Reaction of ferrous lactoperoxidase with hydrogen peroxide and dioxygen: an anaerobic stopped-flow study

Lactoperoxidase (LPO) is found in mucosal surfaces and exocrine secretions including milk, tears, and saliva and has physiological significance in antimicrobial defense which involves (pseudo-)halide oxidation. LPO compound III (a ferrous-dioxygen complex) is known to be formed rapidly by an excess of hydrogen peroxide and could participate in the observed catalase-like activity of LPO. The present anaerobic stopped-flow kinetic analysis was performed in order to elucidate the catalytic mechanism of LPO and the kinetics of compound III formation by probing the reactivity of ferrous LPO with hydrogen peroxide and molecular oxygen. It is shown that ferrous LPO heterolytically cleaves hydrogen peroxide forming water and oxyferryl LPO (compound II). The two-electron oxidation reaction follows second-order kinetics with the apparent bimolecular rate constant being (7.2+/-0.3) x 10(4) M(-1) s(-1) at pH 7.0 and 25 degrees C. The H2O2-mediated conversion of compound II to compound III follows also second-order kinetics (220 M(-1) s(-1) at pH 7.0 and 25 degrees C). Alternatively, compound III is also formed by dioxygen binding to ferrous LPO at an apparent bimolecular rate constant of (1.8+/-0.2) x 10(5) M(-1) s(-1). Dioxygen binding is reversible and at pH 7.0 the dissociation constant (K(D)) of the oxyferrous form is 6 microM. The rate constant of dioxygen dissociation from compound III is higher than conversion of compound III to ferric LPO, which is not affected by the oxygen concentration and follows a biphasic kinetics. A reaction cycle including the redox intermediates compound II, compound III, and ferrous LPO is proposed, which explains the observed (pseudo-)catalase activity of LPO in the absence of one-electron donors. The relevance of these findings in LPO catalysis is discussed.     

Biphasic recombination of photodissociated CO compound of cytochrome o(s) from Vitreoscilla

The soluble cytochrome o from Vitreoscilla contains two identical subunits and two hemes. The reduced form binds 2 mol of CO in a cooperative manner with a Hill coefficient near 2 (Tyree, B., and Webster, D. A. (1978) J. Biol. Chem. 253, 6988-6991). This carbonyl compound was photolysed with a dye laser and recombination followed at 437 or 420 nm where maximal absorbance changes were registered. Recombination kinetics were biphasic, and the fast phase was approximately 10 times the rate of the slow phase. Apparent rate constants of both phases showed a nonlinear dependence on CO concentration, respectively, in conformity with a reaction scheme which assumes the transient formation of an intermediate species in both slow and fast reactions. A study of temperature dependence of the reactions gave EA = 2.7 kcal/mol for the slow reaction and EA = 3.2 kcal/mol for the fast reaction below 23 degrees C; above this temperature the slope of the Arrhenius plot for the fast reaction became positive. Maximal rates for both phases were around pH 6.5 and fell to approximately 40% of maximal at pH 12. The binding reaction was affected by even a low concentration of sodium dodecyl sulfate (0.0025%), which changed both the kinetic constant of each phase and the relative contribution of each phase to the reaction. A model which assumes the existence of fast and slow reaction conformers in equilibrium is proposed.     

Effect of bezafibrate and clofibric acid on the spectroscopic properties of the nitric oxide derivative of ferrous human hemoglobin.

The effect of bezafibrate (BZF) and clofibric acid (CFA) on the spectroscopic (EPR and absorbance) properties of the nitric oxide derivative of ferrous human hemoglobin (HbNO) has been investigated quantitatively. In the presence of BZF and CFA, the X-band EPR spectra and the absorption spectra in the Soret region of HbNO display the same basic characteristics described in the presence of inositol hexakisphosphate (IHP) and 2, 3-diphosphoglycerate (2,3-DPG). Next, in the presence of these allosteric effectors, the oxygen affinity for ferrous human hemoglobin (Hb) is reduced. These findings indicate that BZF and CFA, as already reported for IHP and 2, 3-DPG, induce the stabilization of a low affinity conformation of the ligated hemoprotein (i.e., HbNO). Values of the apparent equilibrium constant for BZF and CFA binding to HbNO (K) are 1.5(+/- 0.2) x 10(-2) M and 2.8(+/- 0.3) x 10(-2) M, respectively, at pH 7.0 (in 0.1 M N-[2-hydroxyethyl]piperazine-N'-[2-ethanesulfonic acid]/NaOH buffer system plus 0.1 M NaCl) and 20 degrees C. The results reported here represent clearcut evidence for BZF and CFA specific (i.e., functionally relevant) binding to a ligated derivative of Hb (i.e., HbNO).     

Kinetic studies on thermal denaturation of C-phycocyanin

The kinetics of thermal denaturation of a biliprotein, C-phycocyanin (C-PC) isolated from Spirulina platensis were studied at different pH values, ranging from 4.0 to 8.0. The denaturation of C-PC follows the first order kinetics and rate constant at pH 5.0 and temperature 55 degrees C is found to be 4.37 x 10(-5) s(-1), which increases to 5.46 x 10(-1) s(-1) at pH 7.0. The denaturation rate is much higher at 65 degrees C and pH 7.0 (7.96 x 10(-4)), as compared to at pH 5.0 (1.46 x 10(-4)). The thermal stability of C-PC is more at pH 5.0, as compared to other pH values. The observed differences in entropy values at pH 5.0, as compared to other pH values indicate a considerably close fit structure of the protein at pH 5.0, which increases the stability of native structure, even at higher temperature (65 degrees C).     

Anticooperative ligand binding properties of recombinant ferric Vitreoscilla homodimeric hemoglobin: a thermodynamic, kinetic and X-ray crystallographic study.

Thermodynamics and kinetics for cyanide, azide, thiocyanate and imidazole binding to recombinant ferric Vitreoscilla sp. homodimeric hemoglobin (Vitreoscilla Hb) have been determined at pH 6.4 and 7.0, and 20.0 degrees C, in solution and in the crystalline state. Moreover, the three-dimensional structures of the diligated thiocyanate and imidazole derivatives of recombinant ferric Vitreoscilla Hb have been determined by X-ray crystallography at 1.8 A (Rfactor=19.9%) and 2.1 A (Rfactor=23.8%) resolution, respectively. Ferric Vitreoscilla Hb displays an anticooperative ligand binding behaviour in solution. This very unusual feature can only be accounted for by assuming ligand-linked conformational changes in the monoligated species, which lead to the observed 300-fold decrease in the affinity of cyanide, azide, thiocyanate and imidazole for the monoligated ferric Vitreoscilla Hb with respect to that of the fully unligated homodimer. In the crystalline state, thermodynamics for azide and imidazole binding to ferric Vitreoscilla Hb may be described as a simple process with an overall ligand affinity for the homodimer corresponding to that for diligation in solution. These data suggest that the ligand-free homodimer, observed in the crystalline state, is constrained in a low affinity conformation whose ligand binding properties closely resemble those of the monoligated species in solution. From the kinetic viewpoint, anticooperativity is reflected by the 300-fold decrease of the second-order rate constant for cyanide and imidazole binding to the monoligated ferric Vitreoscilla Hb with respect to that for ligand association to the ligand-free homodimer in solution. On the other hand, values of the first-order rate constant for cyanide and imidazole dissociation from the diligated and monoligated derivatives of ferric Vitreoscilla Hb in solution are closely similar. As a whole, ligand binding and structural properties of ferric Vitreoscilla Hb appear to be unique among all Hbs investigated to date.     

The conformational dynamic of the tetramer hemoglobin molecule as revealed by hydrogen exchange. I. Influence pH, temperature and ligand binding

The rate of the H-D exchange of the peptide NH atoms of the different forms of human Hb was studied at the range of pH 5-10 and temperature 10-63 degrees C by the IR spectroscopy. The pH-dependence of the H-D exchange rate is accordance with the EX2 mechanism. Two pH-dependent conformers of ligand forms of Hb existes at 10-30 degrees C with lower probability of local fluctuations of the alkaline conformer. The difference between two conformers vanishes at 40 degrees C with the appearance of the third conformer with higher probability of local fluctuations. The deoxyHb at 20 degrees C and pH range 6-9 has no pH-dependent conformers and the probability of local fluctuations is considerably reduced in comparison to the acid conformer of ligand Hb. Upon the destabilization of the ligand Hb structure by the pH decreasing to 5.0 at 20 degrees C or the temperature increasing up to 50-60 degrees C at pH 7.1 the global fluctuations of the native structure are intensified providing the H-D exchange of the slowest exchanging NH atoms. The nature of the local and global fluctuations and possible similarity between the two pH-dependent conformers of ligand Hb and its functional R and R2 states revealed by the X-ray analysis and NMR spectroscopy were discussed.     

Kinetics of oxygen binding to ferrous myeloperoxidase

Myeloperoxidase (MPO), which is involved in host defence and inflammation, is a unique peroxidase in having a globin-like standard reduction potential of the ferric/ferrous couple. Intravacuolar and exogenous MPO released from stimulated neutrophils has been shown to exist in the oxyferrous form, called compound III. To investigate the reactivity of ferrous MPO with molecular oxygen, a stopped-flow kinetic analysis was performed. In the absence of dioxygen, ferrous MPO decays to ferric MPO (0.04 s(-1) at pH 8 versus 1.4 s(-1) at pH 5). At pH 7.0 and 25 degrees C, compound III formation (i.e., binding of dioxygen to ferrous MPO) occurs with a rate constant of (1.1+/-0.1) x 10(4)M(-1)s(-1). The rate doubles at pH 5.0 and oxygen binding is reversible. At pH 7.0, the dissociation equilibrium constant of the oxyferrous form is (173+/-12)microM. The rate constant of dioxygen dissociation from compound III is much higher than conversion of compound III to ferric MPO (which is not affected by the oxygen concentration). This allows an efficient transition of compound III to redox intermediates which actually participate in the peroxidase or halogenation cycle of MPO.     

Double mixing stopped-flow method for the study of equilibria and kinetics of dimer-tetramer association of hemoglobins: studies on Hb Carp, Hb A, and Hb Rothschild.

A double mixing stopped-flow method is described for studying the dimer-tetramer equilibria of oxyhemoglobins and the kinetics of association of unliganded dimers. The three hemoglobins studied were: Hb Carp, Hb A, and Hb Rothschild (Trp beta 37 (C3)----Arg). The new method reproduces the data obtained for oxyHb A by other established methods. In agreement with previous studies, the new method indicates little, if any, dissociation of oxyHb carp into dimers even in 2 M urea solutions (0.1 M Bis-Tris pH 7.0). OxyHb Rothschild, on the other hand, is extensively dissociated into dimers (K(Hb4L4 in equilibrium with 2Hb2) = 37.3 x 10(-6) M) and the rate constant for the association of deoxy dimers of Hb Rothschild is about one-tenth of the value for Hb A indicating that the deoxy tetramer of Hb Rothschild is at least 10 times more dissociated into dimers than deoxyHb A.     

Thermolysis of coenzymes B12 at physiological temperatures: activation parameters for cobalt-carbon bond homolysis and a quantitative analysis of the perturbation of the homolysis equilibrium by the ribonucleoside triphosphate reductase from Lactobacillus leichmannii

The kinetics of the thermolysis of 5'-deoxyadenosylcobalamin (AdoCbl, coenzyme B12) in aqueous solution, pH 7.5, have been studied in the temperature range 30-85 degrees C using AdoCbl tritiated at the adenine C2 position and the method of initial rates. Combined with a careful analysis of the distribution of adenine-containing products, the results permit the dissection of the competing rate constants for carbon-cobalt bond homolysis and heterolysis. After correction for the temperature-dependent occurrence of the much less reactive base-off species of AdoCbl, the activation parameters for homolysis of the base-on species were found to be delta H++homo,on = 33.8 +/- 0.2 kcal mol-1 and delta S++homo,on = 13.5 +/- 0.7 cal mol-1 K-1, values not significantly different from those determined by Hay and Finke (J. Am. Chem. Soc. 108 (1986) 4820), in the temperature range 85-115 degrees C. In contrast, the heterolysis of base-on AdoCbl was characterized by a much smaller enthalpy of activation (delta H++het,on = 18.5 +/- 0.2 kcal mol-1) and a negative entropy of activation (delta S++het,on = -34.0 +/- 0.7 cal mol-1 K-1) so that heterolysis, which is minor pathway at elevated temperatures, is the dominant pathway for AdoCbl decomposition at physiological temperatures. Using literature values for the rate constant for the reverse reaction, the equilibrium constant for AdoCbl homolysis at 37 degrees C was calculated to be 7.9 x 10(-18). Comparison with the equilibrium constant for this homolysis at the active site of the ribonucleoside triphosphate reductase from Lactobacillus leichmannii shows that the enzymes shifts the equilibrium constant towards homolysis products by a factor of 2.9 x 10(12) (17.7 kcal mol-1) by binding the thermolysis products with an equilibrium constant of 7.1 x 10(16) M-2, compared to the bonding constant for AdoCbl of 2.4 x 10(4) M-1.     

Microtubule assembly kinetics. Changes with solution conditions.

The assembly kinetics of microtubule protein are altered by ionic strength, temperature and Mg2+, but not by pH. High ionic strength (I0.2), low temperature (T less than 30 degrees C) and elevated Mg2+ (greater than or equal to 1.2 mM) induce a transition from biphasic to monophasic kinetics. Comparison of the activation energy obtained for the fast biphasic step at low ionic strength (I0.069) shows excellent agreement with the values obtained at high ionic strength, low temperature and elevated Mg2+. From this observation it can be implied that the tubulin-containing reactant of the fast biphasic event is also the species that elongates microtubules during monophasic assembly. Second-order rate constants for biphasic assembly are 3.82(+/- 0.72) x 10(7) M-1.s-1 and 5.19(+/- 1.25) x 10(6) M-1.s-1, and for monophasic assembly the rate constant is 2.12(+/- 0.56) x 10(7) M-1.s-1. The microtubule number concentration is constant during elongation of microtubules for biphasic and monophasic assembly.     

Probing the conformational heterogeneity of the acetylaminofluorene-modified 2'-deoxyguanosine and DNA by 19F NMR spectroscopy.

19F NMR spectroscopy was used to probe the conformation of a DNA adduct derived from the carcinogen 7-fluoro-N-acetyl-2-aminofluorene (FAAF) in three structural contexts: as a monomer and incorporated into single- and double-stranded DNA. The 19F NMR spectrum of dG-C8-FAAF [N-(deoxyguanosin-8-yl)-N-acetyl-7-fluoro-2-aminofluorene] in methanol at -30 degrees C exhibited four interconvertible signals in a 11:52:26:11 ratio. Dynamic NMR analysis indicated that the four torsional isomers arise from restricted rotation about the amide (gamma) (14.4 kcal/mol) and the guanyl-nitrogen (alpha) bonds. The conformational heterogeneity persisted in a single strand FAAF-12-mer, d(CTTCTTG[FAAF]ACCTC), whose 19F NMR spectrum at 22 degrees C and pH 7.0 gave only two signals in a 40:60 ratio, instead of four. The two 19F signals followed a two-site exchange with the rotation barrier of 14.7 kcal/mol about the amide (gamma') bond. A similar conformational theme was observed in the FAAF-12-mer duplex, d(CTTCTTG[FAAF]ACCTC).d(GAGGTCAAGAAG), which revealed two 19F resonances in a 41:59 ratio at 22 degrees C and pH 7.0. According to solvent-induced isotope and magnetic anisotropy effects, the two duplex conformers adopt exclusively a base displacement structure, being different only in their relative acetyl group orientations, cis (gamma' approximately 180 degrees) or trans (gamma' approximately 0 degrees ). Dynamic NMR data indicated that the two conformers do not exchange over a wide range of temperatures. This contrasts with the nonacetylated counterpart, which exhibits an equilibrium between the "B-type" and "stacked" conformers [Zhou, L., et al. (1997) J. Am. Chem. Soc. 119, 5384-5389]. The exclusive stacked nature of the AAF adducts may provide insight into why AAF adducts are more mutagenic and prone to repair than the nonacetylated AF adducts.     

The reaction of neuroglobin with potential redox protein partners cytochrome b5 and cytochrome c

Previously identified, potentially neuroprotective reactions of neuroglobin require the existence of yet unknown redox partners. We show here that the reduction of ferric neuroglobin by cytochrome b(5) is relatively slow (k=6 x 10(2)M(-1)s(-1) at pH 7.0) and thus is unlikely to be of physiological significance. In contrast, the reaction between ferrous neuroglobin and ferric cytochrome c is very rapid (k=2 x 10(7)M(-1)s(-1)) with an apparent overall equilibrium constant of 1 microM. Based on this data we propose that ferrous neuroglobin may well play a role in preventing apoptosis.     

Temperature dependence of the rate constants of the Escherichia coli RNA polymerase-lambda PR promoter interaction. Assignment of the kinetic steps corresponding to protein conformational change and DNA opening

The kinetics of formation and of dissociation of open complexes (RPo) between Escherichia coli RNA polymerase (R) and the lambda PR promoter (P) have been studied as a function of temperature in the physiological range using the nitrocellulose filter binding assay. The kinetic data provide further evidence for the mechanism R + P in equilibrium I1 in equilibrium I2 in equilibrium RPo, where I1 and I2 are kinetically distinguishable intermediate complexes at this promoter which do not accumulate under the reaction conditions investigated. The overall second-order association rate constant (ka) increases dramatically with increasing temperature, yielding a temperature-dependent activation energy in the range 20 kcal (near 37 degrees C) to 40 kcal (near 13 degrees C) (1 kcal = 4.184 kJ). Both isomerization steps (I1----I2 and I2----RPo) appear to be highly temperature dependent. Except at low temperatures (less than 13 degrees C) the step I1----I2, which we attribute to a conformational change in the polymerase with a large negative delta Cp degrees value, is rate-limiting at the reactant concentrations investigated and hence makes the dominant contribution to the apparent activation energy of the pseudo first-order association reaction. The subsequent step I2----RPo, which we attribute to DNA melting, has a higher activation energy (in excess of 100 kcal) but only becomes rate-limiting at low temperature (less than 13 degrees C). The initial binding step R + P in equilibrium I1 appears to be in equilibrium on the time-scale of the isomerization reactions under all conditions investigated; the equilibrium constant for this step is not a strong function of temperature and is approximately 10(7) M-1 under the standard ionic conditions of the assay (40 mM-Tris . HCl (pH 8.0), 10 mM-MgCl2, 0.12 M-KC1). The activation energy of the dissociation reaction becomes increasingly negative at low temperatures, ranging from approximately -9 kcal near 37 degrees C to -30 kcal near 13 degrees C. Thermodynamic (van't Hoff) enthalpies delta H degrees of open complex formation consequently are large and temperature-dependent, increasing from approximately 29 to 70 kcal as the temperature is reduced from 37 to 13 degrees C. The corresponding delta Cp degrees value is approximately -2.4 kcal/deg. We propose that this large negative delta Cp degrees value arises primarily from the burial of hydrophobic surface in the conformational change (I1 in equilibrium I2) in RNA polymerase in the key second step of the mechanism.(ABSTRACT TRUNCATED AT 400 WORDS)     

Reactions of Fe(II)-ATP and Fe(II)-citrate complexes with t-butyl hydroperoxide and cumyl hydroperoxide

Rate constants for the reactions of cumyl hydroperoxide and t-butyl hydroperoxide with ferrous complexes of ATP and citrate were measured at pH 7.4. These ligands are potential chelators of iron(II) in the low-molecular weight iron pool that may catalyze oxidative degradation of biomolecules. The second-order rate constants for the reduction of cumyl hydroperoxide and t-butyl hydroperoxide by ferrous ATP are 3.1 x 10(3) and 1.3 x 10(3) M-1.s-1, respectively, at 25 degrees C and 0.11 M ionic strength. Rates of reduction by ferrous citrate are similar. Activation enthalpies for these reactions average 10 kcal/mol.     

Pre-steady-state and steady-state kinetic analysis of the low molecular weight phosphotyrosyl protein phosphatase from bovine heart.

The complete time course of the hydrolysis of p-nitrophenyl phosphate catalyzed by the low molecular weight (acid) phosphotyrosyl protein phosphatase from bovine heart was elucidated and analyzed in detail. Burst titration kinetics were demonstrated for the first time with this class of enzyme. At pH 7.0, 4.5 degrees C, a transient pre-steady-state "burst" of p-nitrophenol was formed with a rate constant of 48 s-1. The burst was effectively stoichiometric and corresponded to a single enzyme active site/molecule. The burst was followed by a slow steady-state turnover of the phosphoenzyme intermediate with a rate constant of 1.2 s-1. Product inhibition studies indicated an ordered uni-bi kinetic scheme for the hydrolysis. Partition experiments conducted for several substrates revealed a constant product ratio. Vmax was constant for these substrates, and the overall rate of hydrolysis was increased greatly in the presence of alcohol acceptors. An enzyme-catalyzed 18O exchange between inorganic phosphate and water was detected and occurred with kcat = 4.47 x 10(-3) s-1 at pH 5.0, 37 degrees C. These results were all consistent with the existence of a phosphoenzyme intermediate in the catalytic pathway and with the breakdown of the intermediate being the rate-limiting step. The true Michaelis binding constant Ks = 6.0 mM, the apparent Km = 0.38 mM, and the rate constants for phosphorylation (k2 = 540 s-1) and dephosphorylation (k3 = 36.5 s-1) were determined under steady-state conditions with p-nitrophenyl phosphate at pH 5.0 and 37 degrees C in the presence of phosphate acceptors. The energies of activation for the enzyme-catalyzed hydrolysis at pH 5.0 and 7.0 were 13.6 and 14.1 kcal/mol, respectively. The activation energy for the enzyme-catalyzed medium 18O exchange between phosphate and water was 20.2 kcal/mol. Using the available equilibrium and rate constants, an energetic diagram was constructed for the enzyme-catalyzed reaction.     

The folding of staphylococcal nuclease in the presence of methanol or guanidine thiocyanate

The effect of methanol on the folding of staphylococcal nuclease has been investigated. Equilibrium thermal unfolding transitions were monitored by fluorescence emission. The transition was very sensitive to the presence of methanol (at pH 7.0), the Tm decreased from above 50 degrees C for aqueous solution to below 0 degree C for 70% methanol. The transitions were fully reversible and conformed to two-state behavior. A linear relationship was observed between the hydrophobicity of the solvent and both the Tm and the change in delta G for unfolding. The effect of pH on the transition in 50% methanol at 0 degree C was essentially the same as for aqueous solution, with a cooperative transition in the vicinity of apparent pH (pH*) 4. The unfolding transition was determined as a function of guanidine thiocyanate in aqueous and 50% methanol solvents. The midpoints of the transitions were 0.30 and 0.20 M, respectively, at 2.1 degrees C. The kinetics of folding at 0 degree C were compared in aqueous, 50% methanol and 0.30 M guanidine thiocyanate solvents, by monitoring changes in the tryptophan fluorescence intensity. Triphasic kinetics for refolding in both aqueous and 50% methanol solutions were observed in stopped-flow experiments. In both solvent systems the slowest phase is ascribed to proline isomerization. The kinetics of refolding were monitored at subzero temperatures in 50% methanol at pH* 7.0 in manual mixing experiments. Biphasic kinetics were observed at temperatures between 0 and -35 degrees C. A third, faster phase, was inferred from the missing amplitude. The energies of activation were 20.0 and 17.2 kcal mol-1, respectively, for the two slower phases. At -33.8 degrees C, the observed pseudo first-order rate constants were 1.2 x 10(-3) and 2.1 x 10(-5) s-1. At temperatures above -35 degrees C, the sum of the observed amplitudes was essentially constant at 70-75% of the expected total amplitude. At lower temperatures the amplitude of the refolding reaction decreased, and the native state was not formed (unless the temperature was increased), due to the formation of a trapped intermediate state. This intermediate has circular dichroism and fluorescence properties consistent with a compact state with some molten globule characteristics.     

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.     

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)     

Oxidized dimeric Scapharca inaequivalvis. Co-driven perturbation of the redox equilibrium

The dimeric hemoglobin isolated from Scapharca inaequivalvis, HbI, is notable for its highly cooperative oxygen binding and for the unusual proximity of its heme groups. We now report that the oxidized protein, an equilibrium mixture of a dimeric high spin aquomet form and a monomeric low spin hemichrome, binds ferrocyanide tightly which allows for internal electron transfer with the heme iron. Surprisingly, when ferricyanide-oxidized HbI is exposed to CO, its spectrum shifts to that of the ferrous CO derivative. Gasometric removal of CO leads to the oxidized species rather than to ferrous deoxy-HbI. At equilibrium, CO binds with an apparent affinity (p50) of about 10-25 mm of Hg and no cooperativity (20 degrees C, 10-50 mM buffers at pH 6.1). The kinetics of CO binding under pseudo-first order conditions are biphasic (t1/2 of 15-50 s at pH 6.1). The rates depend on protein, but not on CO concentration. The nitrite-oxidized protein is not reduced readily in the presence of CO unless one equivalent of ferrocyanide, but not of ferricyanide, is added. We infer that ferrocyanide, produced in the oxidation reaction, is tightly bound to the protein forming a redox couple with the heme iron. CO shifts the redox equilibrium by acting as a trap for the reduced heme. The equilibrium and kinetic aspects of the process have been accounted for in a reaction scheme where the internal electron transfer reaction is the rate-limiting step.