pH-induced denaturation of proteins: a single salt bridge contributes 3-5 kcal/mol to the free energy of folding of T4 lysozyme

The energetics of a salt bridge formed between the side chains of aspartic acid 70 (Asp70) and histidine 31 (His31) of T4 lysozyme have been examined by nuclear magnetic resonance techniques. The pKa values of the residues in the native state are perturbed from their values in the unfolded protein such that His31 has a pKa value of 9.1 in the native state and 6.8 in the unfolded state at 10 degrees C in moderate salt. Similarly, the aspartate pKa is shifted to a value of about 0.5 in the native state from its value of 3.5-4.0 in the unfolded state. These shifts in pKa show that the salt bridge is stabilized 3-5 kcal/mol. This implies that the salt bridge stabilizes the native state by 3-5 kcal/mol as compared to the unfolded state. This is reflected in the thermodynamic stability of mutants of the protein in which Asp70, His31, or both are replaced by asparagine. These observations and consideration of the thermodynamic coupling of protonation state to folding of proteins suggest a mechanism of acid denaturation in which the unfolded state is progressively stabilized by protonation of its acid residues as pH is lowered below pH 4. The unfolded state is stabilized only if acidic groups in the folded state have lower pKa values than in the unfolded state. When the pH is sufficiently low, the acid groups of both the native and unfolded states are fully protonated, and the apparent unfolding equilibrium constant becomes pH independent. Similar arguments apply to base-induced unfolding.(ABSTRACT TRUNCATED AT 250 WORDS)     

Dimer-tetramer equilibrium of glutathione reductase from the cyanobacterium Spirulina maxima

Glutathione reductase [NAD(P)H:GSSG oxidoreductase; EC 1.6.4.2] from cyanobacterium Spirulina maxima exists as an equilibrium system between a dimer (S20,W = 5.96) and a tetramer (S20,W = 8.49) which has a very slow interconversion rate at neutral pH. Our results showed that the apparent dissociation constant (kd) was 4.61 X 10(-7) M. The proportion of both forms at pH 7.0 did not alter at either 4 or 25 degrees C. However, electrophoretic analysis at various pH values showed that at 25 degrees C a gradual transition takes place between oligomers with an apparent pKa of 7.55. When dimers aggregate to form tetramers, the reaction involves the uptake of eight protons (K = 1.58 X 10(-64) M9). At pH 7.7, the equilibrium shifts completely from dimers-tetramers to dimers when temperature is increased, which would suggest that the dissociation is an endothermic process. Thermodynamic parameters obtained from the temperature study show that the dissociation of glutathione reductase is characterized by positive entropy and enthalpy changes. Neither NADPH nor GSSG have any effect on the dimer-tetramer equilibrium. Measurements of reductase activity indicate that the tetramer is almost certainly active, whereas the dimer is either less active or inactive.     

A convenient method of preparation of high-activity urease from Canavalia ensiformis by covalent chromatography and an investigation of its thiol groups with 2,2''-dipyridyl disulphide as a thiol titrant and reactivity probe.

1. A convenient method of preparation of jack-bean urease (EC3.5.1.5) involving covalent chromatography by thiol-disulphide interchange is described. 2. Urease thus prepared has specific activity comparable with the highest value yet reported (44.5 +/- 1.47 kat/kg, Km = 3.32 +/- 0.05 mM; kcat. = 2.15 X 10(4) +/- 0.05 X 10(4)s-1 at pH7.0 and 38 degrees C). 3. Titration of the urease thiol groups with 2,2'-dipyridyl disulphide (2-Py-S-S-2-Py) and application of the method of Tsou Chen-Lu [(1962) Sci. Sin. 11, 1535-1558] suggests that the urease molecule (assumed to have mol.wt. 483000 and epsilon280 = 2.84 X 10(5) litre-mol-1-cm-1) contains 24 inessential thiol groups of relatively high reactivity (class-I), six 'essential' thiol groups of low reactivity (class-II) and 54 buried thiol groups (class-III) which are exposed in 6M-guanidinium chloride. 4. The reaction of the class-I thiol groups with 2-Py-S-S-2-Py was studied in the pH range 6-11 at 25 degrees C(I = 0.1 mol/l) by stopped-flow spectrophotometry, and the analogous reaction of the class-II thiol groups by conventional spectrophotometry. 5. The class-I thiol groups consist of at least two sub-classes whose reactions with 2-Py-S-S-2-Py are characterized by (a) pKa = 9.1, k = 1.56 X 10(4)M-1-s-1 and (b) pKa = 8.1, k = 8.05 X 10(2)M-1-s-1 respectively. The reaction of the class-II thiol groups is characterized by pKa = 9.15 and k = 1.60 X 10(2)M-1-s-1. 6. At pH values 7-8 the class-I thiol groups consist of approx. 50% class-Ia groups and 50% class-Ib groups. The ratio class Ia/class Ib decreases an or equal to approx. 9.5, and at high pH the class-I thiol groups consist of at most 25% class-Ia groups and at least 75% class-Ib groups. 7. The reactivity of the class-II thiol groups towards 2-Py-S-S-2-Py is insensitive to the nature of the group used to block the class-I thiols. 8. All the 'essential' thiol groups in urease appear to be eeactive only as uncomplicated thiolate ions. The implications of this for the active-centre chemistry of urease relative to that of the thiol proteinases are discussed.     

Reversible modulation of human factor Xa activity with phosphonate esters: media effects

Enantiomers of 4-nitrophenyl 4-X-phenacyl methylphosphonate esters (X = H, PMN; CH3 and CH3O) inactivate human factor Xa with rate constants 8-86 M(-1)s(-1) at pH 6.75 in 0.025 M Hepes buffer, 0.15 M NaCl and 2 mM CaCl2 at 7.0+/-0.1 degrees C. The stereoselectivity of the inactivation of factor Xa is 2-10 and favors the levorotatory enantiomers. The pH-dependence of inactivation of factor Xa by (-)-PMN is sigmoidal and consistent with the participation of a catalytic residue with a pKa of 6.2+/-0.1. Factor Xa reactivates from its phosphonyl adducts through a self-catalyzed intramolecular reaction, which is much influenced by the presence of phospholipids. The rate of reactivation in the absence of phospholipids is not pH dependent at pH <9, but it increases very much at pH >9. In the presence of phospholipids, the pH dependence of the rate constant for reactivation is sigmoidal in the pH 6.5-10.3 range and levels off at pH >9 indicating that the enzyme catalyzes its reactivation. The kinetic pKa for the recovery of factor Xa from its adducts with the PMNs is in the range of 6.7-8.1 and is consistent with the participation of the catalytic His57 in the reactivation process.     

Horseradish peroxidase. XXXII. pH dependence of the oxidation of L-(-)-tyrosine by compound I.

The rate of oxidation of L-(-)-tyrosine by horseradish peroxidase compound 1 has been studied as a function of pH at 25 degrees C and ionic strength 0.11. Over the pH range of 3.20--11.23 major effects of three ionizations were observed. The pKa values of the phenolic (pKa = 10.10) and amino (pKa = 9.21) dissociations of tyrosine and a single enzyme ionization (pKa = 5.42) were determined from nonlinear least squares analysis of the log rate versus pH profile. It was noted that the less acidic form of the enzyme was most reactive; hence, the reaction is described as base catalyzed. The rate of tyrosine oxidation falls rapidly with the deprotonation of the phenolic group.     

Determination of the redox properties of the Rieske [2Fe-2S] cluster of bovine heart bc1 complex by direct electrochemistry of a water-soluble fragment.

The redox potential of the Rieske [2Fe-2S] cluster of the bc1 complex from bovine heart mitochondria was determined by cyclic voltammetry of a water-soluble fragment of the iron/sulfur protein. At the nitric-acid-treated bare glassy-carbon electrode, the fragment gave an immediate and stable quasireversible response. The midpoint potential at pH 7.2, 25 degrees C and I of 0.01 M was Em = +312 +/- 3 mV. This value corresponds within 20 mV to results of an EPR-monitored dye-mediated redox titration. With increasing ionic strength, the midpoint potential decreased linearly with square root of I up to I = 2.5 M. From the cathodic-to-anodic peak separation, the heterogeneous rate constant, k degrees, was calculated to be approximately 2 x 10(-3) cm/s at low ionic strength; the rate constant increased with increasing ionic strength. From the temperature dependence of the midpoint potential, the standard reaction entropy was calculated as delta S degrees = -155 J.K-1.mol-1. The pH dependence of the midpoint potential was followed over pH 5.5-10. Above pH 7, redox-state-dependent pK changes were observed. The slope of the curve, -120 mV/pH above pH9, indicated two deprotonations of the oxidized protein. The pKa values of the oxidized protein, obtained by curve fitting, were 7.6 and 9.2, respectively. A group with a pKa,ox of approximately 7.5 could also be observed in the optical spectrum of the oxidized protein. Redox-dependent pK values of the iron/sulfur protein are considered to be essential for semiquinone oxidation at the Qo center of the bc1 complex.     

1H NMR study of the solution properties of the polypeptide neurotoxin I from the sea anemone Stichodactyla helianthus

The solution properties of the polypeptide neurotoxin I from the sea anemone Stichodactyla helianthus (Sh I) have been investigated by high-resolution 1H nuclear magnetic resonance (NMR) spectroscopy at 300 MHz. The pH dependence of the spectra has been examined over the range 1.1-12.2 at 27 degrees C. Individual pKa values have been obtained for the alpha-ammonium group of Ala-1 (8.6) and the side chains of Glu-8 (3.7), Tyr-36 (10.9), and Tyr-37 (10.8). For the remaining seven carboxyl groups in the molecule (from five Asp, Glu-31, and the C-terminus), four pKa values, viz., 2.8, 3.5, 4.1 and 6.4, can be clearly identified. The five Lys residues titrate in the range 10.5-11, but individual pKa values could not be obtained because of peak overlap. Conformational changes associated with the protonation of carboxylates occur below pH 4, while in the alkaline pH range major unfolding occurs above pH 10. The molecule also unfolds at elevated temperatures, having a transition temperature of ca. 55 degrees C at pH 5.25. Exchange of the backbone amide protons has been monitored at various values of pH and temperature in the ranges pH 4-5 and 12-27 degrees C. Up to 18 slowly exchanging amides are observed, consistent with the existence of a core of hydrogen-bonded secondary structure, most probably beta-sheet.(ABSTRACT TRUNCATED AT 250 WORDS)     

Physico-chemical properties of pregnant mare serum gonadotropin

Pregnant mare serum gonadotropin exhibits a dissociation at acid pH as shown by the drop of s20,w values from 3.52 S at pH 8.1 to 2.52 S at pH 2.0. The dissociation is accompanied by an absorbance change with a maximum at 287 nm and a parallel loss of both follicle-stimulating hormone (FSH) and luteinizing hormone (LH) activities as followed by radioreceptor assays. The apparent pKa of the acid transition is 3.45 with an extremely slow and temperature-dependent rate at pH 2.0 (1.8 . 10(-4) s-1 at 37 degrees C). By gel filtration the molecular weight of the active hormone is estimated to be 45 000 (rather than the previously reported 53 000-64 000). The active conformation of the hormone includes beta sheet structure (34%) as for other gonadotropin hormones with a minor but significative amount of alpha-helix. Four tyrosine residues were titrated, two of pKa = 10.3 and two of pKa = 11 out of a total of seven tyrosines. The parallel changes in FSH and LH activities during the preparation and the acid transition suggest that the two biological activities are intrinsic properties of the same molecular entity.     

Investigation of the haem-nicotinate interaction in leghaemoglobin. Role of hydrogen bonding.

A strategic assessment of the contributions of two active-site hydrogen bonds in the binding of nicotinate to recombinant ferric soybean leghaemoglobin a (rLb) was carried out by mutagenic replacement of the hydrogen-bonding residues (H61A and Y30A variants) and by complementary chemical substitution of the carboxylate functionality on the nicotinate ligand. Dissociation constants, Kd (pH 5.5, mu = 0.10 M, 25.0 +/- 0.1 degrees C), for binding of nicotinate to ferric rLb, H61A and Y30A were 1.4 +/- 0.3 microM, 19 +/- 1 microM and 11 +/- 1 microM, respectively; dissociation constants for binding of nicotinamide were, respectively, 38 +/- 1 mM, 50 +/- 2 mM and 12 +/- 1 mM, and for binding of pyridine were 260 +/- 50 microM, 4.5 +/- 0.5 microM and 66 +/- 8 microM, respectively. Binding of cyanide and azide to the H61A and Y30A variants was unaffected by the mutations. The pH-dependence of nicotinate binding for rLb and Y30A was consistent with a single titration process (pKa values 6.9 +/- 0.1 and 6.7 +/- 0.2, respectively); binding of nicotinate to H61A was independent of pH. Reduction potentials for the rLb and rLb-nicotinate derivatives were 29 +/- 2 mV (pH 5.40, 25.0 degrees C, mu = 0.10 M) and - 65 +/- 2 mV (pH 5.42, 25.0 degrees C, mu = 0.10 M), respectively. The experiments provide a quantitative assessment of the role of individual hydrogen bonds in the binding process, together with a definitive determination of the pKa of His61 and unambiguous evidence that titration of His61 controls binding in the neutral to alkaline region.     

The interaction of a tyrosyl residue and carboxyl groups in the specific interaction between Streptomyces subtilisin inhibitor and subtilisin BPN'. A chemical modification study.

An ultraviolet absorption difference spectrum that is typical of a change in ionization state (pKa 9.7 leads to greater than 11.5) of a tyrosyl residue has been observed on the binding between Streptomyces subtilisin inhibitor (SSI) and subtilisin BPN' [EC 3.4.21.14] at alkaline pH, ionic strength 0.1 M, at 25 degrees C (Inouye, K., Tonomura, B., and Hiromi, K., submitted). When the complex of SSI and subtilisin BPN' is formed at an ionic strength of 0.6 M and pH 9.70, the characteristic features of the protonation of a tyrosyl residue in the difference spectrum are diminished. These results suggest that the pKa-shift of a tyrosyl residue observed at alkaline pH and lower ionic strength results from an electrostatic interaction. Nitration of tyrosyl residues of SSI and of subtilisin BPN' was performed with tetranitromethane (TNM). By measurements of the difference spectra observed on the binding of the tyrosyl-residue-nitrated SSI and the native subtilisin BPN', and on the binding of the native SSI and the tyrosyl-residue-nitrated subtilisin BPN' and alkaline pH, the tyrosyl residue in question was shown to be one out of the five tyrosyl residues of pKa 9.7 of the enzyme. This tyrosyl residue was probably either Tyr 217 or Tyr 104 on the basis of the reactivities of tyrosyl residues of the enzyme with TNM and their locations on the enzyme molecule. Carboxyl groups of SSI were modified by covalently binding glycine methyl ester with the aid of water-soluble carbodiimide, in order to neutralize the negative charges on SSI. In the difference spectrum which was observed on the binding of subtilisin BPN' and the 5.3-carboxyl-group-modified SSI at alkaline pH, the characteristic features of the protonation of a tyrosyl residue were essentially lost, and the difference spectrum is rather similar to that observed on the binding of the native SSI and the enzyme at neutral pH. This phenomenon indicates that the pKa of a tyrosyl residue of the enzyme is shifted upwards by interaction with carboxyl group(s) of SSI on the formation of the enzyme-inhibitor complex.     

The catalytic activity of pig pepsin C towards small synthetic substrates.

A series of small peptides has been synthesized and used to investigate the activity of a minor pig pepsin, pepsin C (EC 3.4.23.3). The peptides had the general formula A-Leu-Val-His-B. B was either OMe, NH2 or OH. With B = NH2 hydrolysis (kcat./Km) at 37 degrees C and pH 2.07 increased as A was Ac-Ala, Ac-Tyr, Ac-Phe and Ac-Ala-Phe. The pH dependence of the hydrolysis of Ac-Phe-Leu-Val-His-NH2 indicated the apparent pKa values of two catalytically important groups on the enzyme as 1.42 and 4.88. Inhibition of the hydrolysis of the same peptide by Ac-Phe at pH 3.01 showed a form of mixed non-competitive inhibition. Hydrolysis of Ac-Tyr-Leu-Val-His-OMe and the corresponding amide showed non-classical kinetics, which are discussed in terms of a substrate-activating mechanism. The results are discussed with reference to observations made by other workers on pig pepsin A.     

Study of the enzymatic hydrolysis of a phosphonic ester using microcalorimetry]

A "Batch" microcalorimeter is used at 30 degrees C for the study of the hydrolysis of 4-nitro-phenylphenylphosphonate with a calf-intestinal phosphonate esterase, in a tris buffer, pH 8. The yield of enzymatic hydrolysis is estimated by spectrophotometric determination of the p--nitrophenol evolved; we have then calculated the apparent molar enthalph of the reaction. (delta Happ = -72,2 kj. mol-1). Phenylphosphonic acid, the second reaction product, is not transphosphonylated on tris. The second acidity of phenylphosphonic acid was studied at 30 degrees C by sodium hydroxide electrotitration (pKa2 = 7,13) and by "Flow" microcalorimetry (delta Hionization = 19,8 kj.mol-1). In the same manner at 30 degrees C, we measured the heat of ionization of p-nitrophenol (delta Hionization = 26,75 kj.mol-1). These findings allow a calculation for the actual heat of hydrolysis of 4-nitro-phenyl-phenylphosphonate (delta Hrho = -29,7 kj.mol-1).     

Characterization of steady state, single-turnover, and binding kinetics of the TaqI restriction endonuclease.

The TaqI restriction endonuclease recognizes and cleaves the duplex DNA sequence T decreases CGA. Steady state kinetic analysis with a small oligodeoxyribonucleotide substrate showed that the enzyme obeyed Michaelis-Menten kinetics (Km = 53 nM, kcat = 1.3 min-1 at 50 degrees C and Km = 0.5 nM, kcat = 2.9 min-1 at 60 degrees C). At 0 degree C, the enzyme was completely inactive, while at 15 degrees C, turnover produced nicked substrate as the major product in excess of enzyme indicating dissociation between nicking events. Above 37 degrees C, both strands in the duplex were cleaved prior to dissociation. In contrast to the tight, temperature-dependent binding of substrate, binding of the Mg2+ cofactor was weak (Kd = 2.5 mM) and the same at either 50 degrees C or 60 degrees C. Single-turnover experiments using oligonucleotide substrate showed that hydrolysis of duplex DNA occurred via two independent nicking events, each with a first order rate constant (kst) of 5.8 min-1 at 60 degrees C and 3.5 min-1 at 50 degrees C. The pH dependence of Km (pKa = 9) and kst (pKa = 7) suggests Lys/Arg and His, respectively, as possible amino acids influencing these constants. Moreover, although kst increased significantly with pH, kcat did not, indicating that at least two steps can be rate-controlling in the reaction pathway. Binding of protein to canonical DNA in the presence of Mg2+ at 0 degree C or in the absence of Mg2+ at 50 degrees C was weak (Kd = 2.5 microM or 5,000-fold weaker than the optimal measured Km) and equal to the binding of noncanonical DNA as judged by retention on nitrocellulose. Similar results were seen in gel retardation assays. These results suggest that both Mg2+ and high temperature are required to attain the correct protein conformation to form the tight complex seen in the steady state analysis. In the accompanying paper (Zebala, J. A., Choi, J., Trainor, G. L., and Barany, F. (1992) J. Biol. Chem. 267, 8106-8116), we report how these kinetic constants are altered using substrate analogues and propose a model of functional groups involved in TaqI endonuclease recognition.     

Thermal unfolding of dodecameric glutamine synthetase: inhibition of aggregation by urea.

Thermal unfolding of dodecameric manganese glutamine synthetase (622,000 M(r)) at pH 7 and approximately 0.02 ionic strength occurs in two observable steps: a small reversible transition (Tm approximately 42 degrees C; delta H approximately equal to 0.9 J/g) followed by a large irreversible transition (Tm approximately 81 degrees C; delta H approximately equal to 23.4 J/g) in which secondary structure is lost and soluble aggregates form. Secondary structure, hydrophobicity, and oligomeric structure of the equilibrium intermediate are the same as for the native protein, whereas some aromatic residues are more exposed. Urea (3 M) destabilizes the dodecamer (with a tertiary structure similar to that without urea at 55 degrees C) and inhibits aggregation accompanying unfolding at < or = 0.2 mg protein/mL. With increasing temperature (30-70 degrees C) or incubation times at 25 degrees C (5-35 h) in 3 M urea, only dodecamer and unfolded monomer are detected. In addition, the loss in enzyme secondary structure is pseudo-first-order (t1/2 = 1,030 s at 20.0 degrees C in 4.5 M urea). Differential scanning calorimetry of the enzyme in 3 M urea shows one endotherm (Tmax approximately 64 degrees C; delta H = 17 +/- 2 J/g). The enthalpy change for dissociation and unfolding agrees with that determined by urea titrations by isothermal calorimetry (delta H = 57 +/- 15 J/g; Zolkiewski M, Nosworthy NJ, Ginsburg A, 1995, Protein Sci 4: 1544-1552), after correcting for the binding of urea to protein sites exposed during unfolding (-42 J/g). Refolding and assembly to active enzyme occurs upon dilution of urea after thermal unfolding.     

Protein chromatography on adsorbents with hydrophobic and ionic groups. Some properties of N-(3-carboxypropionyl)aminodecyl-sepharose and its interaction with wheat-germ aspartate transcarbamoylase.

1. The charge state of two derivatives of Sepharose prepared by the CNBr activation method were studied by acid-base titration and by ion-exchange chromatography. Dodecyl-Sepharose exhibited cationic groups (21mumol/ml of settled gel; pKa=9.6) that were tentatively assigned to the coupling isourea group. 2. CPAD-Sepharose [N-(3-carboxypropionyl)aminodecyl-Sepharose] has anionic (carboxyl) groups (pKa=4.5) and cationic groups (pKa=9.6) in roughly equal concentrations (e coupling group. CPAD-Sepharose is slightly negatively charged at pH 7.0 and substantially negatively charged at pH 8.5. 3. The pKa values of dodecyl-Sepharose and CPAD-Sepharose are unaffected by a 100-fold increase in the concentration of KCl. 4. CPAD-Sepharose has considerable affinity for wheat-germ aspartate transcarbamoylase at pH 8.5 when the adsorbent and enzyme are both negatively charged. The interaction involves the C10 chain but is relatively moderate compared with C10 chains associated only with positive charge. 5. Desorption of the enzyme adsorbed to CPAD-Sepharose can be achieved by raising the pH to increase the electrostatic repulsion, or by introducing the detergent sodium deoxycholate. Acetone and butan-1-ol also weaken the adsorption at pH 8.5. 6. High concentrations of sodium acetate or sodium phosphate induced the enzyme to bind more tightly to CPAD-Sepharose. 7. These results are discussed in terms of a 'repulsion-controlled' model or hydrophobic chromatography.     

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)     

Thermodynamics of melittin tetramerization determined by circular dichroism and implications for protein folding.

The tetramerization of melittin, a 26-amino acid peptide from Apis mellifera bee venom, has been studied as a model for protein folding. Melittin converts from a monomeric random coil to an alpha-helical tetramer as the pH is raised from 4.0 to 9.5, as ionic strength is increased, as temperature is raised or lowered from about 37 degrees C, or as phosphate is added. The thermodynamics of this tetramerization (termed "folding") are explored using circular dichroism. The melittin tetramer has two pKa values of 7.5 and 8.5 corresponding to protonation of the N-terminus and Lys 23, respectively. pKa values calculated with the program DelPhi (Gilson, M.K., Sharp, K.A., & Honig, B.H., 1987, J. Comp. Chem. 9, 327-335; Gilson, M.K. & Honig, B.H., 1988a, Proteins 3, 32-52; Gilson, M.K. & Honig, B.H., 1988b, Proteins 4, 7-18) agree with experimental titration data. Greater electrostatic repulsion of these protonated groups destabilizes the tetramer by 3.6 kcal/mol at pH 4.0 compared to pH 9.5. Increasing the concentration of NaCl in the solution from 0 to 0.5 M stabilizes the tetramer by 5-6 kcal/mol at pH 4.0. The effect of NaCl is modeled with a ligand-binding approach. The melittin tetramer is found to have a temperature of maximum stability ranging from 35.5 to 43 degrees C depending on the pH, unfolding above and below that temperature. delta Cp0 for folding ranges from -0.085 to -0.102 cal g-1 K-1, comparable to that of other small globular proteins (Privalov, P.L., 1979, Adv. Protein Chem. 33, 167-241). delta H0 and delta S0 are found to decrease with temperature, presumably due to the hydrophobic effect (Kauzmann, W., 1959, Adv. Protein Chem. 14, 1-63). Phosphate is found to perturb the equilibrium substantially with a maximal effect at 150 mM, stabilizing the tetramer at pH 7.4 and 25 degrees C by 4.6 kcal/mol. The enthalpy change due to addition of phosphate (-7.5 kcal/mol at 25 degrees C) can be accounted for by simple dielectric screening. Both circular dichroism and crystallographic results suggest that phosphate may bind Lys 23 at the ends of the elongated tetramer. These detailed measurements give insight into the relative importance of various forces for the stability of melittin in the folded form and may provide an experimental standard for future tests of computational energetics on this simple protein system.     

Aquo-pentamine Co(III) complexes as models for carbonic anhydrase

The hydrolysis of 4-nitrophenyl acetate by metal complexes Co(en)2(imH)H2O3+, Co(en)2(bzmH)H2O3+, and Co(en)2(imCH3)H2O3+ (imH = imidazole, bzmH = benzimodazole, imCH3 = methyl imidazole) has been investigated in the pH range 5.4-8.9. The small difference in nucleophilic reactivity in the pH range 5.4-6.7 is assumed to be due to hydrogen bonding abilities of the imidazole and substituted imidazole ligands and small pKa differences (k2(imH) = 2.2 X 10(-2) M-1 sec-1, k2(bzmH) = 5.68 X 10(-2) M-1 sec-1, k2(imCH3) = 1.35 X 10(-2) M-1 sec-1, 40 degrees C, 1 = 0.3 NaClO4, pKa(imH) = 6.2, pKa(imCH3) = 6.2 and pKa(bzmH) = 5.9). In the pH range 7.8-8.9, the differences in nucleophilic reactivity (k3(imH) = 85.5 X 10(-2) M-1 sec-1, k3(bzmH) = 33.4 X 10(-2) M-1 sec-1, 40 degrees C, I = 0.3 NaClO4) are reconciled with a significant steric factor outweighing the acidity of the benzimidazole complex. In the pH region 6.7-7.7, the deviation from linearity is presumably due to both hydroxo and imido ligands functioning as nucleophiles, the latter being about 40 times stronger than the former.     

Characterisation of synthetic beta-haematin and effects of the antimalarial drugs quinidine, halofantrine, desbutylhalofantrine and mefloquine on its formation

Infrared spectroscopy, elemental analysis and X-ray powder diffraction show that the product of 30 min of reaction of haematin in 4.5 M acetate, pH 4.5 at 60 degrees C is identical to beta-haematin prepared in 4.5 M acetic acid at 70 degrees C overnight (pH 2.6). There is no evidence for formation of haem-acetate complex, which could not be isolated, even from 11.4 M acetate solution. The antimalarial drugs quinidine, halofantrine, desbutylhalofantrine and mefloquine were found to inhibit formation of beta-haematin, while 5-, 6- and 8-aminoquinoline and quinoline were found to have no effect. Quinidine was shown to form a complex with ferriprotoporphyrin IX in 40% DMSO with log K = 5.02 +/- 0.03. Log K values for halofantrine and desbutylhalofantrine are 5.29 +/- 0.02 and 5.15 +/- 0.02 respectively (solutions containing 30% acetonitrile in addition to DMSO to solubilise these drugs), which are both stronger than chloroquine under the same conditions (log K = 4.56 +/- 0.02).     

Oxygen activation catalyzed by methane monooxygenase hydroxylase component: proton delivery during the O-O bond cleavage steps

The effects of solvent pH and deuteration on the transient kinetics of the key intermediates of the dioxygen activation process catalyzed by the soluble form of methane monooxygenase (MMO) isolated from Methylosinus trichosporium OB3b have been studied. MMO consists of hydroxylase (MMOH), reductase, and "B" (MMOB) components. MMOH contains a carboxylate- and oxygen-bridged binuclear iron cluster that catalyzes O2 activation and insertion chemistry. The diferrous MMOH-MMOB complex reacts with O2 to form a diferrous intermediate compound O (O) and subsequently a diferric intermediate compound P (P), presumed to be a peroxy adduct. The O decay reaction was found to be pH-independent within error at 4 degrees C (kobs = 22 +/- 2 s-1 at pH 7.7; kobs = 26 +/- 2 s-1 at pH 7.0). In contrast, the P formation rate was found to decrease sharply with increasing pH to near zero at pH 8.6; the observed rate constants fit to a single deprotonation event with a pKa = 7.6 and a maximal formation rate at 4 degrees C of kP = 9.1 +/- 0.9 s-1 achieved near pH 6.5. The formation of P was slower than the disappearance of O, indicating that at least one other undetected intermediate (P) must form in between. P decays spontaneously to the highly chromophoric intermediate, compound Q (Q). The decay rate of P matched the formation rate of Q, and both rates decreased sharply with increasing pH to near zero at pH 8.6; the observed rate constants fit to a single deprotonation event with a pKa = 7.6 and a maximal formation rate at 4 degrees C of kQ = 2.6 +/- 0.1 s-1 achieved near pH 6.5. No pH dependence was observed for the decay of Q. The formation and decay rates of P and the formation rate of Q decreased linearly with mole fraction of D2O in the reaction mixture. Kinetic solvent isotope effect values of kH/kD = 1.3 +/- 0.1 (P formation) and kH/kD = 1.4 +/- 0.1 (P decay and Q formation) were observed at 5 degrees C. The linearity of the proton inventory plots suggests that only a single proton is transferred in the transition state of the formation reaction for each intermediate. If these protons are transferred to the bound oxygen molecule, as formally required by the reaction stoichiometry, the data are consistent with a model in which water is formed concurrently with the formation of the reactive bis mu-oxo-binuclear Fe(IV) species, Q.