Structural mimics for the active site of [NiFe] hydrogenase

Hydrogenases are enzymes that catalyse the reversible two-electron oxidation of H-2. The [NiFe] hydrogenases have been characterised spectroscopically and by single crystal X-ray diffraction, and show an active site incorporating a heterobinuclear [NiFe] centre bridged by two cysteine S-donors. Low molecular weight synthetic complex models, which structurally mimic the dithiolate-bridged [NiFe] centre, serve as important probes of structure and chemistry at the active site and are the subject of this review. (C) 2001 Elsevier Science B.V. All rights reserved.

A new lead for nonpeptidic active-site-directed inhibitors of the severe acute respiratory syndrome coronavirus main protease discovered by a combination of screening and docking methods.

The coronavirus main protease, Mpro, is considered to be a major target for drugs suitable for combating coronavirus infections including severe acute respiratory syndrome (SARS). An HPLC-based screening of electrophilic compounds that was performed to identify potential Mpro inhibitors revealed etacrynic acid tert-butylamide (6a) as an effective nonpeptidic inhibitor. Docking studies suggested a binding mode in which the phenyl ring acts as a spacer bridging the inhibitor's activated double bond and its hydrophobic tert-butyl moiety. The latter is supposed to fit into the S4 pocket of the target protease. Furthermore, these studies revealed etacrynic acid amide (6b) as a promising lead for nonpeptidic active-site-directed Mpro inhibitors. In a fluorimetric enzyme assay using a novel fluorescence resonance energy transfer (FRET) pair labeled substrate, compound 6b showed a Ki value of 35.3 M. Since the novel lead compound does not target the S1', S1, and S2 subsites of the enzyme's substrate-binding pockets, there is room for improvement that underlines the lead character of compound 6b.

Screening of electrophilic compounds yields an aziridinyl peptide as new active-site directed SARS-CoV main protease inhibitor.

The coronavirus main protease, Mpro, is considered a major target for drugs suitable to combat coronavirus infections including the severe acute respiratory syndrome (SARS). In this study, comprehensive HPLC- and FRET-substrate-based screenings of various electrophilic compounds were performed to identify potential Mpro inhibitors. The data revealed that the coronaviral main protease is inhibited by aziridine- and oxirane-2-carboxylates. Among the trans-configured aziridine-2,3-dicarboxylates the Gly-Gly-containing peptide 2c was found to be the most potent inhibitor.

Transesterification of vegetable oils on basic large mesoporous alumina supported alkaline fluorides-Evidences of the nature of the active site and catalytic performances

KF, LiF and CsF/A(2)O(3) catalysts with different loadings from 1 to 20 wt% were prepared using aqueous solutions of the alkaline fluoride compounds by wet impregnation of basic mesoporous MSU-type alumina. The catalysts were activated under At at 400 degrees C for 2 h and monitored by in situ XRD measurements. The catalysts were also characterized using several techniques: N-2 adsorption/desorption isotherms at -196 degrees C, FTIR, DR-UV-vis, CO2-TPD, XRD, Al-27 CP/MAS NMR. These characterizations led to the conclusion that the deposition of alkaline fluorides on the alumina surface generates fluoroaluminates and aluminate species. The process is definitivated at 400 degrees C. The fluorine in these structures is less basic than in the parent fluorides, but the oxygen becomes more basic. The catalysts were tested for the transesterification of fatty esters under different experimental conditions using conventional heating, microwave and Ultrasound irradiation. Recycling experiments showed that these catalysts are stable for a limited number of cycles. (C) 2009 Elsevier Inc. All rights reserved.

Molecular Modeling on Inhibitor Complexes and Active-Site Dynamics of Cytochrome P450 C17, a Target for Prostate Cancer Therapy

A molecular model for the P450 enzyme cytochrome P450 C17 (CYP17) is presented based on sequence alignments of multiple template structures and homology modeling. This enzyme plays a central role in the biosynthesis of testosterone and is emerging as a major target in prostate cancer, with the recently developed inhibitor abiraterone currently in advanced clinical trials. The model is described in detail, together with its validation, by providing structural explanations to available site-directed mutagenesis data. The CYP17 molecule in this model is in the form of a triangular prism, with an edge of similar to 55 angstrom and a thickness of similar to 37 angstrom. It is predominantly helical, comprising 13 alpha helices interspersed by six 3(10) helices and 11 beta-sheets. Multinanosecond molecular dynamics simulations in explicit solvent have been carried out, and principal components analysis has been used to reveal the details of dynamics around the active site. Coarse-grained methods have also been used to verify low-frequency motions, which have been correlated with active-site gating. The work also describes the results of docking synthetic inhibitors, including the drug abiraterone and the natural substrate pregnenolone, in the CYP17 active site together with molecular dynamics simulations on the complexes. (C) 2010 Elsevier Ltd. All rights reserved.

Increased Promiscuity of Human Galactokinase Following Alteration of a Single Amino Acid Residue Distant from the Active Site

Galactokinase catalyses the site-and stereospecific phosphorylation of galactose at the expense of ATP. The specificity of bacterial galactokinase enzymes can be broadened by alteration of a tyrosine residue to a histidine. The effects of altering the equivalent residue in human galactokinase (Tyr379) were investigated by testing all 19 possible variants. All of these alterations, except Y379P, resulted in soluble protein on expression in Escherichia coli and all the soluble variants could catalyse the phosphorylation of galactose, except Y379A and Y379E. The variants Y379C, Y379K, Y379R, Y379S and Y379W were all able to catalyse the phosphorylation of a variety of monosaccharides, including ones that are not acted on by the wild-type enzyme. Novel substrates for these variant galactokinases included D-mannose and D-fructose. The latter monosaccharide is presumed to react in the pyranose configuration. Molecular modelling suggested that the alterations do not cause changes to the overall structure of the enzyme. However, alteration of Tyr379 increases the flexibility of the peptide backbone in regions surrounding the active site. Therefore, it is proposed that alteration of Tyr379 affects the substrate specificity by the propagation of changes in flexibility to the active site, permitting a broader range of compounds to be accommodated.

The role of the active site residues in human galactokinase: implications for the mechanisms of GHMP kinases.

Galactokinase catalyses the phosphorylation of galactose at the expense of ATP. Like other members of the GHMP family of kinases it is postulated to function through an active site base mechanism in which Asp-186 abstracts a proton from galactose. This asparate residue was altered to alanine and to asparagine by site-directed mutagenesis of the corresponding gene. This resulted in variant enzyme with no detectable galactokinase activity. Alteration of Arg-37, which lies adjacent to Asp-186 and is postulated to assist the catalytic base, to lysine resulted in an active enzyme. However, alteration of this residue to glutamate abolished activity. All the variant enzymes, except the arginine to lysine substitution, were structurally unstable (as judged by native gel electrophoresis in the presence of urea) compared to the wild type. This suggests that the lack of activity results from this structural instability, in addition to any direct effects on the catalytic mechanism. Computational estimations of the pK(a) values of the arginine and aspartate residues, suggest that Arg-37 remains protonated throughout the catalytic cycle whereas Asp-186 has an abnormally high pK(a) value (7.18). Quantum mechanics/molecular mechanics (QM/MM) calculations suggest that Asp-186 moves closer to the galactose molecule during catalysis. The experimental and theoretical studies presented here argue for a mechanism in which the C-1-OH bond in the sugar is weakened by the presence of Asp-186 thus facilitating nucleophilic attack by the oxygen atom on the gamma-phosphorus of ATP.

The synthesis and electronic structure of a novel [NiS4Fe2(CO)(6)] radical cluster: Implications for the active site of the [NiFe] hydrogenases

A novel [Ni'S-4'Fe-2(CO)(6)] cluster (1: 'S-4'=(CH3C6H3S2)(2)(CH2)(3)) has been synthesised, structurally characterised and has been shown to undergo a chemically reversible reduction process at -1.31 V versus Fc(+)/Fc to generate the EPR-active monoanion 1(-). Multifrequency Q-, X- and S-band EPR spectra of Ni-61-enriched 1(-) show a well-resolved quartet hyperfine splitting in the low-field region due to the interaction with a single Ni-61 (I = 3/2) nucleus. Simulations of the EPR spectra require the introduction of a single angle of non-coincidence between g, and A(1), and g(3) and A(3) to reproduce all of the features in the S- and X-band spectra. This behaviour provides a rare example of the detection and measurement of non-coincidence effects from frozen-solution EPR spectra without the need for single-crystal measurements, and in which the S-band experiment is sensitive to the non-coincidence. An analysis of the EPR spectra of 1(-) reveals a 24% Ni contribution to the SOMO in 1(-), supporting a delocalisation of the spin-density across the NiFe2 cluster. This observation is supported by IR spectroscopic results which show that the CO stretching frequencies, v(CO), shift to lower frequency by about 70 cm(-1) when 1 is reduced to 1(-). Density functional calculations provide a framework for the interpretation of the spectroscopic properties of 1(-) and suggest that the SOMO is delocalised over the whole cluster, but with little S-centre participation. This electronic structure contrasts with that of the Ni-A, -B, -C and -L forms of [NiFe] hydrogenase in which there is considerable S participation in the SOMO.

CHARACTERIZATION OF THE CYSTEINE PROTEINASES OF THE COMMON LIVER FLUKE FASCIOLA-HEPATICA USING NOVEL, ACTIVE-SITE-DIRECTED AFFINITY LABELS

The excreted/secreted proteinases of adult and juvenile Fasciola hepatica maintained in vitro were found to hydrolyse the fluorogenic substrates Cbz-Phe-Arg- and Cbz-Arg-Arg-NHMec. This activity was demonstrated to have a classical cysteine proteinase inhibitor profile, with turn-over of both substrates being blocked by pre-incubation with E64 and peptidyl diazomethanes. The Cbz-Arg-Arg-NHMec hydrolysing activity of the mature fluke exhibited an alkaline stability not characteristic of its mammalian lysosomal counterparts. Further, the biotinylated affinity reagents biotin-Phe-Ala CHN2 and biotin-Phe-Cys(SBzyl)-CHN2 were used to label and characterize these cysteine proteinases in terms of apparent molecular weight and subsite specificity. Adult fluke media were found to contain four species of molecular weights 66, 58, 50 and 25-26 kDa; juvenile media contained three species of molecular weights 66, 54 and 25-26 kDa. The major 25-26 kDa cysteine proteinase common to both stages was shown to have a subsite specificity similar to that of mammalian cathepsin B.

The M17 leucine aminopeptidase of the malaria parasite Plasmodium falciparum:importance of active site metal ions in the binding of substrates and inhibitors

The M17 leucine aminopeptidase of the intraerythrocytic stages of the malaria parasite Plasmodium falciparum (PfLAP) plays a role in releasing amino acids from host hemoglobin that are used for parasite protein synthesis, growth, and development. This enzyme represents a target at which new antimalarials could be designed since metalloaminopeptidase inhibitors prevent the growth of the parasites in vitro and in vivo. A study on the metal ion binding characteristics of recombinant P. falciparum M17 leucine aminopeptidase (rPfLAP) shows that the active site of this exopeptidase contains two metal-binding sites, a readily exchangeable site (site 1) and a tight binding site (site 2). The enzyme retains activity when the metal ion is removed from site 1, while removal of metal ions from both sites results in an inactive apoenzyme that cannot be reactivated by the addition of divalent metal cations. The metal ion at site 1 is readily exchangeable with several divalent metal ions and displays a preference in the order of preference Zn(2+) > Mn(2+) > Co(2+) > Mg(2+). While it is likely that native PfLAP contains a Zn(2+) in site 2, the metal ion located in site 1 may be dependent on the type and concentration of metal ions in the cytosolic compartment of the parasite. Importantly, the type of metal ion present at site 1 influences not only the catalytic efficiency of the enzyme for peptide substrates but also the mode of binding by bestatin, a metal-chelating inhibitor of M17 aminopeptidases with antimalarial activity.