Structure activity relationship studies of cinnamic acid derivatives as inhibitors of human neutrophil elastase revealed by ligand docking calculations.

Structure-activity relationship of cinnamic acid derivatives as inhibitors of the human neutrophil elastase is reported. Comparison of the inhibitory concentrations (IC50 values) with the results of the ligand docking calculations revealed that the structure element of the aromatic ortho-dihydroxy groups combined with a lipophilic residue seems to be a prerequisite for an optimal binding within the active site.

Sesquiterpene lactones as inhibitors of human neutrophil elastase.

Human neutrophil elastase (HNE) is a serine protease that has been implicated in the abnormal turnover of connective tissue proteins and has been described as an important pathogenic factor in several inflammatory diseases such as rheumatoid arthritis or cystic fibrosis. Here we investigated 17 sesquiterpene lactones (SLs) for their ability to inhibit human neutrophil elastase in an in vitro assay. Podachaenin was the most active compound with an IC(50) value of 7 microM. SLs do not covalently bind to the amino acids of the catalytic triad, thus differing from other elastase inhibitors with a lactone moiety. In contrast to most other biological activities of SLs HNE inhibition is not mediated by alpha,beta-unsaturated carbonyl functions. Ligand binding calculations using the X-ray structure of HNE and the program FlexX revealed structural elements which are a prerequisite for their inhibitory activity.

Photo-induced cyclic electron transfer involving cytochrome bc1 complex and reaction center in the obligate aerobic phototroph Roseobacter denitrificans.

Flash-induced redox changes of b-type and c-type cytochromes have been studied in chromatophores from the aerobic photosynthetic bacterium Roseobacter denitrificans under redox-controlled conditions. The flash-oxidized primary donor P+ of the reaction center (RC) is rapidly re-reduced by heme H1 (Em,7 = 290 mV), heme H2 (Em,7 = 240 mV) or low-potential hemes L1/L2 (Em,7 = 90 mV) of the RC-bound tetraheme, depending on their redox state before photoexcitation. By titrating the extent of flash-induced low-potential heme oxidation, a midpoint potential equal to -50 mV has been determined for the primary quinone acceptor QA. Only the photo-oxidized heme H2 is re-reduced in tens of milliseconds, in a reaction sensitive to inhibitors of the bc1 complex, leading to the concomitant oxidation of a cytochrome c spectrally distinct from the RC-bound hemes. This reaction involves cytochrome c551 in a diffusional process. Participation of the bc1 complex in a cyclic electron transfer chain has been demonstrated by detection of flash-induced reduction of cytochrome b561, stimulated by antimycin and inhibited by myxothiazol. Cytochrome b561, reduced upon flash excitation, is re-oxidized slowly even in the absence of antimycin. The rate of reduction of cytochrome b561 in the presence of antimycin increases upon lowering the ambient redox potential, most likely reflecting the progressive prereduction of the ubiquinone pool. Chromatophores contain approximately 20 ubiquinone-10 molecules per RC. At the optimal redox poise, approximately 0.3 cytochrome b molecules per RC are reduced following flash excitation. Cytochrome b reduction titrates out at Eh < 100 mV, when low-potential heme(s) rapidly re-reduce P+ preventing cyclic electron transfer. Results can be rationalized in the framework of a Q-cycle-type model.

Conformational changes of the H+-ATPase from Escherichia coli upon nucleotide binding detected by single molecule fluorescence.

Using a confocal fluorescence microscope with an avalanche photodiode as detector, we studied the fluorescence of the tetramethylrhodamine labeled F1 part of the H+-ATPase from Escherichia coli, EF1, carrying the gammaT106-C mutation [Aggeler, J.A. and Capaldi, R.A. (1992) J. Biol. Chem. 267, 21355-21359] in aqueous solution upon excitation with a mode-locked argon ion laser at 528 nm. The diffusion of the labeled EF1 through the confocal volume gives rise to photon bursts, which were analyzed with fluorescence correlation spectroscopy, resulting in a diffusion coefficient of 3.3 x 10(-7) cm2 s(-1). In the presence of nucleotides the diffusion coefficient increases by about 15%. This effect indicates a change of the shape and/or the volume of the enzyme upon binding of nucleotides, i.e. fluorescence correlation spectroscopy with single EF1 molecules allows the detection of conformational changes.

Structure of the puf operon of the obligately aerobic, bacteriochlorophyll alpha-containing bacterium Roseobacter denitrificans OCh114 and its expression in a Rhodobacter capsulatus puf puc deletion mutant.

Roseobacter denitrificans (Erythrobacter species strain OCh114) synthesizes bacteriochlorophyll a (BChl) and the photosynthetic apparatus only in the presence of oxygen and is unable to carry out primary photosynthetic reactions and to grow photosynthetically under anoxic conditions. The puf operon of R. denitrificans has the same five genes in the same order as in many photosynthetic bacteria, i.e., pufBALMC. PufC, the tetraheme subunit of the reaction center (RC), consists of 352 amino acids (Mr, 39,043); 20 and 34% of the total amino acids are identical to those of PufC of Chloroflexus aurantiacus and Rubrivivax gelatinosus, respectively. The N-terminal hydrophobic domain is probably responsible for anchoring the subunit in the membrane. Four heme-binding domains are homologous to those of PufC in several purple bacteria. Sequences similar to pufQ and pufX of Rhodobacter capsulatus were not detected on the chromosome of R. denitrificans. The puf operon of R. denitrificans was expressed in trans in Escherichia coli, and all gene products were synthesized. The Roseobacter puf operon was also expressed in R. capsulatus CK11, a puf puc double-deletion mutant. For the first time, an RC/light-harvesting complex I core complex was heterologously synthesized. The strongest expression of the R. denitrificans puf operon was observed under the control of the R. capsulatus puf promoter, in the presence of pufQ and pufX and in the absence of pufC. Charge recombination between the primary donor P+ and the primary ubiquinone Q(A)- was observed in the transconjugant, showing that the M and L subunits of the RC were correctly assembled. The transconjugants did not grow photosynthetically under anoxic conditions.

From uni-site to multi-site ATP synthesis in thylakoid membranes.

The membrane-bound H(+)-ATPase from chloroplasts, CF0F1, was brought into the active, reduced state by illumination in the presence of thioredoxin and dithiothreitol. The endogenous nucleotides were removed by a washing procedure so that the active, reduced enzyme contained one tightly bound ATP per CF0F1. When [14C]ADP was added in substoichiometric amounts during continuous illumination, ADP was bound to the enzyme, phosphorylated and released as [14C]ATP, i.e., the tightly bound ATP was not involved in the catalytic turnover ('uni-site ATP-synthesis'). The rate constant for ADP binding was k = (2.0 +/- 0.5) x 10(6) M-1 s-1. The rate of ATP synthesis was measured as a function of the ADP concentration from 8 nM up to 1 mM in the presence of 2 mM phosphate during continuous illumination. A linear increase of the rate was observed up to 100 nM. Above this concentration a supralinear increase was found, indicating the occupation of a second ADP-binding site. A plateau was reached between 1.5 microM and 2.3 microM ADP with a rate of vpl = 3.7 s-1. The half-maximal rate from this plateau was observed at 780 nM. Above 2.3 microM ADP up to 1 mM ADP the data were described by Michaelis-Menten kinetics (vmax = 80 s-1; apparent KM = 32 microM). These results indicated the participation of at least two different ADP binding sites in ATP synthesis catalyzed by the membrane-bound CF0F1.

Transport protons do not participate in ATP synthesis/hydrolysis at the nucleotide binding site of the H(+)-ATPase from chloroplasts.

The H(+)-ATPase from chloroplasts CFoF1, was brought into the active, reduced state by illumination of thylakoids in the presence of thioredoxin and dithiothreitol. Uni-site ATP synthesis was initiated by the addition of 20 nM [alpha-32P]ADP, and enzyme-bound and free nucleotides were separated by a pressure column. The ratio of enzyme-bound ADP to ATP was 0.55 +/- 0.05. In a second experiment, uni-site ATP hydrolysis under energized conditions was initiated by the addition of 36 nM [alpha-32P]ATP; enzyme-bound and free nucleotides were separated by a pressure column. Both procedures were carried out under continuous illumination. The ratio of enzyme-bound ADP to ATP was 0.46 +/- 0.04. In a third experiment, uni-site ATP hydrolysis under de-energized conditions was initiated by the addition of 39 nM [alpha-32P]ATP and NH4Cl/valinomycin in the absence of illumination. Free and enzyme-bound nucleotides were separated also by a pressure column. The ratio of enzyme-bound ADP to ATP was 0.43 +/- 0.02. This ratio was always the same irrespective of whether the reaction runs in the synthesis or the hydrolysis direction. Furthermore, the ratio does not depend on the membrane energization. We conclude, therefore, that the protons are not directly involved in the reaction at the catalytic site.

Proton transport-coupled unisite catalysis by the H(+)-ATPase from chloroplasts.

Proton transport-coupled unisite catalysis was measured with the H(+)-ATPase from chloroplasts. The reaction was measured in the ATP hydrolysis direction under deenergized conditions and in the ATP synthesis direction under energized conditions. The equilibrium constant of the enzyme does not change upon energization, whereas the dissociation constants of substrates and products change by orders of magnitude. This indicates that the Gibbs free enthalpy derived from proton translocation is used to change binding affinities of substrates and products, and this results in synthesis of free ATP.

Numerical simulation of uni-site and bi-site ATP-hydrolysis catalyzed by the membrane-bound H(+)-ATPase from chloroplasts.

The kinetics of ATP-hydrolysis catalyzed by the H(+)-ATPase from chloroplasts was described by an enzyme kinetic model where either one (uni-site catalysis) or two (bi-site catalysis) sites are operating. Numerical simulations are carried out with the software package LARKIN using measured rate constants as a basis. When the ATP-concentration is increased up to 20 nM (at 20 nM enzyme concentration) a change from uni-site to bi-site catalysis occurs at about 7 nM. The measured effective rate constants change by a factor two. However, the calculated rate constants for the second site are drastically increased.

Uni-site ATP synthesis in thylakoids.

Uni-site ATP synthesis was measured with thylakoids. The membrane-bound ATP-synthase, CF0F1, was brought into the active, reduced state by illumination in the presence of thioredoxin, dithiothreitol and phosphate. This enzyme contains two tightly bound ATP per CF0F1. ATP was released from the enzyme when ADP was added in substoichiometric amounts during illumination. Experiments with [14C]ADP indicated that after binding the same nucleotide was phosphorylated and released as [14C]ATP, i.e. only one site is involved in ATP-synthesis ('uni-site ATP-synthesis'). The two tightly bound ATP are not involved in the catalytic turnover. The rate constant for ADP binding was (4 +/- 2) x 10(6) M-1s-1. Compared to deenergized conditions the rate constant for ADP binding and that for ATP-release were drastically increased, i.e. membrane energization increased the rate constants for the ATP-synthesis direction.