Transition of hemoglobin between two tertiary conformations: The transition constant differs significantly for the major and minor hemoglobins of the Japanese quail (Cortunix cortunix japonica)

We demonstrate that 5,5'-dithiobis(2-nitrobenzoate) - DTNB - reacts with only CysF9[93]beta and CysB5[23]beta among the multiple sulfhydryl groups of the major and minor hemoglobins of the Japanese quail (Cortunix cortunix japonica). K(equ), the equilibrium constant for the reaction, does not differ very significantly between the two hemoglobins. It decreases 430-fold between pH approximately 5.6 and pH approximately 9: from a mean of 7+/-1 to a mean of 0.016+/-0.003. Quantitative analyses of the K(equ) data based on published X-ray and temperature-jump evidence for a tertiary structure transition in liganded hemoglobin enable the calculation of K(rt), the equilibrium constant for the r<---->t tertiary structure transition. K(rt) differs significantly between the two hemoglobins: 0.744+/-0.04 for the major, 0.401+/-0.01 for the minor hemoglobin. The mean pK(a)s of the two groups whose ionizations are coupled to the DTNB reaction are about the same as previously reported for mammalian hemoglobins.     

Reversible reaction of 5,5'-dithiobis(2-nitrobenzoate) with the hemoglobins of the domestic cat: acetylation of NH3+ terminal group of the beta chain transforms the complex pH dependence of the forward apparent second order rate constant to a simple form

We demonstrate kinetically that the reaction of 5,5'-dithiobis(2-nitrobenzoate) with the CysF9[93]beta sulfhydryl group of domestic cat hemoglobins is a reversible process. In the major hemoglobin, in which the NH3+ terminal group of GlyNA1[1]beta is free, kf, the apparent forward second order rate constant, has a complex pH dependence profile. In the minor hemoglobin, the NH3+ terminal group of SerNA1[1]beta is acetylated, and the pH dependence profile of kf is simple. These results support the proposal that the positively charged groups at the organic phosphate binding site are electrostatically linked to CysF9[93]beta. Quantitative analyses of the complex profiles enabled us to estimate pKas of 7.47 +/- 0.3; 6.53 +/- 0.03 and 8.49 +/- 0.3 for GlyNA1[1]beta, HisH21[143]beta and other histidines within 2 nm of the sulfhydryl, and CysF9[93]beta, respectively, of the major hemoglobin. Analyses of the simple profiles gave pKas of 6.33 +/- 0.17 and 8.54 +/- 0.5 for HisH21[143]beta and other histidines within a distance of 2 nm of the sulfhydryl, and CysF9[93]beta of the minor hemoglobin, respectively.     

Reversible reaction of 5,5'-dithiobis(2-nitrobenzoate) with the CysF9[93]beta sulfhydryl groups of the hemoglobins of the domestic cat: variation of the equilibrium and reverse rate constants with pH

We have determined for the first time the equilibrium constant, Keq, for the reaction of Ellman's reagent, 5,5'-dithiobis(2-nitrobenzoate), with the CysF9[93]beta sulfhydryl groups of the hemoglobins of the domestic cat. In the pH range 5.6 to 9.0 Kequ varies over four orders of magnitude--between ca 10 and 10(-3)--for all hemoglobin derivatives. Using these Kequ values and published data on the dependence of the apparent second order forward rate constant, kf, on pH we have calculated the apparent second order reverse rate constant, kr, as a function of pH. This parameter increases strongly with pH, particularly above pH 7.5. Quantitative analyses of the pH dependence profiles of log10kr indicate that the reverse reaction is coupled to the ionization of two groups on the protein with pKas of 7.2+/-0.2 and 9.4+/-0.1 in the major hemoglobin and 6.7+/-0.3 and 8.4+/-0.1 in the minor hemoglobin.     

Ionizable groups linked to the reaction of 2,2'-dithiobispyridine with hemoglobin.

The pH-dependence of the second-order rate-constant for the reaction of 2,2'-dithiobispyridine with the CysF9(93) beta sulphydryl group of hemoglobin in the R quaternary structure is analyzed in terms of a tentative model based on the observation that this sulphydryl exists as a mixture of two tertiary conformations in dynamic equilibrium. For the four aquomethemoglobins studied (human A and S, dog and rabbit), the equation derived from this model gives a better fit than a simpler equation based on the assumption of only one tertiary conformation. For the corresponding carbonmonoxyhemoglobins the simpler equation gives a better fit. The dog and rabbit oxy and azidomet data are better fitted by the model equation, whereas the data for the corresponding human A and S derivatives are better fitted by the simpler equation. From the analysis pKa values of 6.1 and 8.7 are obtained for the ionization of groups coupled to the presumed conformational transition. The pKa of 6.1 is assigned to HisHC3(146) beta; the pKa of 8.7 is assigned to the CysF9(93) beta sulphydryl group in its external conformation. It is estimated that the pKa of this sulphydryl may be as high as 12.9 in its internal conformation.     

Temperature-jump studies on hemoglobin. Kinetic evidence for a non-quaternary isomerization process in deoxy- and carbonmonoxyhemoglobin

Temperature jumps on mixtures of hemoglobin and pH indicators give rise to relaxation signals in the microsecond range. The pH and concentration dependences of the reciprocal relaxation time, 1/tau, may be rationalized on the basis of a reaction scheme in which a slow isomerization process in the protein moiety is coupled to a rapid co-operative ionization of two protons. At 11 degrees C the rate constants of the isomerization are kr = 4.2(+/- 1.8) x 10(4) s-1 and kf = 1.3(+/- 0.1) x 10(4) s-1 in deoxyhemoglobin; in carbonmonoxyhemoglobin they are kr = 3.9(+/- 1.3) x 10(4) s-1 and kf = 5.3(+/- 1.8) x 10(3) s-1. The pKa values of the coupled ionizing groups are 5.3 in deoxy- and 6.0 in carbonmonoxyhemoglobin. Modification of the CysF9(93) beta sulfhydryl group with iodoacetamide abolishes the pH dependence of 1/tau, suggesting that this sulfhydryl is involved in the isomerization process. Consideration of the X-ray structure of oxyhemoglobin allows a structural interpretation of the results. It is concluded that the isomerization may be important for the physiological function of hemoglobin, but that it is not identical with the quaternary structure transition.     

Speciation study of the anti-inflammatory drug tenoxicam (Htenox) with Cu(II): X-ray crystal structure of [Cu(tenox)(2)(py)(2)].EtOH

A speciation study was carried out in aqueous solution of the anti-inflammatory drug tenoxicam (Htenox), under quasi-physiological conditions (temperature of 37 degrees C and ionic strength 0.15 M NaCl) in order to determine the acidity constants from spectrophotometric studies, the pK(a) values found being pK(1)=1.143+/-0.008 and pK(2)=4.970+/-0.004. Subsequently, the spectrophotometrical speciation of the different complexes of Cu(II) with the drug was performed under the same conditions of temperature and ionic strength, observing the formation of Cu(Htenox)(2)(2+) with log beta(212)=20.05+/-0.01, Cu(tenox)(2) with log beta(012)=13.6+/-0.1, Cu(Htenox)(2+) with log beta(111)=10.52+/-0.08, as well as Cu(tenox)(+) with log beta(011)=7.0+/-0.2, all of them in solution, and solid species Cu(tenox)(2)(s) with an estimated value of log beta(012)(s) approximately 18.7. The crystalline structure of the complex [Cu(tenox)(2)(py)(2)]. EtOH, was also determined, and it was observed that tenoxicam employs the oxygen of the amide group and the pyridyl nitrogen to bond to the cation.     

Varying apparent rate constant: determination of uptake and release of protons during tetramer-dimer dissociation in human hemoglobin A

The effect of association-dissociation on the sulphydryl reactivity of human hemoglobin A is reported. The reactivity of CysF9(93)beta towards the sulphydryl reagent, 5,5'-dithiobis(2-nitrobenzoate), is higher at lower concentrations of hemoglobin at all pH values. This is because hemoglobin dimers have higher sulphydryl reactivity than tetramers and it is known that the proportion of dimers increases as the hemoglobin concentration decreases. This study takes advantage of this observation to determine the tetramer-dimer dissociation constant, K(4,2), of hemoglobin A and subsequently the proton uptake and the proton release during this process. The concentration dependence profiles of the apparent second-order rate constants, k(app), show that (between 2 and 20 microM heme) k(app) decreases with increasing hemoglobin concentration. Above 30 M heme k(app) remains fairly constant for all hemoglobin derivatives (oxy, carbonmonoxy and aquomethemoglobin) used. The pH dependence of the negative logarithm of tetramer-dimer dissociation constant, pK(4,2), for oxy- (and for carbonmonoxy-) hemoglobin exhibits a biphasic character with a maximum near pH 7.4 (and 6.6). For aquomethemoglobin, pK(4,20 decreases with increasing pH. The tetramer-dimer dissociation of human oxyhemoglobin A at an ionic strength of 200 mM uptakes 0.87 +/- 0.09 mole of protons between pH 6.2 to 7.4 phase and releases 0.84 0.09 mole of protons between pH 7.4 and 9.0 phase. Under a similar condition carbonmonoxyhemoglobin uptakes 0.54 +/- 0.05 mole of protons between pH 5.8 and 6.6 phase and releases 0.48 +/- 0.05 mole of protons between pH 6.6 and 9.0 phase. Aquomethemoglobin has only a single phase, it releases 0.39 +/- 0.05 mole of protons during tetramer-dimer dissociation.     

The [Ru(Hedta)NO](0.1-) system: structure, chemical reactivity and biological assays

The [Ru(II)(Hedta)NO(+)] complex is a diamagnetic species crystallizing in a distorted octahedral geometry, with the Ru-N(O) length 1.756(4) A and the RuNO angle 172.3(4) degrees . The complex contains one protonated carboxylate (pK(a)=2.7+/-0.1). The [Ru(II)(Hedta)NO(+)] complex undergoes a nitrosyl-centered one-electron reduction (chemical or electrochemical), with E(NO+/NO)=-0.31 V vs SCE (I=0.2 M, pH 1), yielding [Ru(II)(Hedta)NO](-), which aquates slowly: k(-NO)=2.1+/-0.4x10(-3) s(-1) (pH 1.0, I=0.2 M, CF(3)COOH/NaCF(3)COO, 25 degrees C). At pHs>12, the predominant species, [Ru(II)(edta)NO](-), reacts according to [Ru(II)(edta)NO](-)+2OH(-)-->[Ru(II)(edta)NO(2)](3-), with K(eq)=1.0+/-0.4 x 10(3) M(-2) (I=1.0 M, NaCl; T=25.0+/-0.1 degrees C). The rate-law is first order in each of the reactants for most reaction conditions, with k(OH(-))=4.35+/-0.02 M(-1)s(-1) (25.0 degrees C), assignable mechanistically to the elementary step comprising the attack of one OH(-) on [Ru(II)(edta)NO](-), with subsequent fast deprotonation of the [Ru(II)(edta)NO(2)H](2-) intermediate. The activation parameters were DeltaH(#)=60+/-1 kJ/mol, DeltaS(#)=-31+/-3 J/Kmol, consistent with a nucleophilic addition process between likely charged ions. In the toxicity up-and-down tests performed with Swiss mice, no death was observed in all the doses administered (3-9.08 x 10(-5) mol/kg). The biodistribution tests performed with Wistar male rats showed metal in the liver, kidney, urine and plasma. Eight hours after the injection no metal was detected in the samples. The vasodilator effect of [Ru(II)(edta)NO](-) was studied in aortic rings without endothelium, and was compared with sodium nitroprusside (SNP). The times of maximal effects of [Ru(II)(edta)NO](-) and SNP were 2 h and 12 min, respectively, suggesting that [Ru(II)(edta)NO](-) releases NO slowly to the medium in comparison with SNP.     

Kinetics and thermodynamics of complex formation with iron of a new series of dicatecholspermidine siderophore-like ligands

This article deals with the kinetics and thermodynamics of complex formation between Fe(3+) and a series of four synthetic chelators of the 1,2-dicatecholspermidine family (LA5, LA3, LE5 and LE3). LA5 and LA3 bear a carboxylic moiety linked to the central nitrogen by either a C(5) or a C(3) chain, whereas LE5 and LE3 bear an ethyl ester moiety. The following data concern LE5, LE3, LA5 and LA3, respectively. Each species undergoes four acid-base dissociations of the hydroxyls of the catechols with, for the two hydroxyls in position 1; average pK(2a)=7.30, 7.25, 7.45, 7.34 and, for the two hydroxyl in position 2; average pK(3a)=12.35, 12.65, 12.10, 12.60. The LA5 and LA3 species also undergo proton-dissociations of their carboxylic moieties; pK(1a)=5.20 and 5.10. The four species form one-to-one iron complexes with, for the 1-hydroxyl; an average pK(22a)=2.65, 2.25, 2.95, 2.80, for the 2-hydroxyl; pK(33a)=5.20, 5.40, 6.10, 5.40 and, for the carboxylic moieties; pK(11a)=3.90 and 4.45. In the vicinity of pH 5, Fe(3+) is rapidly exchanged between FeNta and the four ligands. This occurs with direct rate constants: k(1)=(1.3+/-0.1)x10(4), (1.4+/-0.2)x10(4), (3.3+/-0.2)x10(4), (1.4+/-0.1)x10(4)M(-1)s(-1), and reverse rate constants: k(-1)=(7+/-0.5)x10(4), (9+/-1)x10(4), (1.15+/-0.15)x10(5), (7+/-0.5)x10(4)M(-1)s(-1). The kinetic data, the pK(a) values of the free ligands, those of the iron complexes and the beta value of FeNta allow us to determine the affinity constants of the four ligands for iron: logbeta(1)=33, 34, 33, 34, and pFe=23.3, 24.6, 22.2, 24.3. This implies that these ligands of the dicatecholspermidine family may act as siderophores. They may also be used as drug carriers which can utilize the bacterial iron-acquisition paths.     

Unraveling the catalytic mechanism of nitrile hydratases

To elucidate a detailed catalytic mechanism for nitrile hydratases (NHases), the pH and temperature dependence of the kinetic constants k(cat) and K(m) for the cobalt-type NHase from Pseudonocardia thermophila JCM 3095 (PtNHase) were examined. PtNHase was found to exhibit a bell-shaped curve for plots of relative activity versus pH at pH 3.2-11 and was found to display maximal activity between pH 7.2 and 7.8. Fits of these data provided pK(E)(S1) and pK(E)(S2) values of 5.9 +/- 0.1 and 9.2 +/- 0.1 (k(cat)' = 130 +/- 1 s(-1)), respectively, and pK(E)(1) and pK(E)(2) values of 5.8 +/- 0.1 and 9.1 +/- 0.1 (k(cat)'/K(m)' = (6.5 +/- 0.1) x 10(3) s(-1) mm(-1)), respectively. Proton inventory studies indicated that two protons are transferred in the rate-limiting step of the reaction at pH 7.6. Because PtNHase is stable at 60 degrees C, an Arrhenius plot was constructed by plotting ln(k(cat)) versus 1/T, providing E(a) = 23.0 +/- 1.2 kJ/mol. The thermal stability of PtNHase also allowed DeltaH(0) ionization values to be determined, thus helping to identify the ionizing groups exhibiting the pK(E)(S1) and pK(E)(S2) values. Based on DeltaH(0)(ion) data, pK(E)(S1) is assigned to betaTyr(68), whereas pK(E)(S2) is assigned to betaArg(52), betaArg(157), or alphaSer(112) (NHases are alpha(2)beta(2)-heterotetramers). A combination of these data with those previously reported for NHases and synthetic model complexes, along with sequence comparisons of both iron- and cobalt-type NHases, allowed a novel catalytic mechanism for NHases to be proposed.     

pH-dependent Soret difference spectra of the deoxy and carbonmonoxy forms of human hemoglobin and its derivatives.

The transition from deoxy to oxystructure of hemoglobin A (Hb) is accompanied by the breaking of the salt bridges formed by C-terminal residues in deoxy-Hb. This, in turn, changes the state of the heme. The switch between these different allosteric forms can be followed by changes in the optical absorbance spectra (Perutz, M. F., Ladner, J. E., Simon, S. R., and Ho, C. (1974), Biochemistry 13, 2163). Using difference spectroscopy in the soret region, pH-dependent spectral changes of Hb and its derivatives (carbamylated at both the alpha-NH2 groups, alpha2cbeta2c; N-ethylsuccinimide hemoglobin, NES-Hb) in their deoxy and carbonmonoxy forms were measured. From these measurements, the pK values of histidine-146beta and valine-1alpha in deoxy-Hb were determined to be 8.6 +/- 0.2 and 7.7 +/- 0.1, respectively. In carbonmonoxy-Hb a pK value of 6.3 +/- 0.1 was found.     

A study of conformational changes in two beta-93 modified hemoglobin A's using a triphosphate spin label.

The binding of oxygen and 1-oxyl-2,2,6,6-tetramethylpiperidine 4-triphosphate (spin-labeled triphosphate) to normal adult human hemoglobin (HbA) covalently labeled at the beta-93 sulfhydryl groups with N-(2,2,6,6-tetramethyl-4-piperidinyl)iodoacetamide (I) was studied. HbA-I was used as a model for HbA labeled at the beta-93 SH groups with N-(1-oxyl-2,2,6,6-tetramethyl-4-piperidinyl)iodoacetamide (II) since the binding of SLTP to HbA-II could not be measured conveniently, in the presence of the paramagnetic resonance signal of II. Both HbA-I and HbA-II can be treated as variant hemoglobins with abnormal beta chains. The oxygen and SLTP binding data from HbA-I and oxygen binding data from HbA-II are consistent with a concerted transition model for cooperativity which assumes nonequivalence between alpha and beta subunits (GCT model). The distribution of environments "seen" by conformation sensitive probes such as II and trifluoracetone (19F NMR probe) attached to the beta-93 sulfhydryl groups of HbA can also be accounted for by the GCT model. It is proposed that the beta-93 probes sense the dramatic change in beta subunit structure resulting from the quaternary structure change (T leads to R) upon heme saturation as well as tertiary structure changes at the alpha1-beta2 contact region resulting from ligand binding to the beta-heme group. Structural changes caused by ligation of the alpha-hemes are not discussed.     

The immune response to GHRH, relationship to conformation

Antibodies to GHRH1-44, GHRH1-29, and proinsulin were induced in guinea pigs. GHRH1-44 forms 7 S and 10 S complexes with antibodies. It is a divalent antigen. The sequence 30-44 bound 85% of the antibodies to GHRH1-44 with high affinity (3.8 +/- 0.9 x 10(9) l/mol). The fragment 1-29 bound with low affinity (0.6 +/- 0.3 x 10(9) l/mol) 15% of the antibodies (2p less than 0.001). Antibodies to GHRH1-29 had low affinity towards the native hormone (0.4 +/- 0.2 x 10(9) l/mol) and the region 1-29 (0.3 +/- 0.2 x 10(9) l/mol). Antibodies to proinsulin bound linear C-peptide with lower affinity (0.3 +/- 0.2 x 10(9) l/mol) than the C-peptide loop in proinsulin (3.4 +/- 0.9 x 10(9) l/mol). It is concluded that the conformation of the epitopes on the sequence 1-29, recognized during the immune response, i.e. on the cell membrane, is different from the conformation of GHRH1-29 or GHRH1-44 in aqueous solution.     

Symmetry distortion in the human hemoglobin tetramer induced by asymmetric ligation

To investigate the conformational changes in human tetrameric (αβ)(2) hemoglobin upon binding of the first two ligands, we have measured the kinetics of reactions between 4,4'-dithiodipyridine and β93Cys sulfhydryl groups of four diliganded hemoglobins by using CO-bound Fe(II)-Ni(II) hybrids with and without β-β cross-linking. The data show that all the diliganded intermediates have high sulfhydryl reactivities, which are greater than or equal to that for the fully-liganded end state, especially when containing liganded α subunit(s). The results also reveal that both the asymmetrically (α1β1 and α1β2) diliganded species show similar high rates of sulfhydryl reactivity and biphasic kinetics, suggesting a new conformation but only slight functional distortion caused by asymmetric ligation.     

Cooperative oxygen binding, subunit assembly, and sulfhydryl reaction kinetics of the eight cyanomet intermediate ligation states of human hemoglobin.

Correlations between the energetics of cooperativity and quaternary structural probes have recently been made for the intermediate ligation states of Hb [Daugherty et al. (1991) Proc. Natl. Acad. Sci. US 88, 1110-1114]. This has led to a "molecular code" which translates configurations of the 10 ligation states into switch points of quaternary transition according to a "symmetry rule"; T-->R quaternary structure change is governed by the presence of at least one heme-site ligand on each of the alpha beta dimeric half-molecules within the tetramer [see Ackers et al. (1992) Science 255, 54-63, for summary]. In order to further explore this and other features of the cooperative mechanism, we have used oxygen binding to probe the energetics and cooperativities for the vacant sites of the cyanomet ligation species. We have also probed structural aspects of all eight cyanomet ligation intermediates by means of sulfhydryl reaction kinetics. Our oxygen binding results, obtained from a combination of direct and indirect methods, demonstrate the same combinatorial aspect to cooperativity that is predicted by the symmetry rule. Overall oxygen affinities of the two singly-ligated species (alpha +CN beta)(alpha beta) and (alpha beta +CN)(alpha beta) were found to be identical (pmedian = 2.4 Torr). In contrast, the doubly-ligated species exhibited two distinct patterns of oxygen equilibria: the asymmetric species (alpha +CN beta +CN)(alpha beta) showed very high cooperativity (nmax = 1.94) and low affinity (pmedian = 6.0 Torr), while the other three doubly-ligated species showed diminished cooperativity (nmax = 1.23) and considerably higher oxygen affinity (pmedian = 0.4 Torr). Extremely high oxygen affinities were found for the triply-ligated species (alpha +CN beta +CN)(alpha beta +CN) and (alpha +CN beta +CN)(alpha +CN beta) (pmedian = 0.2 Torr). Their oxygen binding free energies are considerably more favorable than those of the alpha and beta subunits within the dissociated alpha beta dimer, demonstrating directly the quaternary enhancement effect, i.e., enhanced oxygen affinity at the last binding step of tetramer relative to the dissociated protomers. Oxygen binding free energies measured for the alpha subunit within the isolated (alpha beta +CN) dimer and for the beta subunit within the isolated (alpha +CN beta) dimer sum to the free energy for binding two oxygens to normal hemoglobin dimers (-16.3 +/- 0.2 versus -16.7 +/- 0.2, respectively), arguing against cooperativity in the isolated dimer. Correlations were established between cooperative free energies of the 10 cyanomet ligation microstates and the kinetics for reacting their free sulfhydryl groups.(ABSTRACT TRUNCATED AT 400 WORDS)     

Jack bean (Canavalia ensiformis) urease. Probing acid-base groups of the active site by pH variation.

A pH-variation study of jack bean (Canavalia ensiformis) urease steady-state kinetic parameters and of the inhibition constant of boric acid, a urease competitive inhibitor, was performed using both noninhibitory organic (MES, HEPES and CHES) and inhibitory inorganic (phosphate) buffers, in an effort to elucidate the functions exercised in the catalysis by the ionizable groups of the enzyme active site. The results obtained are consistent with the requirement for three groups utilized by urease with pK(a)s equal to 5.3+/-0.2, 6.6+/-0.2 and 9.1+/-0.4. Based on the appearance of the ionization step with pK(a)=5.3 in v(max)-pH, K(M)-pH and K(i)-pH profiles, we assigned this group as participating both in the substrate binding and catalytic reaction. As shown by its presence in v(max)-pH and K(M)-pH curves, the obvious role of the group with pK(a)=9.1 is the participation in the catalytic reaction. One function of the group featuring pK(a)=6.6, which was derived from a two-maxima v(max)-pH profile obtained upon increasing phosphate buffer concentration, an effect the first time observed for urease-phosphate systems, is the substrate binding, another possible function being modulation of the active site structure controlled by the ionic strength. It is also possible that the pK(a)=6.6 is a merger of two pK(a)s close in value. The study establishes that regular bell-shaped activity-pH profiles, commonly reported for urease, entail more complex pH-dependent behavior of the urease active site ionizable groups, which could be experimentally derived using species interacting with the enzyme, in addition to changing solution pH and ionic strength.     

Basis for the equilibrium constant in the interconversion of l-lysine and l-beta-lysine by lysine 2,3-aminomutase

l-beta-lysine and beta-glutamate are produced by the actions of lysine 2,3-aminomutase and glutamate 2,3-aminomutase, respectively. The pK(a) values have been titrimetrically measured and are for l-beta-lysine: pK(1)=3.25 (carboxyl), pK(2)=9.30 (beta-aminium), and pK(3)=10.5 (epsilon-aminium). For beta-glutamate the values are pK(1)=3.13 (carboxyl), pK(2)=3.73 (carboxyl), and pK(3)=10.1 (beta-aminium). The equilibrium constants for reactions of 2,3-aminomutases favor the beta-isomers. The pH and temperature dependencies of K(eq) have been measured for the reaction of lysine 2,3-aminomutase to determine the basis for preferential formation of beta-lysine. The value of K(eq) (8.5 at 37 degrees C) is independent of pH between pH 6 and pH 11; ruling out differences in pK-values as the basis for the equilibrium constant. The K(eq)-value is temperature-dependent and ranges from 10.9 at 4 degrees C to 6.8 at 65 degrees C. The linear van't Hoff plot shows the reaction to be enthalpy-driven, with DeltaH degrees =-1.4 kcal mol(-1) and DeltaS degrees =-0.25 cal deg(-1) mol(-1). Exothermicity is attributed to the greater strength of the bond C(beta)-N(beta) in l-beta-lysine than C(alpha)-N(alpha) in l-lysine, and this should hold for other amino acids.     

Energetic basis of molecular recognition in a DNA aptamer

The thermal stability and ligand binding properties of the L-argininamide-binding DNA aptamer (5'-GATCGAAACGTAGCGCCTTCGATC-3') were studied by spectroscopic and calorimetric methods. Differential calorimetric studies showed that the uncomplexed aptamer melted in a two-state reaction with a melting temperature T(m)=50.2+/-0.2 degrees C and a folding enthalpy DeltaH(0)(fold)=-49.0+/-2.1 kcal mol(-1). These values agree with values of T(m)=49.6 degrees C and DeltaH(0)(fold)=-51.2 kcal mol(-1) predicted for a simple hairpin structure. Melting of the uncomplexed aptamer was dependent upon salt concentration, but independent of strand concentration. The T(m) of aptamer melting was found to increase as L-argininamide concentrations increased. Analysis of circular dichroism titration data using a single-site binding model resulted in the determination of a binding free energy DeltaG(0)(bind)=-5.1 kcal mol(-1). Isothermal titration calorimetry studies revealed an exothermic binding reaction with DeltaH(0)(bind)=-8.7 kcal mol(-1). Combination of enthalpy and free energy produce an unfavorable entropy of -TDeltaS(0)=+3.6 kcal mol(-1). A molar heat capacity change of -116 cal mol(-1) K(-1) was determined from calorimetric measurements at four temperatures over the range of 15-40 degrees C. Molecular dynamics simulations were used to explore the structures of the unligated and ligated aptamer structures. From the calculated changes in solvent accessible surface areas of these structures a molar heat capacity change of -125 cal mol(-1) K(-1) was calculated, a value in excellent agreement with the experimental value. The thermodynamic signature, along with the coupled CD spectral changes, suggest that the binding of L-argininamide to its DNA aptamer is an induced-fit process in which the binding of the ligand is thermodynamically coupled to a conformational ordering of the nucleic acid.     

Cloning and characterization of histamine dehydrogenase from Nocardioides simplex

Histamine dehydrogenase (NSHADH) can be isolated from cultures of Nocardioides simplex grown with histamine as the sole nitrogen source. A previous report suggested that NSHADH might contain the quinone cofactor tryptophan tryptophyl quinone (TTQ). Here, the hdh gene encoding NSHADH is cloned from the genomic DNA of N. simplex, and the isolated enzyme is subjected to a full spectroscopic characterization. Protein sequence alignment shows NSHADH to be related to trimethylamine dehydrogenase (TMADH: EC 1.5.99.7), where the latter contains a bacterial ferredoxin-type [4Fe-4S] cluster and 6-S-cysteinyl FMN cofactor. NSHADH has no sequence similarity to any TTQ containing amine dehydrogenases. NSHADH contains 3.6+/-0.3 mol Fe and 3.7+/-0.2 mol acid labile S per subunit. A comparison of the UV/vis spectra of NSHADH and TMADH shows significant similarity. The EPR spectrum of histamine reduced NSHADH also supports the presence of the flavin and [4Fe-4S] cofactors. Importantly, we show that NSHADH has a narrow substrate specificity, oxidizing only histamine (K(m)=31+/-11 microM, k(cat)/K(m)=2.1 (+/-0.4)x10(5)M(-1)s(-1)), agmatine (K(m)=37+/-6 microM, k(cat)/K(m)=6.0 (+/-0.6)x10(4)M(-1)s(-1)), and putrescine (K(m)=1280+/-240 microM, k(cat)/K(m)=1500+/-200 M(-1)s(-1)). A kinetic characterization of the oxidative deamination of histamine by NSHADH is presented that includes the pH dependence of k(cat)/K(m) (histamine) and the measurement of a substrate deuterium isotope effect, (D)(k(cat)/K(m) (histamine))=7.0+/-1.8 at pH 8.5. k(cat) is also pH dependent and has a reduced substrate deuterium isotope of (D)(k(cat))=1.3+/-0.2.     

Association of the AB and CD-EF domains from rat alpha- and beta-parvalbumin

Association of the parvalbumin AB and CD-EF domains was examined in Hepes-buffered saline, pH 7.4, employing fragments from rat alpha and beta. All of the interactions require Ca(2+). In saturating Ca(2+), the alpha AB/alpha CD-EF (alpha/alpha) complex displays an association constant of (7.6 +/- 0.4) x 10(7) M(-1). Ca(2+)-binding data for a mixture of the alpha fragments are compatible with an identical two-site model, yielding an average binding constant of (8.5 +/- 0.2) x 10(5) M(-1). The beta/beta interaction is significantly weaker, exhibiting an association constant of (3.0 +/- 0.6) x 10(6) M(-1). The Ca(2+)-binding constants for beta/beta are likewise diminished, at (1.0 +/- 0.1) x 10(5) and (2.3 +/- 0.2) x 10(4) M(-1). The magnitude of the apparent DeltaDeltaG(degree)' for Ca(2+) binding by alpha/alpha and beta/beta, at 3.4 kcal/mol, approaches that measured for the intact proteins (3.6 kcal/mol) and is substantially larger than the 1.5 kcal/mol value previously measured for the isolated CD-EF domains. This result suggests that the AB domain can modulate the Ca(2+) affinities of the CD and EF sites. Interestingly, the heterologous alpha/beta complex displays a larger association constant [(6.6 +/- 0.4) x 10(6) M(-1)] than the homologous beta/beta complex and heightened Ca(2+) affinity [binding constants of (1.3 +/- 0.1) x 10(6) and (8.8 +/- 0.2) x 10(4) M(-1)]. By contrast, beta/alpha associates more weakly than alpha/alpha and exhibits sharply reduced affinity for Ca(2+). Thus, the interaction between the beta AB domain and beta CD-EF domain may act to attenuate Ca(2+) affinity in the intact protein.