The amine capture strategy for peptide bond formation—an outline of progress

A review is presented of the general features of amide bond formation by prior capture of the amine-bearing peptide fragment. Capture by formation of a bond to the N-terminal nitrogen is shown in eight model systems to result in slow intramolecular acyl transfer, accompanied by anomalously large Gly/Ala and Gly/Val rate ratios. Two examples are given of rapid intramolecular acyl transfer via rings of 9 and 12 members. The transfer efficiency as estimated by the effective molarity of reactive amine at the acyl carbon in the latter case is 18M. Capture at the thiol function of an N-terminal cysteine residue is shown to be feasible in mercurithio and disulfide systems. In the latter case, effective concentrations of 3M and intramolecular transfer half-times of 10 min are demonstrated.     

Light scattering from wormlike chains with excluded volume effects

This paper reports a calculation of the angular dependence of light scattering from wormlike chains with excluded volume effects. The Daniels distribution function, modified for excluded volume effects, is used to compute averages for scattering elements separated by contour lengths which are long with respect to the persistence length of the chain. An expansion in terms of exactly known moments of the distribution for the wormlike coil without excluded volume is used for short contour lengths. The results are applied to scattering from calf thymus (M = 18.1 × 10⁶) and T7 (M = 25.4 × 10⁶) DNA. It is demonstrated that the same values of excluded volume parameter (ε = 0.11) and statistical segment length (1/λ′ = 900 Å) which explain the sedimentation and viscosity behavior of DNA also account satisfactorily for the scattering behavior. Molecular weights and root-mean-square radii estimated by extrapolation from scattering data obtained in the angular region from 10° to 25° will be 5–10% too large for DNA of molecular weight 20 × 10⁶–30 × 10⁶.     

Thiol activation of a model O-aryl diazeniumdiolate prodrug in phospholipid vesicle media

Thiolysis of the model diazeniumdiolate prodrug, O²-(2,4-dinitrophenyl) 1-(N,N-diethylamino)diazen-1-ium-1,2-diolate (DNP-DEA/NO, 1), by glutathione (GSH), cysteine (CYSH) and 1-heptanethiol (heptylmercaptan, HM) has been examined in anionic (DOPG), neutral (DPPC, DOPE) and cationic (DOTAP) vesicle media and in glycine buffered aqueous solutions. DOTAP vesicles accelerate the bimolecular reaction with glutathione, cysteine and 1-heptanethiol by factors of 81, 8.2 and 4630, respectively, while reaction is inhibited 5- to 10-fold in the presence of neutral and anionic vesicles. The intrinsic nucleophilicity of the thiols has been compared through the second-order rate constants, 22.9, 5.24 and 43.1 M⁻¹ s⁻¹, for nucleophilic attack on 1 by GS⁻, CYS⁻ and M⁻, respectively, obtained in buffered aqueous media. Analysis of the catalysis by DOTAP vesicles, using pseudophase ion-exchange formalism, suggests that the rate increase is due to reactant concentration in the bilayer and interfacial region coupled with enhanced dissociation of the thiol at the vesicle surface. Some contribution from enhanced nucleophilic reactivity at the vesicle interface may also contribute to the greater catalysis by HM. Inhibition of the thiolysis reaction by phospholipid liposomes is attributed to repulsion of the thiolate anions by the negatively charged acyl phosphate of the lipid head group. DOPG = 1,2-dioleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)], DPPC = 1,2-dipalmitoyl-sn-glycero-3-phosphocholine, DOPE = 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, DOTAP = 1,2-dioleoyl-3-trimethylammonium-propane.     

Histidine-containing cyclic dipeptides as catalysts in the hydrolysis of carbonic acid p-nitrophenyl esters

A histidine-containing cyclic dipeptide, cyclo(D -Leu-L -His), was almost 20 times as efficient a catalyst as imidazole in the hydrolysis of p-nitrophenyl laurate. The effect of dioxane on the hydrolysis showed that hydrophobic interaction between the cyclic dipeptide and the ester is very important. This reaction obeyed the Michaelis-Menten kinetics, and the Michaelis constant K_m was as low as 9.98 × 10^?5M. Since the linear dipeptide having D -Leu-L -His sequence was nearly inactive in the hydrolysis, the functional groups of cyclo(D -Leu-L -His) in a specific arrangement held by the rigid backbone must have cooperated in the fast hydrolysis. Very weak catalysis by the diasteremeric cyclic dipeptide, cyclo(L -Leu-L -His), in the hydrolysis supported the above view.     

The dehalogenation of the 5-halo-5,6-dihydrouracils: Uracil formation from 5-iodo- and 5-bromo-5,6-dihydrouracil

The elimination of halide ion from either 5-bromo- or 5-iodo-5,6-dihydrouracil to yield uracil is a slow reaction which, in the case of 5-iodo-5,6-dihydrouracil, is 400 times slower than the enzymatic release of ¹²⁵I^? from 5-[¹²⁵I]iodouracil. The elimination of HBr from 5-bromo-5,6-dihydrouracil is subject to general base catalysis by tris(hydroxymethyl)aminomethane (k₂^{Tris base} = 11 × 10^?4M^?1 min^?1, 37°C, ionic strength 1.0 M). At pH values near and above physiological, both the bromo- and iododihydropyrimidines are subject to hydrolysis of the dihydropyrimidine ring, a reaction which parallels halide elimination to yield uracil. The resulting 2-halo-3-ureidopropionate then cyclizes via intramolecular attack of the ureido oxygen atom to yield halide ion and 2-amino-2-oxazoline-5-carboxylic acid as final products. In dilute hydroxide ion, the kinetics of 5-bromo-5,6-dihydrouracil hydrolysis (25°C, ionic strength 1.0 M) show a change in rate-determining step as a function of increasing hydroxide ion concentration, a result which, as in the case of 5,6-dihydrouracil, can be explained in terms of the formation of a tetrahedral addition intermediate. The data are discussed relative to enzymatically catalyzed halopyrimidine dehalogenation.     

Intramolecular participation of the amide group in the hydrolysis of p-nitrophenyl N-(bromoacetyl) anthranilate

The rate of hydrolysis of p-nitrophenyl N-(bromoacetyl) anthranilate (Ib) has been measured in aqueous solution between pH 1 and 6.5 has been found to increase linearly with pH at pH higher than 3. An abnormally large apparent alkaline rate constant of 3.8 × 10⁶M^?1 sec^?1 has been determined. Intramolecular nucleophilic displacement by the amide group at the carbonyl carbon of the ester occurred and a cyclic intermediate was formed. This intermediate has been detected by direct isolation and by measurements of the proton release accompanying the reaction. The rates of hydrolysis of analogous derivatives (IIb-IIIb-IV), for which this intramolecular assistance was not possible, were slower by a factor of about 5 × 10⁵. Such an example of intramolecular catalysis may be useful for a better understanding of the enzymatic catalysis.     

Kinetics of oxidation of tyrosine by a model alkoxyl radical

Abstract

Oxidation of tyrosine moieties by radicals involved in lipid peroxidation is of current interest; while a rate constant has been reported for reaction of lipid peroxyl radicals with a tyrosine model, little is known about the reaction between tyrosine and alkoxyl radicals (also intermediates in the lipid peroxidation chain reaction). In this study, the reaction between a model alkoxyl radical, the tert-butoxyl radical and tyrosine was followed using steady-state and pulse radiolysis. Acetone, a product of the β-fragmentation of the tert-butoxyl radical, was measured; the yield was reduced by the presence of tyrosine in a concentration- and pH-dependent manner. From these data, a rate constant for the reaction between tert-butoxyl and tyrosine was estimated as 6?±?1 × 10⁷ M^?1 s^?1 at pH 10. Tyrosine phenoxyl radicals were also monitored directly by kinetic spectrophotometry following generation of tert-butoxyl radicals by pulse radiolysis of solutions containing tyrosine. From the yield of tyrosyl radicals (measured before they decayed) as a function of tyrosine concentration, a rate constant for the reaction between tert-butoxyl and tyrosine was estimated as 7?±?3 × 10⁷ M^?1 s^?1 at pH 10 (the reaction was not observable at pH 7). We conclude that reaction involves oxidation of tyrosine phenolate rather than undissociated phenol; since the pK_a of phenolic hydroxyl dissociation in tyrosine is ～ 10.3, this infers a much lower rate constant, about 3 × 10⁵ M^?1 s^?1, for the reaction between this alkoxyl radical and tyrosine at pH 7.4.     

Amine-catalyzed elimination of beta-phosphoric esters from aldehydes derived from ribonucleic acid and model substrates.

The kinetics have been measured for several steps of the diamine-catalyzed elimination of the terminal nucleoside from periodate-oxidized RNA and from several model substrates. The general-acid-catalyzed, rate-determining step has a k_HA of 0.13 M^?1 min^?1 (HA = RNH₃⁺) for primary amines, and the specific-base-catalyzed reaction has a k_HH of 0.35 min^?1 (0.2 mm RNA) with ornithine catalysis and a k_HH of 0.077 min^? (0.2 mm RNA) with lysine catalysis. Lysine has a third catalysis component, with a k_AH of 12 min^?1 M^?2. The diamino acid α,γ-diaminobutyrate is not effective as a catalyst, due to cyclic gem diamine formation. Substituents on the 5′-phosphoric ester group do not affect the kinetics unless the substituent is a proton (e.g., as in AMP); thus, AMP is not an accurate model for this type of sequential degradation of RNA.There are two degradative pathways, the β-elimination path and a route that involves cleavage of the C-1′-0-C-4′ ether linkage before the phosphoric ester is eliminated. The direct β-elimination path predominates below pH 7.5, with a maximum near pH 6, and yields only one set of end products. Because of its rapid and predictable course, the latter reaction is preferred for sequential degradation of RNA. The structure of the catalytically active intermediate (general-acid-catalyzed reaction series) involves the primary amino group of ornithine (lysine) condensed with the dialdehyde terminus to form the carbinolamine, aldimine, and enamine intermediates leading to the elimination.The ether cleavage path is controlled by a specific-base (k_HB) intramolecular catalysis above pH 7, and a side reaction leads to lowered yields of phosphoric ester cleavage. A primary amine group is required, since 3-dimethylamino propylamine does not catalyze the ether cleavage.     

Tyrosine substitution of a conserved active‐site histidine residue activates Plasmodium falciparum peroxiredoxin 6

Peroxiredoxins efficiently remove hydroperoxides and peroxynitrite in pro‐ and eukaryotes. However, isoforms of one subfamily of peroxiredoxins, the so‐called Prx6‐type enzymes, usually have very low activities in standard peroxidase assays in vitro. In contrast to other peroxiredoxins, Prx6 homologues share a conserved histidyl residue at the bottom of the active site. Here we addressed the role of this histidyl residue for redox catalysis using the Plasmodium falciparum homologue PfPrx6 as a model enzyme. Steady‐state kinetics with tert‐butyl hydroperoxide (tBuOOH) revealed that the histidyl residue is nonessential for Prx6 catalysis and that a replacement with tyrosine can even increase the enzyme activity four‐ to six‐fold in vitro. Stopped‐flow kinetics with reduced PfPrx6^WT, PfPrx6^C128A, and PfPrx6^H39Y revealed a preference for H₂O₂ as an oxidant with second order rate constants for H₂O₂ and tBuOOH around 2.5 × 10⁷ M^?1 s^?1 and 3 × 10⁶ M^?1 s^?1, respectively. Differences between the oxidation kinetics of PfPrx6^WT, PfPrx6^C128A, and PfPrx6^H39Y were observed during a slower second‐reaction phase. Our kinetic data support the interpretation that the reductive half‐reaction is the rate‐limiting step for PfPrx6 catalysis in steady‐state measurements. Whether the increased activity of PfPrx6^H39Y is caused by a facilitated enzyme reduction because of a destabilization of the fully folded enzyme conformation remains to be analyzed. In summary, the conserved histidyl residue of Prx6‐type enzymes is non‐essential for catalysis, PfPrx6 is rapidly oxidized by hydroperoxides, and the gain‐of‐function mutant PfPrx6^H39Y might provide a valuable tool to address the influence of conformational changes on the reactivity of Prx6 homologues.     

Fluctuation analysis in small chemically reacting systems

A theory of noise fluctuations is developed which is applicable to systems of any size in which unimolecular or bimolecular reactions are occurring. The main difference between small and large reacting systems is that in the former the probability of finding a particle in a particular state does not obey a Gaussian distribution, but satisfies a distribution which reflects the mechanism of the chemical reaction. This difference is reflected in the main result of the theory: an autocorrelation function that is expressible as a sum of exponentials, the amplitudes of which are explicit functions of the moments of the distribution. Thus, by using small systems, the autocorrelation function,in principle, allows the elucidation of reaction mechanisms. Numerical simulations indicate that for reacting systems having ten or fewer particles, the deviation of the autocorrelation function from a single exponential should be easily detectable, and that estimates of the first four moments of the distribution should be possible. Accurate inference of the distribution, however, will require further mathematical and experimental advances.     

Study of the reactivity of quinohemoprotein alcohol dehydrogenase with heterocycle-pentacyanoferrate(III) complexes and the electron transfer path calculations

The reactivity of alcohol dehydrogenase IIG (ADH IIG) from Pseudomonas putida HK5 with new heterocycle-pentacyanoferrate(III) complexes and hexacyanoferrate(III) was determined at pH 7.2. The pentacyanoferrate(III) complexes contained imidazole, pyrazole, pyridine, their derivatives and 2-aminobenzothiazole as the sixth ligand. The largest reactivity of the complexes with ADH IIG was estimated for the complex containing pyridine. An apparent bimolecular constant (k _ox ) for this complex was 8.7 × 10⁵ M⁻¹s⁻¹. The lowest value of k _ox was estimated for the complex with benzotriazole (k _ox = 3.1 × 10⁴ M⁻¹s⁻¹). The investigation of the hexacyanoferrate(III) enzymatic reduction rate at different ionic strength gave a single negative charge of reduced ADH IIG. Docking calculations revealed two binding sites of the complexes in ADH-IIG structure. The first one is located at the entrance to the PQQ pocket, and the second is at the site of cytochrome domain. The calculations of electron transfer (ET) path indicated that the most effective ET takes place from heme to the complex docked at the entrance to the PQQ pocket. This shortest path is constructed of amino acids Ser607 and Cys606.     

Zinc ion-catalyzed ester hydrolysis of O-acetyl-2-acetylpyridineketoxime: bimolecular participation of hydroxozinc(II) ion

The Zn²⁺-catalyzed ester hydrolysis of O-acetyl-2-acetylpyridineketoxime (A) proceeds through three paths. The first two paths, the water path and the hydroxide path, have been observed in related systems. The third path, whose rate is bimolecular with respect to hydroxozinc(II) ion, is newly observed. The methyl group of A raises reaction rates through steric compression. The rate data obtained with A provide further evidences for the intramolecular nucleophilic attack of the metal-bound water molecule or hydroxide ion at the complexed ester. The third path is attributed either to the general base participation of free hydroxozinc(II) ion in the nucleophilic reaction of the Zn(II)-bound hydroxide ion or to the intramolecular reaction of dimeric hydroxozinc(II) ions. Implications of the present results to the actions of metalloenzymes, especially carbonic anhydrase and carboxypeptidase A, are also discussed.     

Interactions of superoxide anion with enzyme radicals: Kinetics of reaction with lysozyme tryptophan radicals and corresponding effects on tyrosine electron transfer

The kinetics of O^·-₂ reaction with semi-oxidized tryptophan radicals in lysozyme, Trp^·(Lyz) have been investigated at various pHs and conformational states by pulse radiolysis. The Trp^·(Lyz) radicals were formed by Br^·-₂ oxidation of the 3–4 exposed Trp residues in the protein. At pH lower than 6.2, the apparent bimolecular rate is about 2 × 10⁸M^-1s^-1; but drops to 8 × 10⁷M^-1s^-1 or less above pH 6.3 and in CTAC micelles. Similarly, the apparent bimolecular rate constant for the intermolecular Trp^·(Lyz) + Trp^·(Lyz) recombination reaction is about (4-7 × 10⁶M^-1s^-1) at/or below pH 6.2 then drops to 1.3-1.6 × 10⁶M^-1s^-1 at higher pH or in micelles. This behavior suggests important conformational and/or microenvironmental rearrangement with pH, leading to less accessible semioxidized Trp^· residues upon Br^·-₂ reaction. The kinetics of Trp^·(Lyz) with ascorbate, a reducing species rather larger than O^·-₂ have been measured for comparison. The well-established long range intramolecular electron transfer from Tyr residues to Trp radicals-leading to the repair of the semi-oxidized Trp^·(Lyz) and formation of the tyrosyl phenoxyl radical is inhibited by the Trp^·(Lyz)+O^·-₂ reaction, as is most of the Trp^·(Lyz)+Trp^·(Lyz) reaction. However, the kinetic behavior of Trp^·(Lyz) suggests that not all oxidized Trp residues are involved in the intermolecular recombination or reaction with O^·-₂. As the kinetics are found to be quite pH sensitive, this study demonstrates the effect of the protein conformation on O^·-₂ reactivity. To our knowledge, this is the first report on the kinetics of a protein-O^·-₂ reaction not involving the detection of change in the redox state of a prosthetic group to probe the reactivity of the superoxide anion.     

Effects of molecular association on structure and dynamics of a collagenous peptide

Peptide GVKGDKGNPGWPGAPY from the triple-helix domain of type IV collagen aggregates in solution at a critical aggregation concentration of 18 mM. This molecular self association process is investigated by ¹H- and ¹³C-nmr spectroscopy. As a function of increasing peptide concentration, selective ¹H resonances are cooperatively chemically shifted by up to 0.04 ppm to apparently saturable values at high concentration. Pulsed field gradient nmr was used to derive translation diffusion constants that, as the peptide concentration is increased, also cooperatively and monotonically decrease to an apparent limiting value. An average number of 6 monomer units per aggregate have been estimated from diffusion constant and ¹³C relaxation data. Comparative ¹H nuclear Overhauser effect spectroscopy (NOESY) spectra accumulated at high and low peptide concentrations suggest that average internuclear distances are decreased as a result of peptide association. ¹³C-nmr multiplet spin-lattice relaxation and ¹³C- {¹H} NOE effects on ¹³C-enriched glycine methylene positions in the peptide demonstrate that overall molecular tumbling and backbone internal motions are attenuated in the aggregate state. Lowering the solution pD from pD 6 to pD 2 disrupts the aggregate state, suggesting a role for electrostatic interactions in the association process. Based on thermodynamic considerations, hydrophobic interactions also probably act to stabilize the aggregate state. These data are discussed in terms of an nmr/NOE constrained computer-modeled structure of the peptide. © 1993 John Wiley & Sons, Inc.     

Chemical modification ofSemele solida arginase by diethyl pyrocarbonate: Evidence for a critical histidine residue

Arginase from the gills of the bivalveSemele solida was inactivated by diethyl pyrocarbonate (DEPC) in a pseudo-first-order reaction with a bimolecular rate constant of 160 M⁻¹ min⁻¹. The reaction order with respect to DEPC concentration was 1, the inactivation followed a titration curve for a residue with a pK_a of 6.4 at 25°C and the enzymatic activity was restored by hydroxylamine. It is concluded that inactivation results from the modification of a single histidine residue. Borate, a noncompetitive inhibitor with respect to arginine, protected the enzyme from inactivation by DEPC.     

Kinetics and thermodynamics of peroxidase- and laccase-catalyzed oxidation of N-substituted phenothiazines and phenoxazines

N -substituted phenothiazines (PTs) and phenoxazines (POs) catalyzed by fungal Coprinus cinereus peroxidase and Polyporus pinsitus laccase were investigated at pH 4–10. In the case of peroxidase, an apparent bimolecular rate constant (expressed as k _cat/K _m) varied from 1 ×10⁷M⁻¹s⁻¹to 2.6×10⁸M⁻¹s⁻¹ at pH 7.0. The constants for PO oxidation were higher in comparison to PT. pH dependence revealed two or three ionizable groups with pK _a values of 4.9–5.7 and 7.7–9.7 that significantly affected the activity of peroxidase. Single-turnover experiments showed that the limiting step of PT oxidation was reduction of compound II and second-order rate constants were obtained which were consistent with the constants at steady-state conditions. Laccase-catalyzed PT and PO oxidation rates were lower; apparent bimolecular rate constants varied from 1.8×10⁵M⁻¹s⁻¹ to 2.0×10⁷M⁻¹s⁻¹ at pH 5.3. PO constants were higher in comparison to PT, as was the case with peroxidase. The dependence of the apparent bimolecular constants of compound II or copper type 1 reduction, in the case of peroxidase or laccase, respectively, was analyzed in the framework of the Marcus outer-sphere electron-transfer theory. Peroxidase-catalyzed reactions with PT, as well as PO, fitted the same hyperbolic dependence with a maximal oxidation rate of 1.6×10⁸M⁻¹s⁻¹ and a reorganization energy of 0.30 eV. The respective parameters for laccase were 5.0×10⁷M⁻¹s⁻¹ and 0.29 eV. Received: 20 September 1999 / Accepted: 24 February 2000     

Infinite-dilution viscoelastic properties of tobacco mosaic virus.

The storage and loss shear moduli, G′ and G″, have been measured for dilute solutions of unaggregated and aggregated tobacco mosaic virus samples in glycerol–water mixtures, by the Birnboim–Schrag multiple-lumped resonator modified for use with aqueous solvents. The frequency range was 100–5800 Hz, the concentration range 0.6–2.1 × 10^?3 g/ml, and the temperatures 25.0° and 37.8°C. The number-average and weight-average molecular weights of the aggregated sample were estimated as 1.4 and 2.0 × 10⁸, respectively, from electron microscopy. The extrapolated intrinsic moduli [G′] and [G″] were compared with the predictions of the Kirkwood–Auer theory for rigid rodlike molecules. For the unaggregated sample, the frequency dependence of [G′] and [G″] agreed well with the theory assuming the intrinsic viscosity to be 27 ml/g, though the asymptotic limit of [G′]M/RT at higher frequencies was slightly larger than the theoretical value of 3/5. For the aggregated sample, the data agreed with theory for rigid rods as modified to account for molecular-weight distribution.     

Absence of kinetic barrier for transfer of protons from aqueous phase to membrane-water interface

The kinetics of interfacial proton transfer reaction is an important factor in proton transport across membranes. The following experimental system was designed in order to measure this kinetics. Sonicated liposomes having the protonophore SF6847 was suspended in Tris buffer. Application of a temperature jump (in ∼ 3 μs) caused a drop in the aqueous phase pH which was subsequently sensed by the membrane-bound SF6847. The kinetics of this interfacial proton transfer reaction was monitored on μs timescales. The estimated bimolecular rate constant of 2×10¹¹ M⁻¹ s_#x2212;1 for this process show that there is no kinetic barrier for the transfer of protons from the aqueous phase to the membrane-water interface.     

Dynamic light scattering of aqueous solutions of linear aggregates induced by thermal denaturation of ovalbumin

Dynamic light scattering measurements were performed on dilute aqueous solutions of native ovalbumin (OA) and on those of linear OA aggregates induced by thermal denaturation at low ionic strength and neutral pH. The weight-average molecular weight M_w of four aggregates tested ranged from 1,700,000 to 5,500,000. The translational diffusion coefficient D₀ of native OA at infinite dilution was estimated as 8.70 × 10 ^?7 cm²/s, which gave 56.0 Å as the diameter of the rigid spherical particle. The intensity autocorrelation function of linear OA polymers was analyzed with the cumulant method to obtain the first cumulant Λ_e. The dependence of Λ_e on the scattering vector q at very low polymer concentration was found intermediate between those of a flexible chain and a rigid rod. The translational diffusion coefficient D_tr [≡ (T_e/q²)_{q → 0}] was in proportion to M, and the magnitude was in good agreement with a value calculated from the wormlike cylinder model with values of three parameters determined in an earlier study, M_L = 1600 Å^?1, d = 120 Å, and Q = 230 Å, where M_L, d, and Q are the molecular weight per unit length, diameter, and persistence length, respectively. Based on these results, a new model, to be called as the dimer model, was proposed to interpret the formation mechanism of linear OA polymers induced by thermal denaturation. © 1993 John Wiley & Sons, Inc.