biochem. 55, 287-315] were observed for a series of beta-D-galactosides differing at the beta-glycosidic position. A decrease in delta H degrees of approximately 6 kcal/mol upon replacement of the O-methyl substituent by S-methyl indicates a substantial increase in van der Waals' interactions and/or hydrogen bonding in this region of the ligand binding site. The more favorable free energy change for the binding of the S-isopropyl vs S-methyl compound is due mainly to more positive entropic contributions, consistent with an increase in apolar interactions.(ABSTRACT TRUNCATED AT 250 WORDS)