Redox control in heme proteins: electrostatic substitution in the active site of leghemoglobin

The effects of electrostatic substitutions on the spectroscopic, ligand binding, and redox properties of the heme in leghemoglobin have been examined by replacement of the proximal leucine 88 residue with an aspartic acid residue (Leu88Asp). Electronic and resonance Raman spectra of the ferric derivative of Leu88Asp indicate a mixture of 6-coordinate, high-spin and 6-coordinate, low-spin hemes, analogous to that observed in the recombinant wild-type protein (rLb). At alkaline pH, formation of hydroxide-bound heme is indicated for Leu88Asp; the pK(a) for this transition (8.7 +/- 0.2, micro = 0.10 M, 25.0 degrees C) is 0.4 pH units higher than for rLb. Equilibrium dissociation constants (sodium phosphate, pH 7.0, micro = 0.10 M, 25.0 +/- 0.1 degrees C) for binding of anionic ligands (N(-)(3), nicotinate) to Leu88Asp are higher (K(d,nicotinate) = 6.8 +/- 0.2 microM; K(d,azide) = 33 +/- 0.6 microM) than the corresponding values for rLb (K(d,nicotinate) = 1.4 +/- 0.3 microM (pH 5.5, micro = 0.10 M, 25.0 +/- 0.1 degrees C); K(d,azide) = 4.8 +/- 0.2 microM). Resonance Raman spectra (sodium phosphate, pH 7.0, micro = 0.10 M) for the ferrous derivatives of Leu88Asp and rLb exhibit a strong nu(Fe-His) stretching frequency at 223 cm(-1) in both cases, indicating that the hydrogen bonding structure on the proximal side is not substantially altered in the variant. The reduction potential of Leu88Asp is -14 +/- 2 mV vs standard hydrogen electrode (SHE) (25.0 degrees C, micro = 0.10 M, pH 7.0), a decrease of 35 mV over the corresponding value for the wild-type protein under the same conditions (21 +/- 3 mV vs SHE). An assessment of these data in terms of electrostatic and hydrogen bonding considerations is presented.     

Two uncompetitive, activated, and transport sites of the Na+/H+ exchanger for pH regulation in perfused rat kidney

The purpose of this study is to assess the effect of an apparent alteration in intracellular pH and the effect of amiloride on the activity of the Na+/H+ antiporter in perfused rat kidney. Rat kidney-Na+ retention was determined using tracer 22Na in perfusate composed of HCl-glycine buffer (pH 3.80 to pH 5.92) or NH4OH-glycine buffer (pH 6.22-7.95) containing Na+ to match physiologic concentrations. Plotting renal Na+ retention for 10 min versus pH in absence of amiloride showed two classical uncompetitive activator curves for H+, one curve from pH 4.19 to 5.10 and another from pH 6.22 to 7.95. H+ acts as an uncompetitive reversible binding substrate with the receptor triggering activation of the exchanger already sequestered with Na+, thus yielding two Ka values for the exchanger suggesting non-first order kinetics. Using an equation derived for uncompetitive-activation binding of Nao+ and Hi+, plotting [mM Na+ mg protein-1 10 min-1]-1 versus [H+], two linear plots are observed on Cartesian coordinates with abscissa intersecting at 47 +/- 1 microM, pKa = 4.32 +/- 0.02 (pH 4.19-5.10) and 4.21 +/- 0.02 microM, pKa = 5.38 +/- 0.01 (pH 6.22-7.95), respectively. Perfusing buffer containing 2 mM amiloride, completely inactivated the antiporter showing stronger inhibition between pH 3.80 and 5.92. Results suggest the presence of two uncompetitive binding sites for H+ with the Na+/H+ exchanger. One is a high affinity binding site at physiological intracellular apparent pH, and another is a low affinity binding site at ischaemic apparent pH, implying the existence of two titration sites for intracellular pH regulation.     

Kinetics and equilibria of cyanide binding to prostaglandin H synthase

Cyanide binding to prostaglandin H (PGH) synthase results in a spectral shift in the Soret region. This shift was exploited to determine equilibrium and kinetic parameters of the cyanide binding process. At pH 8.0, ionic strength 0.22 M, 4 degrees C, the cyanide dissociation constant, determined from equilibrium experiments, is (65 +/- 10) microM. The binding rate constant is (2.8 +/- 0.2) x 10(3) M-1 s-1, and the dissociation rate constant is zero within experimental error. Through a kinetic study of the binding process as a function of pH, from pH 3.96 to 8.00, it was possible to determine the pKa of a heme-linked acid group on the enzyme of 4.15 +/- 0.10 with citrate buffer. An apparent pKa of 4.75 +/- 0.03 was determined with acetate buffer; this different value is attributed to complexation of the enzyme with one of the components of the acetate buffer.     

Role of histidine 42 in ascorbate peroxidase. Kinetic analysis of the H42A and H42E variants.

To examine the role of the distal His42 residue in the catalytic mechanism of pea cytosolic ascorbate peroxidase, two site-directed variants were prepared in which His42 was replaced with alanine (H42A) or glutamic acid (H42E). Electronic spectra of the ferric derivatives of H42A and H42E (pH 7.0, mu = 0.10 m, 25.0 degrees C) revealed wavelength maxima [lambda(max) (nm): 397, 509, approximately equal to 540(sh), 644 (H42A); 404, 516, approximately equal to 538(sh), 639 (H42E)] consistent with a predominantly five-co-ordinate high-spin iron. The specific activity of H42E for oxidation of L-ascorbate (8.2 +/- 0.3 U.mg(-1)) was approximately equal to 30-fold lower than that of the recombinant wild-type enzyme (rAPX); the H42A variant was essentially inactive but activity could be partially recovered by addition of exogenous imidazoles. The spectra of the Compound I intermediates of H42A [lambda(max) (nm) = 403, 534, 575(sh), 645] and H42E [lambda(max) (nm) = 404, 530, 573(sh), 654] were similar to those of rAPX. Pre-steady-state data for formation of Compound I for H42A and H42E were consistent with a mechanism involving accumulation of a transient enzyme intermediate (K(d)) followed by conversion of this intermediate into Compound I (k'(1)). Values for k'(1) and K(d) were, respectively, 4.3 +/- 0.2 s(-1) and 30 +/- 2.0 mM (H42A) and 28 +/- 1.0 s(-1) and 0.09 +/- 0.01 mM (H42E). Photodiode array experiments for H42A revealed wavelength maxima for this intermediate at 401 nm, 522 nm and 643 nm, consistent with the formation of a transient [H42A-H(2)O(2)] species. Rate constants for Compound I formation for H42A were independent of pH, but for rAPX and H42E were pH-dependent [pKa = 4.9 +/- 0.1 (rAPX) and pK(a) = 6.7 +/- 0.2 (H42E)]. The results provide: (a) evidence that His42 is critical for Compound I formation in APX; (b) confirmation that titration of His42 controls Compound I formation and an assignment of the pK(a) for this group; (c) mechanistic and spectroscopic evidence for an intermediate before Compound I formation; (d) evidence that a glutamic acid residue at position 42 can act as the acid-base catalyst in ascorbate peroxidase.     

Haloalkane dehalogenases: steady-state kinetics and halide inhibition

The substrate specificities and product inhibition patterns of haloalkane dehalogenases from Xanthobacter autotrophicus GJ10 (XaDHL) and Rhodococcus rhodochrous (RrDHL) have been compared using a pH-indicator dye assay. In contrast to XaDHL, RrDHL is efficient toward secondary alkyl halides. Using steady-state kinetics, we have shown that halides are uncompetitive inhibitors of XaDHL with 1, 2-dichloroethane as the varied substrate at pH 8.2 (Cl-, Kii = 19 +/- 0.91; Br-, Kii = 2.5 +/- 0.19 mM; I-, Kii = 4.1 +/- 0.43 mM). Because they are uncompetitive with the substrate, halide ions do not bind to the free form of the enzyme; therefore, halide ions cannot be the last product released from the enzyme. The Kii for chloride was pH dependent and decreased more than 20-fold from 61 mM at pH 8.9 to 2.9 mM at pH 6.5. The pH dependence of 1/Kii showed simple titration behavior that fit to a pKa of approximately 7.5. The kcat was maximal at pH 8.2 and decreased at lower pH. A titration of kcat versus pH also fits to a pKa of approximately 7.5. Taken together, these data suggest that chloride binding and kcat are affected by the same ionizable group, likely the imidazole of a histidyl residue. In contrast, halides do not inhibit RrDHL. The Rhodococcus enzyme does not contain a tryptophan corresponding to W175 of XaDHL, which has been implicated in halide ion binding. The site-directed mutants W175F and W175Y of XaDHL were prepared and tested for halide ion inhibition. Halides do not inhibit either W175F or W175Y XaDHL.     

Catalytic oxidation of p-cresol by ascorbate peroxidase

Transient and steady state kinetics, together with a range of chromatographic and spectroscopic techniques, have been used to establish the mechanism and the products of the H(2)O(2)-dependent oxidation of p-cresol by ascorbate peroxidase (APX). HPLC, GC-MS, and NMR analyses are consistent with the formation of 2, 2'-dihydroxy-5,5'-dimethylbiphenyl (II) and 4alpha,9beta-dihydro-8, 9beta-dimethyl-3(4H)-dibenzofuranone (Pummerer's ketone, III) as the major products of the reaction. In the presence of cumene hydroperoxide, two additional products were observed which, from GC and MS analyses, were shown to be 1,1-dimethylbenzylalcohol (IV) and bis-(1-methyl-1-phenyl-ethyl)-peroxide (V). The product ratio II:III was dependent on enzyme concentration: at low concentrations Pummerer's ketone (III) predominates and at high concentrations formation of the biphenyl compound (II) is favored. Steady-state data showed a sigmoidal dependence on [p-cresol] that was consistent with the presence of 2.01 +/- 0.15 binding sites for the substrate (25.0 degrees C, sodium phosphate, pH 7.0, mu = 2.2 mM) and independent of ionic strength in the range 2.2-500 mM. Single turnover kinetic experiments (pH 7.0, 5.0 degrees C, mu = 0.10 M) yielded second-order rate constants for Compound I reduction by p-cresol, k(2), of 5.42 +/- 0.10 x 10(5) M(-1) s(-1), respectively. Rate-limiting reduction of Compound II by p-cresol, k(3), showed saturation kinetics, giving values for K(d) = 1.54 +/- 0.12 x 10(-3) M and k(3) = 18.5 +/- 0.7 s(-1). The results are discussed in the more general context of APX-catalyzed aromatic oxidations.     

Exploiting the conformational flexibility of leghemoglobin: a framework for examination of heme protein axial ligation

We have exploited the intrinsic conformational flexibility of leghemoglobin to reengineer the heme active site architecture of the molecule by replacement of the mobile His61 residue with tyrosine (H61Y variant). The electronic absorption spectrum of the ferric derivative of H61Y is similar to that observed for the phenolate derivative of the recombinant wild-type protein (rLb), consistent with coordination of Tyr61 to (high-spin) iron. EXAFS data clearly indicate a 6-coordinate heme geometry and a Fe-O bond length of 185pm. MCD and EPR spectroscopies are consistent with this assignment and support ligation by an anionic (tyrosinate) group. The alteration in heme ligation leads to a 148mV decrease in the reduction potential for H61Y (-127+/-5mV) compared to rLb and destabilisation of the functional oxy-derivative. The results are discussed in terms of our wider understanding of other heme proteins with His-Tyr ligation.     

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.     

Effect of ionic strength and pH on the activity of pyruvate dehydrogenase complex from pig kidney cortex.

The activity of pyruvate dehydrogenase complex (PDC) purified from pig kidney cortex is sensitive to changes in ionic strength (mu). At low ionic strength (mu = 0.04 M) the specific activity of PDC was 12.22 mumol/min/mg, whereas at high ionic strength (mu = 0.15 M) the measured activity of the complex decreased to 4.88 mumol/min/mg. The optimum activity of PDC was achieved within a small range of ionic strength, mu = 0.035-0.040 M. Increasing the ionic strength from mu = 0.05 to mu = 0.15 M decreased the s0.5 for pyruvate from 125 to 72 microM and increased the Hill coefficient from 1.0 to 1.3. The effect of pH on PDC activity also was dependent upon ionic strength. At pH 7.2 the activity of PDC at mu = 0.05 and mu = 0.15 M was 90 and 55% of the maximal activity, respectively. Furthermore, the effects of Na+, K+, HCO3-, Cl-, and HPO4(2-) on PDC activity were dependent on ionic strength and pH. The addition of K+ (80 mM) at mu = 0.10 and mu = 0.15 M increased the activity of PDC by 12 and 42%, respectively. Lowering the pH from 8.2 to 7.5 resulted in a decrease in the s0.5 for pyruvate from 179 to 110 microM and from 110 to 35 microM in the presence and absence of K+ (80 mM), Na+ (20 mM), Cl- (20 mM), HCO3- (20 mM), and HPO4(2-) (10 mM), respectively. The observed changes in the properties of PDC in response to changes in ionic strength likely was a result of changes in the intramolecular electrostatic interactions within the complex. In this regard it was determined using two-dimensional agarose gel electrophoresis of the intact multienzyme complex that increasing the ionic strength to which PDC is exposed decreased the measured radius of PDC and may have decreased the electronegative surface charge of the complex.     

Kinetic, thermodynamic and statistical studies on the inhibition of adenosine deaminase by aspirin and diclofenac

The kinetic and thermodynamic effects of aspirin and diclofenac on the activity of adenosine deaminase (ADA) were studied in 50 mM phosphate buffer pH = 7.5 at 27 and 37 degrees C, using UV-Vis spectrophotometry and isothermal titration calorimetry (ITC). Aspirin exhibits competitive inhibition at 27 and 37 degrees C and the inhibition constants are 42.8 and 96.8 microM respectively, using spectrophotometry. Diclofenac shows competitive behavior at 27 degrees C and uncompetitive at 37 degrees C with inhibition constants of 56.4 and 30.0 microM, at respectively. The binding constant and enthalpy of binding, at 27 degrees C are 45 microM, - 64.5 kJ/mol and 61 microM, - 34.5 kJ/mol for aspirin and diclofenac. Thermodynamic data revealed that the binding process for these ADA inhibitors is enthalpy driven. QSAR studies by principal component analysis implemented in SPSS show that the large, polar, planar, and aromatic nucleoside and small, aromatic and polar non-nucleoside molecules have lower inhibition constants.     

Binding of thiocyanate to lactoperoxidase: 1H and 15N nuclear magnetic resonance studies

The binding of thiocyanate to lactoperoxidase (LPO) has been investigated by 1H and 15N NMR spectroscopy. 1H NMR of LPO shows that the major broad heme methyl proton resonance at about 61 ppm is shifted upfield by addition of the thiocyanate, indicating binding of the thiocyanate to the enzyme. The pH dependence of line width of 15N resonance of SC15N- in the presence of the enzyme has revealed that the binding of the thiocyanate to the enzyme is facilitated by protonation of an ionizable group (with pKa of 6.4), which is presumably distal histidine. Dissociation constants (KD) of SC15N-/LPO, SC15N-/LPO/I-, and SC15N-/LPO/CN- equilibria have been determined by 15N T1 measurements and found to be 90 +/- 5, 173 +/- 20, and 83 +/- 6 mM, respectively. On the basis of these values of KD, it is suggested that the iodide ion inhibits the binding of the thiocyanate but cyanide ion does not. The thiocyanate is shown to bind at the same site of LPO as iodide does, but the binding is considerably weaker and is away from the ferric ion. The distance of 15N of the bound thiocyanate ion from the iron is determined to be 7.2 +/- 0.2 A from the 15N T1 measurements.     

Manganese (II) And spin-labeled uridine 5'-diphosphate binding to bovine galactosyltransferase.

The kinetically observed Mn(II) activation as well as inhibition has been clarified for bovine galactosyltransferase. An electron spin resonance (ESR) titration of MnCl2 with galactosyltransferase alone at pH 8.0 clearly shows the existence of at least two metal ion binding sites with microscopic dissociation constants of 0.84 +/- 0.1 and 9.0 +/- 1.0 mM, respectively. The second site corresponds with either published kinetic constant for Mn(II) of 8.5 mM (inhibition) or 3.40 mM (activation). The contribution of the binary complex Mn(II)-UDPGal is of lesser significance, as concluded by its ESR measured Kdiss of 14.5 +/- 1.1 mM at pH 8.0. A spin-labeled inhibitor analog of UDPgalactose, UDP-4-O-(2,2,6,6-tetramethyl-4-piperidinyl-1-oxy), or UDP-R, was synthesized as a competitive inhibitor for UDPGal. It was shown from inhibition kinetics to be almost as potent an inhibitor as UDPGlu. The Ki values at pH 8.0 in the N-acetyllactosamine and lactose reactions were 0.38 +/- 0.04 and 0.63 +/- 0.06 mM, respectively, as compared with 0.10 +/- 0.01 and 0.094 +/- 0.009 mM for UDPGlu. An ESR titration of UDP-R with galactosyltransferase at pH 8.0 yielded direct physical dissociation constants of 0.40 +/- 0.07 and 0.53 +/- 0.08 mM in the absence and presence of alpha-lactalbumin, respectively. No other substrates (glucose of N-acetylglucosamine) nor Mn(II) were present.     

Observation and characterization of the interaction between a single immunoglobulin binding domain of protein L and two equivalents of human kappa light chains

Detailed stopped-flow studies in combination with site-directed mutagenesis, isothermal titration calorimetry data and x-ray crystallographic knowledge have revealed that the biphasic pre-equilibrium fluorescence changes reported for a single Ig-binding domain of protein L from Peptostreptococcus magnus binding to kappa light chain are due to the binding of the kappa light chain at two separate sites on the protein L molecule. Elimination of binding site 2 through the mutation A66W has allowed the K(d) for kappa light chain binding at site 1 to be measured by stopped-flow fluorescence and isothermal titration calorimetry techniques, giving values of 48.0 +/- 8.0 nM and 37.5 +/- 7.3 nM respectively. Conversely, a double mutation Y53F/L57H eliminates binding at site 1 and has allowed the K(d) for binding at site 2 to be determined. Stopped-flow fluorimetry suggests this to be 3.4 +/- 0.8 microM in good agreement with the value of 4.6 +/- 0.8 microM determined by isothermal titration calorimetry. The mutation Y53F reduces the affinity of site 1 to approximately that of site 2.     

Kinetics and energetics of the binding between barley alpha-amylase/subtilisin inhibitor and barley alpha-amylase 2 analyzed by surface plasmon resonance and isothermal titration calorimetry

The kinetics and energetics of the binding between barley alpha-amylase/subtilisin inhibitor (BASI) or BASI mutants and barley alpha-amylase 2 (AMY2) were determined using surface plasmon resonance and isothermal titration calorimetry (ITC). Binding kinetics were in accordance with a 1:1 binding model. At pH 5.5, [Ca(2+)] = 5 mM, and 25 degrees C, the k(on) and k(off) values were 8.3 x 10(+4) M(-1) s(-1) and 26.0 x 10(-4) s(-1), respectively, corresponding to a K(D) of 31 nM. K(D) was dependent on pH, and while k(off) decreased 16-fold upon increasing pH from 5.5 to 8.0, k(on) was barely affected. The crystal structure of AMY2-BASI shows a fully hydrated Ca(2+) at the protein interface, and at pH 6.5 increase of [Ca(2+)] in the 2 microM to 5 mM range raised the affinity 30-fold mainly due to reduced k(off). The K(D) was weakly temperature-dependent in the interval from 5 to 35 degrees C as k(on) and k(off) were only increasing 4- and 12-fold, respectively. A small salt dependence of k(on) and k(off) suggested a minor role for global electrostatic forces in the binding and dissociation steps. Substitution of a positively charged side chain in the mutant K140L within the AMY2 inhibitory site of BASI accordingly did not change k(on), whereas k(off) increased 13-fold. ITC showed that the formation of the AMY2-BASI complex is characterized by a large exothermic heat (Delta H = -69 +/- 7 kJ mol(-1)), a K(D) of 25 nM (27 degrees C, pH 5.5), and an unfavorable change in entropy (-T Delta S = 26 +/- 7 kJ mol(-1)). Calculations based on the thermodynamic data indicated minimal structural changes during complex formation.     

AMP interaction sites in glycogen phosphorylase b. A thermodynamic analysis

The binding of AMP to activator site N and to inhibitor site I in glycogen phosphorylase b has been characterized by calorimetry, potentiometry and ultracentrifugation in the pH range 6.5-7.5 at 25 degrees C (mu = 0.1). Calorimetric titration data of phosphorylase b with adenosine 5'-phosphoramidate are also reported at pH 6.9 (T = 25 degrees C, mu = 0.1). Calorimetric curves have been analyzed on the basis of potentiometric and sedimentation velocity results to determine thermodynamic quantities for AMP binding to the enzyme. The comparison of calorimetric titration data of AMP and adenosine 5'-phosphoramidate at pH 6.9 supports the hypothesis previously suggested that the dianionic phosphate form of the nucleotide preferentially binds to the allosteric activator site. The thermodynamic parameters for AMP binding to site N are as follows: delta G0 = -22 kJ mol-1, delta H0 = -34 kJ mol-1 and delta S0 = -40 J mol-1 K-1. The binding of the nucleotide to site I was found to be strongly dependent on the pH. This behaviour may be explained in terms of coupled protonations of three groups having pKa values of 6.0, 6.0 and 6.1 in the unbound form and 7.0, 7.5 and 7.2 in the enzyme-nucleotide complex. The thermodynamic parameters for nucleotide binding to site I for the enzymatic form in which all the modified groups are completely deprotonated or protonated have been calculated to be: delta G0 = -7.7 kJ mol-1, delta H0 = -28 kJ mol-1 and delta S0 = -68 J mol-1 K-1 and delta G0 = -28 kJ mol-1, delta H0H = -10 kJ mol-1 and delta S0H = 61 J mol-1 K-1, respectively. These results suggest that attractive dispersion forces are of primary significance for AMP binding to activator site N, although electrostatic interactions act as a stabilizing factor in the nucleotide binding. The protonation states of those residues of which the pKa values are modified by AMP binding to site I highly influence the thermodynamic parameters for the nucleotide binding to this site.     

Adenine nucleotides affect the binding of 3-phosphoglycerate to pig muscle 3-phosphoglycerate kinase

Pig muscle 3-phosphoglycerate kinase was complexed with 1-anilino-8-naphthalenesulfonate (ANS) in order to monitor the binding of substrates to the enzyme. The enzyme-dye interaction did not influence the enzymic activity under the experimental conditions used. By measuring the substrate-dependent change in the fluorescence emission of ANS molecules tightly bound to the enzyme (Kd less than or equal to 0.05 mM), fluorimetric titrations were carried out in 0.1 M Tris/HCl buffer pH 7.5, containing 5 mM mercaptoethanol, at 20 degrees C. The dissociation constants obtained for the separate bindings of 3-phosphoglycerate, MgATP, 1,3-bisphosphoglycerate and MgADP were 0.03 +/- 0.01 mM, 0.15 +/- 0.10 mM, 0.00005 +/- 0.00001 mM and 0.15 +/- 0.10 mM respectively. binding of 3-phosphoglycerate is weakened when MgATP is also bound to the enzyme: the dissociation constant of 3-phosphoglycerate in this ternary complex (0.25 +/- 0.08 mM) is comparable to its Km value (0.38 +/- 0.10 mM). The same weakening can be observed in the non-productive ternary complexes where MgATP is replaced by MgADP (Kd = 0.20 +/- 0.10 mM) or AMP (Kd = 0.12 +/- 0.05 mM), whereas adenosine has no such effect. This indicates the importance of the negatively charged phosphate(s) of nucleotides in influencing the binding of 3-phosphoglycerate. In contrast to 3-phosphoglycerate, the binding of the substrate analogue, glycerol 3-phosphate is practically not affected by the presence of MgATP: the dissociation constant to the free enzyme (0.40 +/- 0.10 mM) is comparable to its inhibitory constant (0.70 +/- 0.20 mM). This finding and the similarity of the dissociation constant of glycerol 3-phosphate binding (0.40 +/- 0.10 mM) and the Km value of 3-phosphoglycerate (0.38 +/- 0.10 mM) suggest that, during the enzymic reaction, binding of 3-phosphoglycerate occurs probably without involvement of the carboxyl group.     

Binding of NAD+ to pertussis toxin.

The equilibrium dissociation constant of NAD+ and pertussis toxin was determined by equilibrium dialysis and by the quenching of the protein's intrinsic fluorescence on titration with NAD+. A binding constant, Kd, of 24 +/- 2 microM at 30 degrees C was obtained from equilibrium dialysis, consistent with the previously determined value for the Michaelis constant, Km, of 30 +/- 5 microM for NAD+ (when the toxin is catalysing the ADP-ribosylation of water and of dithiothreitol). The intrinsic fluorescence of pertussis toxin was quenched by up to 60% on titration with NAD+, and after correction for dilution and inner filter effects, a Kd value of 27 microM at 30 degrees C was obtained, agreeing well with that found by equilibrium dialysis. The binding constants were measured at a number of temperatures using both techniques, and from this the enthalpy of binding of NAD+ to toxin was determined to be 30 kJ.mol-1, a typical value for a protein-ligand interaction. There is one binding site for NAD+ per toxin molecule.     

Determination of the affinity of each component of a composite quaternary transition-state analogue complex of creatine kinase

Recombinant rabbit muscle creatine kinase (CK) was titrated with MgADP in 50 mM Bicine and 5 mM Mg(OAc)2, pH 8.3, at 30.0 degrees C by following a decrease in the protein's intrinsic fluorescence. In the presence of 50 mM NaOAc, but in the absence of added creatine or nitrate, MgADP has an apparent K(d) of 135 +/- 7 microM, and the total change in fluorescence on saturation (Delta%F) is 15.3 +/- 0.3%. Acetate was used as the anion in this experiment because it does not promote the formation of a CK.MgADP.anion.creatine transition-state analogue complex (TSAC) [Millner-White and Watts (1971) Biochem. J. 122, 727-740]. In the presence of 80 mM creatine, but no nitrate, the apparent K(d) for MgADP remains essentially unchanged at 132 +/- 10 microM, while Delta%F decreases slightly to 13.2 +/- 0.3%. In the presence of 10 mM nitrate, but no creatine, the apparent K(d) is once again essentially unchanged at 143 +/- 23 microM, but the Delta%F is markedly reduced to 4.2 +/- 0.2%. The presence of both 10 mM nitrate and 80 mM creatine during titration reduces the apparent K(d) for MgADP 10-fold to 13.7 +/- 0.7 microM, and Delta%F increases to 20.6 +/- 0.3%, strongly suggesting that the simultaneous presence of saturating levels of creatine and nitrate increases the affinity of CK for MgADP and promotes the formation of the enzyme*MgADP*nitrate*creatine TSAC. When the fluorescence of CK was titrated with MgADP in the presence of 80 mM creatine and fixed saturating concentrations of various anions, apparent K(d) values for MgADP of 132 +/- 10 microM, 25.2 +/- 1.3 microM, 18.8 +/- 0.9 microM, 13.7 +/- 0.7 microM, and 6.4 +/- 0.7 microM were observed as the anion was changed from acetate to formate to chloride to nitrate to nitrite, respectively. This is the same trend reported by Millner-White and Watts for the effectiveness of various monovalent anions in forming the CK.MgADP.anion.creatine TSAC. On titration of CK with MgADP in the presence of 80 mM creatine and various fixed concentrations of NaNO3, the apparent K(d) for MgADP decreases with increasing fixed concentrations of nitrate. A plot of the apparent K(d) for MgADP vs [NO3-] suggests a K(d) for nitrate from the TSAC of 0.39 +/- 0.07 mM. Similarly, titration with MgADP in the presence of 10 mM NaNO3 and various fixed concentrations of creatine gives a value of 0.9 +/- 0.4 mM for the dissociation of creatine from the TSAC. The data were used to calculate K(TDAC), the dissociation constant of the quaternary TSAC into its individual components, of 3 x 10(-10) M3. To our knowledge this is the first reported dissociation constant for a ternary or quaternary TSAC.     

The ferric form of the three human embryonic hemoglobins and their reactions with azide ions

The ferric forms of the three human embryonic hemoglobins exhibit pH titration's with pKa values in the range 8.25-8.6, which correlate with the reduced proteins affinity for oxygen. Azide ions bind to each ferric protein in a process consisting of two separate binding steps. The equilibrium constants for this process are in the 10-30 microM range and can be assigned to the individual chain within the proteins as reactivity appears independent of the nature of the partner chain. The kinetics of the azide binding reactions occur on the second time scale at mM ligand concentrations and also indicate the presence of two distinct processes that have been assigned to each of the two chains within the proteins. Non of these processes indicate any evidence of heme-heme interactions within the ferric form of the proteins. These findings are discussed in comparison with previously reported properties of adult ferric hemoglobin.     

Multiple role of hydrophobicity of tryptophan-108 in chicken lysozyme: structural stability, saccharide binding ability, and abnormal pKa of glutamic acid-35.

Trp108 of chicken lysozyme is in van der Waals contact with Glu35, one of two catalytic carboxyl groups. The role of Trp108 in lysozyme function and stability was investigated by using mutant lysozymes secreted from yeast. By the replacement of Trp108 with less hydrophobic residues, Tyr (W108Y lysozyme) and Gln (W108Q lysozyme), the activity, saccharide binding ability, stability, and pKa of Glu35 were all decreased with a decrease in the hydrophobicity of residue 108. Namely, at pH 5.5 and 40 degrees C, the activities of W108Y and W108Q lysozymes against glycol chitin were 17.3 and 1.6% of that of wild-type lysozyme, and their dissociation constants for the binding of a trimer of N-acetyl-D-glucosamine were 7.4 and 309 times larger than that of wild-type lysozyme, respectively. For the reversible unfolding at pH 3.5 and 30 degrees C, W108Y and W108Q lysozymes were less stable than wild-type lysozyme by 1.4 and 3.6 kcal/mol, respectively. As for the pKa of Glu35, the values for W108Y and W108Q lysozymes were found to be lower than that for wild-type lysozyme by 0.2 and by 0.6 pKa unit, respectively. The pKa of Glu35 in lysozyme was also decreased from 6.1 to 5.4 by the presence of 1-3 M guanidine hydrochloride, or to 5.5 by the substitution of Asn for Asp52, another catalytic carboxyl group. Thus, both the hydrophobicity of Trp108 and the electrostatic interaction with Asp52 are equally responsible for the abnormally high pKa (6.1) of Glu35, compared with that (4.4) of a normal glutamic acid residue.(ABSTRACT TRUNCATED AT 250 WORDS)