An improved fluorescent substrate for assaying soluble and membrane-associated ADAM family member activities

A fluorescent resonance energy transfer substrate with improved sensitivity for ADAM17, −10, and −9 (where ADAM represents a disintegrin and metalloproteinase) has been designed. The new substrate, Dabcyl-Pro-Arg-Ala-Ala-Ala-Homophe-Thr-Ser-Pro-Lys(FAM)-NH₂, has specificity constants of 6.3 (±0.3) × 10⁴ M⁻¹ s⁻¹ and 2.4 (±0.3) × 10³ M⁻¹ s⁻¹ for ADAM17 and ADAM10, respectively. The substrate is more sensitive than widely used peptides based on the precursor tumor necrosis factor-alpha (TNF-alpha) cleavage site, PEPDAB010 or Dabcyl-Ser-Pro-Leu-Ala-Gln-Ala-Val-Arg-Ser-Ser-Lys(FAM)-NH₂ and Mca-Pro-Leu-Ala-Gln-Ala-Val-Dpa-Arg-Ser-Ser-Arg-NH₂. ADAM9 also processes the new peptide more than 18-fold better than the TNF-alpha-based substrates. The new substrate has a unique selectivity profile because it is processed less efficiently by ADAM8 and MMP1, −2, −3, −8, −9, −12, and −14. This substrate provides a unique tool in which to assess ADAM17, −10, and −9 activities.     

Novel internally quenched substrate of the trypsin-like subunit of 20S eukaryotic proteasome

This article describes the synthesis, using combinatorial chemistry, of internally quenched substrates of the trypsin-like subunit of human 20S proteasome. Such substrates were optimized in both the nonprime and prime regions of the peptide chain. Two were selected as the most susceptible for proteasomal proteolysis with excellent kinetic parameters: (i) ABZ-Val-Val-Ser-Arg-Ser-Leu-Gly-Tyr(3-NO₂)-NH₂ (k_cat/K_M = 934,000 M⁻¹ s⁻¹) and (ii) ABZ-Val-Val-Ser-GNF-Ala-Met-Gly-Tyr(3-NO₂)-NH₂ (k_cat/K_M = 1,980,000 M⁻¹ s⁻¹). Both compounds were efficiently hydrolyzed by the 20S proteasome at picomolar concentrations, demonstrating significant selectivity over other proteasome entities.     

The base exchange reaction of NAD+ glycohydrolase: identification of novel heterocyclic alternative substrates

ADP-ribosyl cyclase and NAD⁺ glycohydrolase (CD38, E.C.3.2.2.5) efficiently catalyze the exchange of the nicotinamidyl moiety of NAD⁺, nicotinamide adenine dinucleotide phosphate (NADP⁺) or nicotinamide mononucleotide (NMN⁺) with an alternative base. 4′-Pyridinyl drugs (amrinone, milrinone, dismerinone and pinacidil) were efficient alternative substrates (k_cat/K_M = 0.9-10 μM⁻¹ s⁻¹) in the exchange reaction with ADP-ribosyl cyclase. When CD38 was used as a catalyst the k_cat/K_M values for the exchange reaction were reduced two or more orders of magnitude (0.015-0.15 μM⁻¹ s⁻¹). The products of this reaction were novel dinucleotides. The values of the equilibrium constants for dinucleotide formation were determined for several drugs. These enzymes also efficiently catalyze the formation of novel mononucleotides in an exchange reaction with NMN⁺, k_cat/K_M = 0.05-0.4 μM⁻¹ s⁻¹. The k_cat/K_M values for the exchange reaction with NMN⁺ were generally similar (0.04-0.12 μM⁻¹ s⁻¹) with CD38 and ADP-ribosyl cyclase as catalysts. Several novel heterocyclic alternative substrates were identified as 2-isoquinolines, 1,6-naphthyridines and tricyclic bases. The k_cat/K_M values for the exchange reaction with these substrates varied over five orders of magnitude and approached the limit of diffusion with 1,6-naphthyridines. The exchange reaction could be used to synthesize novel mononucleotides or to identify novel reversible inhibitors of CD38.     

Promiscuity, stability and cold adaptation of a newly isolated acylaminoacyl peptidase

We report on the characterisation of a member of the acylaminoacyl peptidase family, the first isolated from bacteria. The enzyme was obtained from the psychrophilic bacterium Sporosarcina psychrophila and shows the typical features of cold adaptation (low T_m, optimal temperature of 40 °C, poor thermal stability). It was also tested for substrate specificity, effect of metals, temperature dependence and structure stability and revealed promiscuous catalytic activity on at least two chemically distinct substrates, with k_cat/K_m values for ester hydrolysis and acylamino acids cleavage of 1.7 × 10⁴ s⁻¹ M⁻¹ and 6.2 × 10³ s⁻¹ M⁻¹, respectively. Despite some properties cannot be explained with current models, results report on the relevance of structural and catalytic properties for the successful adaptation to cold temperatures.     

Rationalizing 5000-fold differences in receptor-binding rate constants of four cytokines

The four cytokines erythropoietin (EPO), interleukin-4 (IL4), human growth hormone (hGH), and prolactin (PRL) all form four-helix bundles and bind to type I cytokine receptors. However, their receptor-binding rate constants span a 5000-fold range. Here, we quantitatively rationalize these vast differences in rate constants by our transient-complex theory for protein-protein association. In the transient complex, the two proteins have near-native separation and relative orientation, but have yet to form the short-range specific interactions of the native complex. The theory predicts the association rate constant as where k_a0 is the basal rate constant for reaching the transient complex by random diffusion, and the Boltzmann factor captures the rate enhancement due to electrostatic attraction. We found that the vast differences in receptor-binding rate constants of the four cytokines arise mostly from the differences in charge complementarity among the four cytokine-receptor complexes. The basal rate constants (k_a0) of EPO, IL4, hGH, and PRL were similar (5.2 × 10⁵ M⁻¹s⁻¹, 2.4 × 10⁵ M⁻¹s⁻¹, 1.7 × 10⁵ M⁻¹s⁻¹, and 1.7 × 10⁵ M⁻¹s⁻¹, respectively). However, the average electrostatic free energies () were very different (−4.2 kcal/mol, −2.4 kcal/mol, −0.1 kcal/mol, and −0.5 kcal/mol, respectively, at ionic strength = 160 mM). The receptor-binding rate constants predicted without adjusting any parameters, 6.2 × 10⁸ M⁻¹s⁻¹, 1.3 × 10⁷ M⁻¹s⁻¹, 2.0 × 10⁵ M⁻¹s⁻¹, and 7.6 × 10⁴ M⁻¹s⁻¹, respectively, for EPO, IL4, hGH, and PRL agree well with experimental results. We uncover that these diverse rate constants are anticorrelated with the circulation concentrations of the cytokines, with the resulting cytokine-receptor binding rates very close to the limits set by the half-lives of the receptors, suggesting that these binding rates are functionally relevant and perhaps evolutionarily tuned. Our calculations also reproduced well-observed effects of mutations and ionic strength on the rate constants and produced a set of mutations on the complex of hGH with its receptor that putatively enhances the rate constant by nearly 100-fold through increasing charge complementarity. To quantify charge complementarity, we propose a simple index based on the charge distribution within the binding interface, which shows good correlation with . Together these results suggest that protein charges can be manipulated to tune k_a and control biological function.     

Single-Molecule Kinetics Reveal Cation-Promoted DNA Duplex Formation Through Ordering of Single-Stranded Helices

In this work, the kinetics of short, fully complementary oligonucleotides are investigated at the single-molecule level. Constructs 6–9 bp in length exhibit single exponential kinetics over 2 orders of magnitude time for both forward (k_on, association) and reverse (k_off, dissociation) processes. Bimolecular rate constants for association are weakly sensitive to the number of basepairs in the duplex, with a 2.5-fold increase between 9 bp (k′_on = 2.1(1) × 10⁶ M⁻¹ s⁻¹) and 6 bp (k′_on = 5.0(1) × 10⁶ M⁻¹ s⁻¹) sequences. In sharp contrast, however, dissociation rate constants prove to be exponentially sensitive to sequence length, varying by nearly 600-fold over the same 9 bp (k_off = 0.024 s⁻¹) to 6 bp (k_off = 14 s⁻¹) range. The 8 bp sequence is explored in more detail, and the NaCl dependence of k_on and k_off is measured. Interestingly, k_onincreases by >40-fold (k_on = 0.10(1) s⁻¹ to 4.0(4) s⁻¹ between [NaCl] = 25 mM and 1 M), whereas in contrast, k_offdecreases by fourfold (0.72(3) s⁻¹ to 0.17(7) s⁻¹) over the same range of conditions. Thus, the equilibrium constant (K_eq) increases by ≈160, largely due to changes in the association rate, k_on. Finally, temperature-dependent measurements reveal that increased [NaCl] reduces the overall exothermicity (ΔΔH° > 0) of duplex formation, albeit by an amount smaller than the reduction in entropic penalty (−TΔΔS° < 0). This reduced entropic cost is attributed to a cation-facilitated preordering of the two single-stranded species, which lowers the association free-energy barrier and in turn accelerates the rate of duplex formation.     

The new fluorogenic substrates of neutrophil proteinase 3 optimized in prime site region

Previously selected by the combinatorial chemistry approach, potent fluorogenic substrate of proteinase 3 was used as the starting structure to design new substrates. The general formula of the synthesized peptides is as follows: ABZ-Tyr-Tyr-Abu-ANB-X-NH₂, where ANB (5-amino-2-nitrobenzoic acid) served as a chromophore and an acceptor of fluorescence, ABZ (aminobenzoic acid) is a donor of fluorescence in these fluorescence resonance energy transfer (FRET) peptides, and X is a proteinogenic amino acid (except Cys). The introduced modifications influenced substrate activity of the synthesized peptides. The highest value of specificity constant for proteinase 3 was obtained for the single peptide with Gln in the discussed position (k_cat/K_M = 275,000 M⁻¹ s⁻¹), which was nearly twice as active as the reference compound (lacking a substituent in the X position). In addition, more efficient energy transfer was observed, due mainly to the bathochromic effect for the introduced modification. This approach opens a new possibility to design potent and highly specific substrates of proteinase 3 and other proteinases optimized in the prime site region.     

Synthesis and characterization of novel fluorogenic substrates of coagulation factor XIII-A

Further development of our recently published Glu(pNA)-containing peptides (Anal. Biochem. 428 (2012) 73–80) provided new fluorogenic substrates for the activated blood coagulation factor XIII. A first series was designed by incorporation of Glu(AMC) at the penultimate position from the N terminus. For the best derivative H-Tyr-Glu(AMC)-Val-Lys-Val-Ile-NH₂, a moderate k_cat/K_m value of 34 s⁻¹ M⁻¹ was determined, which is more than 100-fold reduced compared with the previously reported Glu(pNA) substrates. Furthermore, two fluorescence resonance energy transfer (FRET) substrates were prepared by incorporation of an N-methyl-anthraniloyl fluorophore and a 2,4-dinitrophenyl quencher. Both substrates were excellently cleaved by FXIII-A₂^∗, which is generated from its zymogen by activation of thrombin in the presence of calcium ions. In the absence and presence of H-Gly-ethyl ester, k_cat/K_m values of 8010 and 8660 s⁻¹ M⁻¹, respectively, were found for the conversion of H-Lys(N(Me)Abz)-Glu(NH-(CH₂)₄-NH-Dnp)-Val-Lys-Val-Ile-Gly-NH₂ (substrate 8). These values are more than 200-fold improved compared with the Glu(AMC) substrates. Substrate 8 is suitable for the measurement of FXIII-A₂^∗ activities in plasma samples as well as for in vitro measurements. Furthermore, it was used for the determination of the inhibitory potency of a newly synthesized chloromethyl ketone derivative, Cbz-Phe-Glu(CMK)-Val-Lys-Val-Ile-Gly-NH₂, which was found to be a potent irreversible inhibitor of FXIII-A₂^∗.     

Acrylonitrile Quenching of Trp Phosphorescence in Proteins: A Probe of the Internal Flexibility of the Globular Fold

Quenching of Trp phosphorescence in proteins by diffusion of solutes of various molecular sizes unveils the frequency-amplitude of structural fluctuations. To cover the sizes gap between O₂ and acrylamide, we examined the potential of acrylonitrile to probe conformational flexibility of proteins. The distance dependence of the through-space acrylonitrile quenching rate was determined in a glass at 77 K, with the indole analog 2-(3-indoyl) ethyl phenyl ketone. Intensity and decay kinetics data were fitted to a rate, k(r) = k₀ exp[−(r − r₀)/r_e], with an attenuation length r_e = 0.03 nm and a contact rate k₀ = 3.6 × 10¹⁰ s⁻¹. At ambient temperature, the bimolecular quenching rate constant (kq) was determined for a series of proteins, appositely selected to test the importance of factors such as the degree of Trp burial and structural rigidity. Relative to kq = 1.9 × 10⁹ M⁻¹s⁻¹ for free Trp in water, in proteins kq ranged from 6.5 × 10⁶ M⁻¹s⁻¹ for superficial sites to 1.3 × 10² M⁻¹s⁻¹ for deep cores. The short-range nature of the interaction and the direct correlation between kq and structural flexibility attest that in the microsecond-second timescale of phosphorescence acrylonitrile readily penetrates even compact protein cores and exhibits significant sensitivity to variations in dynamical structure of the globular fold.     

Recombinant glucose oxidase from Penicillium amagasakiense for efficient bioelectrochemical applications in physiological conditions

GOX is the most widely used enzyme for the development of electrochemical glucose biosensors and biofuel cell in physiological conditions. The present work describes the production of a recombinant glucose oxidase from Penicillium amagasakiense (yGOXpenag) displaying a more efficient glucose catalysis (k_cat/K_M(glucose) = 93 μM⁻¹ s⁻¹) than the native GOX from Aspergillus niger (nGOXaspng), which is the most industrially used (k_cat/K_M(glucose) = 27 μM⁻¹ s⁻¹). Expression in Pichia pastoris allowed easy production and purification of the recombinant active enzyme, without overglycosylation. Its biotechnological interest was further evaluated by measuring kinetics of ferrocinium-methanol (FM_ox) reduction, which is commonly used for electron transfer to the electrode surface. Despite their homologies in sequence and structure, pH-dependant FM_ox reduction was different between the two enzymes. At physiological pH and temperature, we observed that electron transfer to the redox mediator is also more efficient for yGOXpenag than for nGOXaspng(k_cat/K_M(FM_ox) = 27 μM⁻¹ s⁻¹ and 17 μM⁻¹ s⁻¹ respectively). In our model system, the catalytic current observed in the presence of blood glucose concentration (5 mM) was two times higher with yGOXpenag than with nGOXaspng. All our results indicated that yGOXpenag is a better candidate for industrial development of efficient bioelectrochemical devices used in physiological conditions.     

Mechanism and biological implications of the NO release of cis-[Ru(bpy)2L(NO)](n+) complexes: a key role of physiological thiols

Nitric oxide (NO) has a critical role in several physiological and pathophysiological processes. In this paper, the reactions of the nitrosyl complexes of [Ru(bpy)₂L(NO)]ⁿ⁺ type, where L = SO₃²⁻ and imidazole and bpy = 2,2′-bipiridine, with cysteine and glutathione were studied. The reactions with cysteine and glutathione occurred through the formation of two sequential intermediates, previously described elsewhere, [Ru(bpy)₂L(NOSR)]ⁿ⁺ and [Ru(bpy)₂L(NOSR)₂] (SR = thiol) leading to the final products [Ru(bpy)₂L(H₂O)]ⁿ⁺ and free NO. The second order rate constant for the second step of this reaction was calculated for cysteine k₂(SR⁻) = (2.20 ± 0.12) × 10⁹ M^− 1 s^− 1 and k_2(RSH) = (154 ± 2) M^− 1 s^− 1 for L = SO₃²⁻ and k₂(SR⁻) = (1.30 ± 0.23) × 10⁹ M^− 1 s^− 1 and k₂(RSH) = (0.84 ± 0.02) M^− 1 s^− 1 for L = imidazole; while for glutathione they were k₂(SR⁻) = (6.70 ± 0.32) × 10⁸ M^− 1 s^− 1 and k₂(RSH) = 11.8 ± 0.3 M^− 1 s^− 1 for L = SO₃²⁻ and k₂(SR⁻) = (2.50 ± 0.36) × 10⁸ M^− 1 s^− 1 and k₂(RSH) = 0.32 ± 0.01 M^− 1 s^− 1 for L = imidazole. In all reactions it was possible to detect the release of NO from the complexes, which it is remarkably distinct from other ruthenium metallocompounds described elsewhere with just N₂O production. These results shine light on the possible key role of NO release mediated by physiological thiols in reaction with these metallonitrosyl ruthenium complexes.     

Simple phosphonic inhibitors of human neutrophil elastase

Herein, we describe the synthesis and resulting activity of a complex series of α-aminophosphonate diaryl esters as irreversible human neutrophil elastase inhibitors and their selectivity preference for human neutrophil elastase over several other serine proteases such as porcine pancreatic elastase, trypsin, and chymotrypsin. We synthesized and examined the inhibitory potency of several new simple Cbz-protected α-aminoalkylphosphonate diaryl esters that yielded several new HNE inhibitors, where one of the obtained compounds Cbz-Val^P(OC₆H₄-4-COOMe)₂ displayed an apparent second-order inhibition value at 33,015 M⁻¹ s⁻¹.     

Mononuclear and dinuclear mechanisms for catalysis of phosphodiester cleavage by alkaline earth metal ions in aqueous solution

In biological systems, enzymes often use metal ions, especially Mg²⁺, to catalyze phosphodiesterolysis, and model aqueous studies represent an important avenue of examining the contributions of these ions to catalysis. We have examined Mg²⁺ and Ca²⁺ catalyzed hydrolysis of the model phosphodiester thymidine-5′-p-nitrophenyl phosphate (T5PNP). At 25 °C, we find that, despite their different Lewis acidities, these ions have similar catalytic ability with second-order rate constants for attack of T5PNP by hydroxide (k_OH) of 4.1 × 10⁻⁴ M⁻¹s⁻¹ and 3.7 × 10⁻⁴ M⁻¹s⁻¹ in the presence of 0.30 M Mg²⁺ and Ca²⁺, respectively, compared to 8.3 × 10⁻⁷ M⁻¹s⁻¹ in the absence of divalent metal ion. Examining the dependence of k_OH on [M²⁺] at 50 °C indicates different kinetic mechanisms with Mg²⁺ utilizing a single ion mechanism and Ca²⁺ operating by parallel single and double ion mechanisms. Association of the metal ion(s) occurs prior to nucleophilic attack by hydroxide. Comparing the k_OH values reveals a single Mg²⁺ catalyzes the reaction by 1800-fold whereas a single Ca²⁺ ion catalyzes the reaction by only 90-fold. The second Ca²⁺ provides an additional 10-fold catalysis, significantly reducing the catalytic disparity between Mg²⁺ and Ca²⁺.     

Dissecting a bimolecular process of MgATP²- binding to the chaperonin GroEL

Although allosteric transitions of GroEL by MgATP²⁻ have been widely studied, the initial bimolecular step of MgATP²⁻ binding to GroEL remains unclear. Here, we studied the equilibrium and kinetics of MgATP²⁻ binding to a variant of GroEL, in which Tyr485 was replaced by tryptophan, via isothermal titration calorimetry (ITC) and stopped-flow fluorescence spectroscopy. In the absence of K⁺ at 4-5 °C, the allosteric transitions and the subsequent ATP hydrolysis by GroEL are halted, and hence, the stopped-flow fluorescence kinetics induced by rapid mixing of MgATP²⁻ and the GroEL variant solely reflected MgATP²⁻ binding, which was well represented by bimolecular noncooperative binding with a binding rate constant, k_on, of 9.14 × 10⁴ M^− 1 s^− 1 and a dissociation rate constant, k_off, of 14.2 s^− 1, yielding a binding constant, K_b (= k_on/k_off), of 6.4 × 10³ M^− 1. We also successfully performed ITC to measure binding isotherms of MgATP²⁻ to GroEL and obtained a K_b of 9.5 × 10³ M^− 1 and a binding stoichiometric number of 6.6. K_b was thus in good agreement with that obtained by stopped-flow fluorescence. In the presence of 10-50 mM KCl, the fluorescence kinetics consisted of three to four phases (the first fluorescence-increasing phase, followed by one or two exponential fluorescence-decreasing phases, and the final slow fluorescence-increasing phase), and comparison of the kinetics in the absence and presence of K⁺ clearly demonstrated that the first fluorescence-increasing phase corresponds to bimolecular MgATP²⁻ binding to GroEL. The temperature dependence of the kinetics indicated that MgATP²⁻ binding to GroEL was activation-controlled with an activation enthalpy as large as 14-16 kcal mol^− 1.     

[H,N] NMR studies of the aquation of cis-diamine platinum(II) complexes

Two ¹⁵N-labelled cis-Pt(II) diamine complexes with dimethylamine (¹⁵N-dma) and isopropylamine (¹⁵N-ipa) ligands have been prepared and characterised. [¹H,¹⁵N] HSQC NMR spectroscopy is used to obtain the rate and equilibrium constants for the aquation of cis-[PtCl₂(¹⁵N-dma)₂] at 298 K in 0.1 M NaClO₄ and to determine the pK_a values of cis-[PtCl(H₂O)(¹⁵N-dma)₂]⁺ (6.37) and cis-[Pt(H₂O)₂(¹⁵N-dma)₂]²⁺ (pK_a1 = 5.17, pK_a2 = 6.47). The rate constants for the first and second aquation steps (k₁ = (2.12 ± 0.01) × 10⁻⁵ s⁻¹, k₂ = (8.7 ± 0.7) × 10⁻⁶ s⁻¹) and anation steps (k₋₁ = (6.7 ± 0.8) × 10⁻³ M⁻¹ s⁻¹, k₋₂ = 0.043 ± 0.004 M⁻¹ s⁻¹) are very similar to those reported for cisplatin under similar conditions, and a minor difference is that slow formation of the hydroxo-bridged dimer is observed. Aquation studies of cis-[PtCl₂(¹⁵N-ipa)₂] were precluded by the close proximity of the NH proton signal to the ¹H₂O resonance.     

The hormone-sensitive lipase from Psychrobacter sp. TA144: New insight in the structural/functional characterization

Cold-adapted esterases and lipases have been found to be dominant activities throughout the cold marine environment, indicating their importance in bacterial degradation of the organic matter. lip2 Gene from Psychrobacter sp. TA144, a micro-organism isolated from the Antarctic sea water, was cloned and over-expressed in Escherichia coli. The recombinant protein (PsyHSL) accumulated in the insoluble fraction from which it was recovered in active form, purified to homogeneity and deeply characterised. Temperature dependence of PsyHSL activity was typical of psychrophilic enzymes, with an optimal temperature of 35 °C at pH 8.0. The enzyme resulted to be active on pNP-esters of fatty acids with acyl chain length from C₂ to C₁₂ and the preferred substrate was pNP-pentanoate showing a k_cat = 26.2 ± 0.1 s⁻¹, K_M = 0.122 ± 0.006 mM and a k_cat/K_M = 215 ± 11 mM⁻¹ s⁻¹. The enzyme was strongly inhibited by Hg²⁺, Zn²⁺, Cu²⁺, Fe³⁺, Mn²⁺ ions and it resulted to be activated in presence of methanol and acetonitrile, with calculated C₅₀ values of 1.98 M and 0.92 M, respectively.     

Inhibition studies with anions and small molecules of two novel β-carbonic anhydrases from the bacterial pathogen Salmonella enterica serovar Typhimurium

Two new β-carbonic anhydrases (CAs, EC 4.2.1.1) from the bacterial pathogen Salmonella enterica serovar Typhimurium, stCA 1 and stCA 2, were characterized kinetically. The two enzymes possess appreciable activity as catalysts for the hydration of CO₂ to bicarbonate, with k_cat of 0.79 × 10⁶ s⁻¹ and 1.0 × 10⁶ s⁻¹, and k_cat/K_m of 5.2 × 10⁷ M⁻¹ s⁻¹ and of 8.3 × 10⁷ M⁻¹ s⁻¹, respectively. A large number of simple/complex inorganic anions as well as other small molecules (sulfamide, sulfamic acid, phenylboronic acid, phenylarsonic acid, dialkyldithiocarbamates) showed interesting inhibitory properties towards the two new enzymes, with several low micromolar inhibitors discovered. As many strains of S. enterica show extensive resistance to classical antibiotics, inhibition of the β-CAs investigated here may be useful for developing lead compounds for novel types of antibacterials.     

A Phos-tag-based fluorescence resonance energy transfer system for the analysis of the dephosphorylation of phosphopeptides

Fluorescence resonance energy transfer (FRET) is a distance-dependent interaction between the electronic excited states of two dye molecules. Here we introduce a novel FRET system for the detection of phosphopeptides using a phosphate-binding tag molecule, Zn²⁺-Phos-tag (1,3-bis[bis(pyridin-2-ylmethyl)amino]propan-2-olato dizinc(II) complex) attached with a 7-amino-4-methylcoumarin-3-acetic acid (AMCA). Carboxyfluorescein (FAM)-labeled phospho- and nonphosphopeptides were prepared as the target molecules for the FRET system. A set of FAM (a fluorescent acceptor, λ_em 520 nm) and AMCA (a fluorescent donor, λ_ex 345 nm) is frequently used for a FRET system. The AMCA-labeled Zn²⁺-Phos-tag specifically captured the FAM-labeled phosphopeptide to form a stable 1:1 complex, resulting in efficient FRET. After the FAM-labeled phosphopeptide was dephosphorylated with alkaline phosphatase, the FRET disappeared. Using this FRET system, we demonstrated the detection of the time-dependent dephosphorylation of the FAM-labeled protein-tyrosine phosphatase 1B substrate.     

Cyanobacterial cytochrome cM: Probing its role as electron donor for CuA of cytochrome c oxidase

It is well known that efficient functioning of photosynthetic (PET) and respiratory electron transport (RET) in cyanobacteria requires the presence of either cytochrome c₆ (Cytc₆) or plastocyanin (PC). By contrast, the interaction of an additional redox carrier, cytochrome c_M (Cytc_M), with either PET or RET is still under discussion. Here, we focus on the (putative) role of Cytc_M in cyanobacterial respiration. It is demonstrated that genes encoding the main terminal oxidase (cytochrome c oxidase, COX) and cytochrome c_M are found in all 44 totally or partially sequenced cyanobacteria (except one strain). In order to check whether Cytc_M can act as electron donor to COX, we investigated the intermolecular electron transfer kinetics between Cytc_M and the soluble Cu_A domain (i.e. the donor binding and electron entry site) of subunit II of COX. Both proteins from Synechocystis PCC6803 were expressed heterologously in E. coli. The forward and the reverse electron transfer reactions were studied yielding apparent bimolecular rate constants of (2.4 ± 0.1) × 10⁵ M^− 1 s^− 1 and (9.6 ± 0.4) × 10³ M^− 1 s^− 1 (5 mM phosphate buffer, pH 7, 50 mM KCl). A comparative analysis with Cytc₆ and PC demonstrates that Cytc_M functions as electron donor to Cu_A as efficiently as Cytc₆ but more efficient than PC. Furthermore, we demonstrate the association of Cytc_M with the cytoplasmic and thylakoid membrane fractions by immunobloting and discuss the potential role of Cytc_M as electron donor for COX under stress conditions.     

Bicarbonate-catalyzed hydrogen peroxide oxidation of cysteine and related thiols

The effect of bicarbonate on the rates of the H₂O₂ oxidation of cysteine, gluthathione, and N-acetylcysteine to the corresponding disulfides was investigated. The relative oxidation rates at pH 8 for the different thiols are inversely related to the pK_a values of the thiol groups, and the reactive nucleophiles are identified as the thiolate anions or their kinetic equivalents. The second-order rate constants at 25 °C for the reaction of the thiolate anions with hydrogen peroxide are 17 ± 2 M⁻¹ s⁻¹ for all three substrates. In the presence of bicarbonate (>25 mM), the observed rate of thiolate oxidation is increased by a factor of two or more, and the catalysis is proposed to be associated with the formation of peroxymonocarbonate from the equilibrium reaction of hydrogen peroxide with bicarbonate (via CO₂). The calculated second-order rate constants for the direct reaction of the three thiolate anions with peroxymonocarbonate fall within the range of 900-2000 M⁻¹ s⁻¹. Further oxidation of disulfides by peroxymonocarbonate results in the formation of thiosulfonate and sulfonate products. These results strongly suggest that peroxymonocarbonate should be considered as a reactive oxygen species in aerobic metabolism with relevance in thiol oxidations.