Thermodynamic parameters of the interaction between Co(II) bovine carbonic anhydrase and anionic inhibitors.

The pH dependence of the apparent affinity constants of perchlorate for cobalt(II)bovine carbonic anhydrase II has been measured by electronic absorption spectroscopy. The obtained data have been analyzed in terms of the ionization of two acidic groups of CoBCAII, and the affinity of perchlorate for the two water-containing species of the enzyme have been estimated. Furthermore, the affinity constants of nitrate, perchlorate, and azide for CoBCAII in the temperature range 5 degrees C-30 degrees C have been determined by spectrophotometric titrations at pH 7. The affinity constants for these ligands decrease with increasing temperatures. The temperature dependence of binding was used to estimate the enthalpy and entropy parameters for the formation of the corresponding 1:1 adducts. The obtained results indicate that binding of these anions to the cobalt enzyme is an enthalpy driven process which is opposed by a moderate entropy change.     

Isomerization of the muscarinic receptor . antagonist complex.

The mechanism of binding of two antagonists, 3-quinuclidinyl benzilate and N-methyl-4-piperidinyl benzilate, to the muscarinic receptor was studied. The pseudo-first order rate constant of association showed a hyperbolic dependence on the concentration of the antagonist(s) indicating that the interaction involves two equilibria. The first binding equilibrium is reached rapidly and is characterized by dissociation constants 2.7 +/- 0.4 nM and 6.7 +/- 2.5 nM in phosphate buffer (0.05 M, pH = 7.4) for 3-quinuclidinyl benzilate and N-methyl-4-piperidinyl benzilate, respectively. The first binding equilibrium is followed by a slower isomerization step of the receptor . antagonist complex. The equilibrium constants for the isomerization step of the complex for both ligands were about 0.15. The overall constant of binding obtained as the product of the above constants shows good agreement with the results of equilibrium binding studies.     

Thermodynamics of 2,3-diphosphoglycerate association with human oxy- and deoxyhemoglobin

The association of 2,3-diphosphoglycerate with oxy- and deoxyhemoglobin was studied by means of ultrafiltration and microcalorimetry. It was found that in addition to parameters that are known to influence the binding of 2,3-diphosphoglycerate to both species of hemoglobin (such as pH, temperature and concentration of competing anion), the association is also strongly dependent on the hemoglobin concentration. The difference between the apparent association constants for the formation of the complex of the organic phosphate with oxy- and deoxyhemoglobin is relatively small. At pH 7.3, 25° C and 0.154 M chloride this difference is only 0.6 kcal/mole of free energy favoring the Hb·DPG complex. This free energy difference increases with decreasing pH but is not strongly affected by hemoglobin concentration. The enthalpy change for the formation of the 2,3-diphosphoglycerate complex with deoxyhemoglobin is 8–10 kcal/mole more exothermic than the complex with oxyhemoglobin.     

The association of human erythrocyte catalase with the cell membrane

A specific interaction of human erythrocyte catalase with the inner surface of the red cell membrane was demonstrated. The dependency of catalase affinity on pH and ionic strength implies that the interaction is dominated by electrostatic forces. Scatchard analysis of the binding at pH 6.0 and 5 mm Mes buffer reveals a single class of approximately 10⁶ binding sites/ghost with an association constant of 2.5 × 10⁷m^?1. The membrane-bound catalase retains its enzymatic activity. Competition binding studies of catalase and other proteins known to associate with the membrane inner surface were carried out. It was found that the binding of catalase is inhibited by aldolase, glyceraldehyde-3-phosphate dehydrogenase as well as by hemoglobin. The advantage of membrane-bound catalase in protection of the cell membrane against peroxidative damages is discussed.     

pH dependency of kinetic parameters and reaction mechanism of beef liver catalase.

The pH-dependence of the kinetic parameters in H2O2 decomposition by beef liver catalase was investigated. At pH 7.0, the ternary complex (ESS) decomposition rate was about 100 times faster than ESS formation (42 microM H2O2), and the value of the Michaelis constant was 0.025 M. From ethanol competition experiments, two different proton dissociation constants of the enzyme (pKe1 = 5.0, pKes2 = 5.9) were obtained for the binding of first and second H2O2 molecules. Another pKa value (pKes1) of 4.2 was obtained from the pH dependence of overall rate constant (ko). The reaction mechanism of catalase was discussed in relation to these ionizable groups.     

Kinetics of binding of bovine trypsin-killikrein inhibitor (K unitz) in which the reactive-site peptide bond Lys-15--Ala-16 is cleaved, to alpha-chymotrypsin and beta-trypsin.

Equilibrium measurements of the binding of reactive-site-cleaved (modified) bovine trypsin-kallikrein inhibitor (Kunitz) to alpha-chymotrypsin and beta-trypsin show a stoichiometric 1:1 association with high binding constants. At least in the case of chymotrypsin much evidence is presented that the reaction with modified inhibitor leads to the same complex as the reaction with virgin inhibitor does. The association rate constant of modified inhibitor with chymotrypsin at pH 7, 22.5 degrees C is 15.8 M-1 S-1. This is about 2 x 10(4) times slower than the binding of virgin inhibitor to that enzyme. In the analogous reaction of modified inhibitor with beta-trypsin, however, the association rate constant (1.2 x 10(4) M-1 s-1 at pH 6.9, 22.5 degrees C) is of about the same order of magnitude as it is in the reaction of virgin inhibitor and trypsin. These and analogous phenomena observed in the reactions of virgin and modified soybean trypsin inhibitor (Kunitz) with alpha-chymotrypsin and beta-trypsin suggest that the specificity of both inhibitors to trypsin is strongly reflected in the association rate constants of the modified forms. The dissociation rate constants of the complexes of trypsin-kallikrein inhibitor with chymotrypsin or with trypsin towards the modified inhibitor are estimated to be unmeasurably slow (half-life times of 45 or 1.5 x 10(4) years, respectively).     

Role of isomerization of initial complexes in the binding of inhibitors to dihydrofolate reductase

Stopped-flow measurements of protein fluorescence quenching when methotrexate (MTX) binds to dihydrofolate reductase (isoenzyme II) of Streptococcus faecium (SFDHFR II) analyze as the sum of two differentials: a rapid binding phase and a second phase for which the observed rate constant is independent of methotrexate concentration. Analysis of variation of the ratio of the amplitude of the fast and slow phases with methotrexate concentration indicates that the second phase is an isomerization of the initial binary complex. At pH 7.3, the equilibrium constant for this isomerization is 21.9, and the forward and reverse rate constants are 0.57 and 0.026 s-1, respectively. Similar results were obtained for binding of 3-deazamethotrexate to SFDHFR II, but the forward rate constant is greater (2.9 s-1 at pH 7.3). The equilibrium constants for these isomerizations are pH independent, but the rate constants decrease as the pH is raised, probably due to deprotonation of one or more groups on the enzyme. Analysis of progress curves obtained by the development of inhibition when SFDHFR II is added last to reaction mixtures containing dihydrofolate, NADPH, and MTX gives an association constant for initial reactions of 4.3 X 10(7) M-1. Since a preliminary estimate of the association constant for the binding reaction is 7.6 X 10(5) M-1, this suggests an isomerization of the ternary complex(es) with an equilibrium constant of about 56. In addition, analysis of the progress of development of inhibition indicates a further very slow isomerization with equilibrium constant 419 and forward rate constant 2.6 min-1.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mechanism of binding of horse liver alcohol dehydrogenase and nicotinamide adenine dinucleotide

The binding of NAD+ to liver alcohol dehydrogenase was studied by stopped-flow techniques in the pH range from 6.1 to 10.9 at 25 degrees C. Varying the concentrations of NAD+ and a substrate analogue used to trap the enzyme-NAD+ complex gave saturation kinetics. The same maximum rate constants were obtained with or without the trapping agent and by following the reaction with protein fluorescence or absorbance of a ternary complex. The data fit a mechanism with diffusion-controlled association of enzyme and NAD+, followed by an isomerization with a forward rate constant of 500 s-1 at pH 8: E E-NAD+ *E-NAD+. The isomerization may be related to the conformational change determined by X-ray crystallography of free enzyme and enzyme-coenzyme complexes. Overall bimolecular rate constants for NAD+ binding show a bell-shaped pH dependence with apparent pK values at 6.9 and 9.0. Acetimidylation of epsilon-amino groups shifts the upper pK to a value of 11 or higher, suggesting that Lys-228 is responsible for the pK of 9.0. Formation of the enzyme-imidazole complex abolishes the pK value of 6.9, suggesting that a hydrogen-bonded system extending from the zinc-bound water to His-51 is responsible for this pK value. The rates of isomerization of E-NAD+ and of pyrazole binding were maximal at pH below a pK of about 8, which is attributable to the hydrogen-bonded system. Acetimidylation of lysines or displacement of zinc-water with imidazole had little effect on the rate of isomerization of the E-NAD+ complex.(ABSTRACT TRUNCATED AT 250 WORDS)     

Studies on lectins. XLIV. The pH dependence of lectin interactions with sugars as determined by affinity electrophoresis.

The pH dependence of association constants of the lectin-sugar complexes was determined by means of affinity electrophoresis. All the lectins studied (from the seeds of Dolichos biflorus, Glycine soja, Lens esculenta and Vicia cracca and of the fruiting body of Marasmius oreades) were characterized by a similar course of pH dependence of the association constants, with the maximum values at pH 7--9. For concanavalin A and the L-fucose binding Ulex europaeus lectin only the association constants at three selected pH values were determined. Concanavalin A does not interact with immobilized alpha-D-mannosyl residues at pH 2.3. The association constants vs. pH curves measured for lectins isolated from two different lentil varieties slightly differ in accordance with the differences observed in the interaction of these lectins with the Sephadex gel.     

Influence of various fluorescent and non-fluorescent labels on the kinetics of the complex formation of alpha-chymotrypsin with basic pancreatic trypsin inhibitor (Kunitz).

The association of alpha-chymotrypsin with basic pancreatic trypsin inhibitor was studied using extrinsic signals produced by fluorescent and nonfluorescent labels. The reactive dyes were covalently bound to the proteins in the complexed state, in which the binding region was protected. The signals were sufficiently large to measure the complex formation at protein concentrations of 10(-9)M by fluorescence and down to 10(-6)M by absorption. Therefore, the association and dissociation could be followed over a broad range of concentration. Good correspondence was observed between data which were obtained with different labels and with published values for the unlabeled proteins. Existing differences could be explained by different buffer conditions used by the different authors. Also the pH dependence of the dissociation rate constants was essentially unaltered by the introduction of the labels. The large signals allowed a direct measurement of the equilibrium constants of dissociation, even at high pH, at which they are in the range of 10(-8)M. The experimentally determined binding constants were in agreement with those calculated from the rate constants. The temperature dependence of the binding constants revealed a small positive and pH-dependent enthalpy change [deltaHo = 4.0 kcal/mol (16.7 kJ) at H 7.0[. The results prove that the labeling can be performed in such a way that the equilibrium and kinetic parameters of the system studied are not significantly influenced.     

Effect of pH on coenzyme binding to liver alcohol dehydrogenase.

1. The transient-state kinetics of ligand-displacement reactions have been analyzed. Methods based on this analysis have been used to obtain reliable estimates of on-velocity and off-velocity constants for coenzyme binding to liver alcohol dehydrogenase at different pH values between 6 and 10. 2. The rate of NADH dissociation from the enzyme shows no pronounced dependence on pH. The rate of NAD+ dissociation is controlled by a group with a pKa of 7.6, agreeing with the pKa reported to regulate the binding of certain inhibitory substrate analogues to the enzyme . NAD+ complex. 3. Critical experiments have been performed to test a recent proposal that on-velocity constants for the binding of NADH and NAD+ are controlled by proton equilibria exhibiting different pKa values. The results show that association rates for NADH and NAD+ exhibit the same pH dependence corresponding to a pKa of 9.2. Titrimetric evidence is presented indicating that the latter effect of pH derives from ionization of a group which affects the anion-binding capacity of the coenzyme-binding site.     

Soluble high molecular weight derivatives of pancreatic inhibitors. Kinetic-thermodynamic studies of inhibitors linked to differently charged matrices]

The effects of electrostatic charge of the matrix on the pH-dependence of interactions of commercial trypsin with preparations of pancreatic inhibitor modified by soluble polysaccharide coupling were studied. It was shown that the rate constants of trypsin association with native and modified pancreatic inhibitor preparations as well as the rate constants of dissociation of their complexes and, consequently, the inhibition constants are identical. The invariability of the rate constants for the association reaction after the increase in the molecular weight of pancreatic inhibitor may be probably accounted for by the fact that the limiting step of a stable trypsin-inhibitor complex formation is not controlled by diffusion. Thermal denaturation of pancreatic inhibitor preparations modified by binding to polysaccharides (pH 4.7--8.0, 97 degrees C) suggests an essential role of the negative charge of matrix in stabilization of the protein inhibitor globule.     

The kinetics of cyanide binding by human erythrocyte catalase

The kinetics of the binding reaction of cyanide by human erythrocyte catalase at 25 °C has been studied over the pH range 4.2 to 10.2 by means of temperature jump and stopped flow techniques. Catalase reacts with cyanide at a constant rate in the range pH 4.2 to 8.1 which decreases at higher pH. This is most simply explained by the reaction of catalase with unionized hydrogen cyanide molecules. The pH-independent rate constant for the formation of the catalase-cyanide complex is (1.3 ± 0.1) × 10⁶m^?1 s^?1. The association equilibrium constant and the dissociation rate constant for the catalase-cyanide complex were determined from the relaxation amplitudes of temperature jump experiments and by spectrophotometric titration and are (3.1 ± 0.2) × 10⁵m^?1 and 4.2 ± 0.6 s^?1, respectively in the pH-independent region.     

Metal binding to angiotensin converting enzyme: implications for the metal binding site

Angiotensin converting enzyme interacts with the chelator, 1,10-phenanthroline (OP) to form an OP-Zn-ACE ternary complex, which subsequently dissociates to OP-Zn and apoenzyme. The association and dissociation rate constants for the reaction OP + Zn-ACE in equilibrium OP-Zn-ACE have been determined and compared with those of known OP-metal complexes. Such constants were also used to calculate the rate constant for formation of the OP-Zn complex from OP-Zn-ACE. The rate of dissociation of zinc from ACE has been measured in the presence of EDTA (which acts only as a metal scavenger) as a function of chelator concentration, at different pH values, and with different buffers. The stability constant for the binding of zinc to apoACE log Kc = 8.2, determined by equilibrium dialysis using atomic absorption spectroscopy to assess metal concentration, is much smaller than that for Zn-carboxypeptidase A. Zn-thermolysin, or Zn-carbonic anhydrase. This weak binding is attributable to the zinc dissociation rate constant of ACE, 7.5 X 10(-3) sec-1 at pH 7.0, which is much greater than that of the other zinc metalloenzymes. These results lead to inferences regarding the metal binding site of ACE.     

Mechanistic studies of glutamine synthetase from Escherichia coli: kinetics of ADP and orthophosphate binding to the unadenylylated enzyme.

The kinetics of protein fluorescence change exhibited by ADP or orthophosphate addition to the Mg2+-or Mn2+-activated unadenylylated glutamine synthetase from Escherichia coli were studied. The kinetic patterns of these reactions are incompatible with a simple bimolecular binding process and a mechanism which required protein isomerization prior to substrate binding. They are consistent with a mechanism in which direct substrate binding is followed by a substrate-induced conformational change step, ES in equilibrium ES. At pH 7.0 and 15 degrees C, the association constants for the direct binding (K1) of ADP to MnE1.0 and of Pi to MnE1.0ADP are 3.9 X 10(4) and 2.28 X 10(2) M(-1), respectively. The association constant for the direct binding of ADP to MnE1.0Pi is 2.3 X 10(4) M(-1) at pH 7.0 and 19 degrees C. The deltaG degrees for the substrate-induced conformational step are -3.5 and -1.3 kcal mol(-1) due to ADP binding to MnE1.0Pi and MnE1.0, respectively, and -1.4 kcal mol(-1) due to Pi binding to MnE1.0ADP. Rate constants, k2 and k(-2), for the isomerization step are: 90 and 9.5 s(-1) for ADP binding to MnE1.0, 440 and 0.36 s(-1) for ADP binding to MnE1.0Pi, and 216 and 1.8 s(-1) for Pi binding to MnE1.0ADP. Due to low substrate affinity, the association constant for direct Pi binding to MnE1.0 was roughly estimated to be 230 M(-1) and k2 = 750 s(-1), k(-2) = 250 s(-1). At 9 degrees C and pH 7.0, the estimated association constants for the direct ADP binding to MgE1.0 and MgE1.0 Pi are 1.8 X 10(4) and 1.6 X 10(4) M(-1), respectively; and the rate constants for the isomerization step associated with the corresponding reaction are k2 = 550 s(-1), k(-2) = 500 s(-1), and k2 = 210 s(-1), k(-2) = 100 s(-1). From the kinetic analysis it is evident that the inability of Mn2+ to support biosynthetic activity of the unadenylylated enzyme is due to the slow rate of ADP release from the MnE1.0PiADP complex. In contrast the large k(-2) obtained for ADP release from the MgE1.0ADP or MgE1.0PiADP complex indicates that this step is not rate limiting in the biosynthesis of glutamine since the k catalysis obtained under the same conditions is 7.2 s(-1).     

Site selectivity in the binding of inorganic anions to serum transferrin

Equilibrium constants for the sequential binding of two anions at the specific metal-binding sites of apotransferrin have been measured by difference ultraviolet spectroscopy in 0.1 M N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (Hepes) at pH 7.4 and 25 degrees C. Log K1 values for phosphate, phosphite, sulfate, and arsenate fall in the narrow range of 3.5-4.0, while the log K1 for bicarbonate is 2.73. No binding is observed for nitrate, perchlorate, or borate. A dinegative charge appears to be the most important criterion for anion binding. Equilibrium constants have also been measured for binding of anions to both forms of mono(ferric)transferrin. There appears to be a very small site selectivity (0.2 to 0.4 log units) for phosphate, arsenate, and phosphite that favors binding to the N-terminal site, but there is no detectable selectivity for binding of sulfate or bicarbonate. Comparison of the binding affinities and anion selectivity with literature data on anion-binding to protonated macrocyles and cryptates strongly supports the existence of specific anion-binding sites on the protein. Binding constants were also measured in 0.01 M Hepes. The anionic sulfonate group of the buffer appears to have a small effect on anion binding.     

Nonequivalence of the metal binding sites in vanadyl-labeled human serum transferrin.

Vanadyl ion, VO(IV), has been used as an electron paramagnetic resonance (EPR) spin label to study the metal-binding properties of human serum transferrin in the presence of bicarbonate. Iron-saturated transferrin does not bind the vanadyl ion. Room temperature titrations of apotransferrin with VO(IV) as monitored by EPR indicate the extent of binding to be pH dependent, with a full 0.2 VO(IV) ions per transferrin molecule bound at pH 7.5 and 9, but only about 1.2 VO(IV) ions bound at pH 6. The EPR spectra of frozen solutions with or without 0.1 M NaCUO4 at 77 K show that there are two spectroscopically nonequivalent binding sites (A and B) with a slight difference in binding constants. One site (A site) exhibits essentially constant binding capacity in the pH range 6-9, but the other (B site) becomes less avialable as the pH is reduced below 7. Results with mixed Fe(III)-VO(IV) transferrin complexes suggest that iron shows a slight tendency to bind at the B site over the A site pH 7.5 and 9.0. Only the B site in both vanadyl and iron transferrins is perturbed by the presence of perchlorate.     

Interaction of bovine (BSA) and human (HSA) serum albumins with ionic surfactants: spectroscopy and modelling

The binding of several different categories of small molecules to bovine (BSA) and human (HSA) serum albumins has been studied for many years through different spectroscopic techniques to elucidate details of the protein structure and binding mechanism. In this work we present the results of the study of the interactions of BSA and HSA with the anionic sodium dodecyl sulfate (SDS), cationic cethyltrimethylammonium chloride (CTAC) and zwitterionic N-hexadecyl-N,N-dimethyl-3-ammonium-1-propanesulfonate (HPS) monitored by fluorescence spectroscopy of the intrinsic tryptophans at pH 5.0. Similarly to pH 7.0 and 9.0, at low concentrations, the interaction of BSA with these surfactants shows a quenching of fluorescence with Stern-Volmer quenching constants of (1.1+/-0.1)x10(4) M(-1), (3.2+/-0.1)x10(3) M(-1) and (2.1+/-0.1)x10(3) M(-1) for SDS, HPS and CTAC, respectively, which are associated to the 'effective' association constants to the protein. On the interaction of these surfactants with HSA, an opposite effect was observed as compared to BSA, i.e., an enhancement of fluorescence takes place. For both proteins, at low surfactant concentrations, a positive cooperativity was observed and the Hill plot model was used to estimate the number of surfactant binding sites, as well as the association constants of the surfactants to the proteins. It is worthy of notice that the binding constants for the surfactants at pH 5.0 are lower as compared to pH 7.0 and 9.0. This is probably due to fact that the protein at this acid pH is quite compact reducing the accessibility of the surfactants to the hydrophobic cavities in the binding sites. The interaction of myristic acid with both proteins shows a similar fluorescence behaviour, suggesting that the mechanism of the interaction is the same. Recently published crystallographic studies of HSA-myristate complex were used to perform a modelling study with the aim to explain the fluorescence results. The crystallographic structure reveals that a total of five myristic acid molecules are asymmetrically bound in the macromolecule. Three of these sites correspond to higher affinity ones and correlate with high association constants described in the literature. Our models for BSA and HSA with bound SDS suggest that the surfactant could be bound at the same sites as those reported in the crystal structure for the fatty acid. The differences in tryptophan vicinity upon surfactant binding are explored in the models in order to explain the observed spectroscopic changes. For BSA the quenching is due to a direct contact of a surfactant molecule with the indole of W131 residue. It is clear that the binding site in BSA which is very close, in contact with tryptophan W131, corresponds to a lower affinity site, explaining the lower binding constants obtained from fluorescence studies. In the case of HSA the enhancement of fluorescence is due to the removal of static quenching of W214 residue in the intact protein caused by nearby residues in the vicinity of this tryptophan.     

A two-state thermodynamic and kinetic analysis of the allosteric functioning of the haemoglobin of an extreme poikilotherm.

The blood of the extreme poikilotherm Trematomus borchgrevinki contains one major haemoglobin component, which may be separated from the minor species by ion-exchange chromatography. This haemoglobin shows co-operative CO-binding isotherms at pH 8.2. An analysis of the temperature-dependence of the binding curves has allowed the thermodynamic constants associated with the two-state allosteric parameters L, KR and KT to be measured. The binding of CO at lower pH (6.2) is characterized by the maintenance of the T-state to relatively high degrees of saturation. Kinetic investigations with the use of flash photolysis of the haemoglobin-CO complex under various conditions has allowed the determination of the thermodynamic parameters association with the T-state and R-state rate constants. An amalgamation of these data has allowed the mathematical simulation of predicted time courses for CO binding to this haemoglobin, by using the two-state model, which very closely represents those obtained experimentally. The overall findings show that this haemoglobin closely follows the two-state model of co-operative interaction.