Predominant Protection of D2-Protein against Photodestruction in Isolated D1/D2/Cytochrome b 559 by K15, a Phenolic-Type Inhibitor of Electron Transfer in Photosystem 2

A protective action of K15 (4-[methoxy-bis(trifluoromethyl)methyl]-2,6-dinitrophenylhydrazone methyl ketone), an inhibitor of electron transport in photosystem 2 (PS 2), against photoinactivation of the PS 2 reaction center (RC) D1/D2/cytochrome b(559) complex, isolated from pea chloroplasts, by red light (0.7 mmol photons/sec per m(2)) has been investigated under aerobic conditions. The inhibitor K15 causing cyclic electron transfer around PS 2 and thus prohibiting stabilization of separated charges has been shown to effectively protect RC both against the loss of photochemical activity (measured as reversible photoinduced absorbance changes related to photoreduction of pheophytin) and aggregation and degradation of the proteins D2 and D1 during photoinactivation. Comparison of the protective action of K15 and of another inhibitor of electron transfer in PS 2, diuron, against light-induced destruction of proteins D1 and D2 shows that diuron stabilizes protein D1 and K15 stabilizes protein D2. The preferential protection of D2 against photoinduced destruction revealed in our work is in accord with the concept of a specific binding of K15 with this protein. It is proposed that this binding site may be that of the primary quinone electron acceptor Q(A) located on the D2 protein (in contrast to diuron, which is known to replace the secondary electron acceptor Q(B) from its binding site on D1).     

Snake inhibitors of phospholipase A2 enzymes

Phospholipase A₂ (PLA₂) enzymes consist of a large family of proteins which share the same enzymatic function and display considerable sequence homology. These enzymes have been identified and characterised in mammalian tissue and snake venoms. Numerous physiological functions have been attributed to mammalian PLA₂s and they are nontoxic. In comparison, venom PLA₂s are toxic and induce a variety of pharmacological effects that are probably mediated via membrane receptors. Snake PLA₂ inhibitors (PLIα), with a similar structure to the M-type receptor, have been identified as soluble complexes in the serum of viperinae and crotalinae snakes. These inhibitors showed selective binding to crotalid group II PLA₂s and appeared to be restricted to the serum of this snake family. Analysis of PLA₂ binding to recombinant fragments of PLIα indicated that the CRD region was most likely responsible for enzyme inhibition. A second type of inhibitor, PLIβ, has been identified in serum from one viperid snake and consists of a leucine-rich structure. The third type of inhibitor, PLIγ, was found in the serum of five snake families and contains a pattern of cysteine residues that define a three-finger structure. PLIγ inhibitors isolated from the serum of Elapidae, Hydrophidae, Boidae and Colubridae families were able to inhibit a broad range of enzymes including the nontoxic mammalian group IB and IIA PLA₂s, and bee venom group III PLA₂. However, differences in the binding affinities indicated specificity for particular PLA₂s. A different representation has emerged for crotalid and viperid snakes. Their PLIγs did not inhibit bee venom group III, mammalian group IB and IIA enzymes. Furthermore, inhibition data for the γ-type inhibitor from Crotalus durissus terrificus (CICS) showed that this inhibitor was specific for viperid β-neurotoxins and did not inhibit β-neurotoxins from elapids [1]. Further studies are required to determine if this phenomenon is true for all γ-type inhibitors from Crotalidae snakes. The relative distribution of these inhibitors, their specificities and the structural features involved in binding are discussed in this review.     

Field-dispersion profiles of the proton spin-lattice relaxation rate in chloroplast suspensions. Effect of manganese extraction by EDTA,Tris, and hydroxylamine

Proton spin-lattice relaxation rates (R₁) have been measured in a variety of dark-adapted chloroplast suspensions over a range of field strengths between 1 and 15 kG (4–5 MHz). When the effects of EDTA or Tris washing on chloroplast relaxivities are compared, the pool of Mn associated with oxygen evolution is seen not to contribute significantly to relaxivity. Instead, nearly all of the observed relaxivity, which is characterized by a paramagnetic maximum near 20.7 MHz in the field dispersion profile of R₁, appears to arise from contaminating non-functional Mn(II) that can be removed by EDTA during the isolation procedure. These observations, which contradict previous reports ascribing chloroplast relaxivity to the water-oxidizing system, require a reevaluation of proposed models, derived from NMR studies, of the state of Mn in the water-splitting reaction.Chloroplasts from which loosely bound non-functional Mn has been removed by EDTA washing do show an enhancement of relaxivity when exposed to NH₂OH at concentrations known to inactivate water oxidation. This NH₂OH-induced relaxivity is comprised of Mn(II) in two distinct paramagnetic sites. One site is chelatable by EDTA, whereas the other site is not. This finding suggests that some Mn(II) tightly bound to thylakoid membranes can contribute to relaxivity after inactivation of the oxygen-evolving reaction.     

Analysis of the gastric H,K ATPase for ion pathways and inhibitor binding sites

New models of the gastric H,K ATPase in the E1K and E2P states are presented as the first structures of a K+ counter-transport P2-type ATPase exhibiting ion entry and exit paths. Homology modeling was first used to generate a starting conformation from the srCa ATPase E2P form (PDB code 1wpg) that contains bound MgADP. Energy minimization of the model showed a conserved adenosine site but nonconserved polyphosphate contacts compared to the srCa ATPase. Molecular dynamics was then employed to expand the luminal entry sufficiently to allow access of the rigid K+ competitive naphthyridine inhibitor, Byk99, to its binding site within the membrane domain. The new E2P model had increased separation between transmembrane segments M3 through M8, and addition of water in this space showed not only an inhibitor entry path to the luminal vestibule but also a channel leading to the ion binding site. Addition of K+ to the hydrated channel with molecular dynamics modeling of ion movement identified a pathway for K+ from the lumen to the ion binding site to give E2K. A K+ exit path to the cytoplasm operating during the normal catalytic cycle is also proposed on the basis of an E1K homology model derived from the E12Ca2+ form of the srCa ATPase (PDB code 1su4). Autodock analyses of the new E2P model now correctly discriminate between high- and low-affinity K+ competitive inhibitors. Finally, the expanded luminal vestibule of the E2P model explains high-affinity ouabain binding in a mutant of the H,K ATPase [Qiu et al. (2005) J. Biol. Chem. 280, 32349-32355].     

Thermodynamic characterization of cytosolic phospholipase A2 alpha inhibitors

Cytosolic phospholipase A₂ alpha (cPLA₂α, type IVA phospholipase) acts at the membrane surface to release free arachidonic acid, which is metabolized into inflammatory mediators, including leukotrienes and prostaglandins. Thus, specific cPLA₂α inhibitors are predicted to have antiinflammatory properties. However, a key criterion in the identification and development of such inhibitors is to distinguish between compounds that bind stoichiometrically to cPLA₂α and nonspecific membrane perturbants. In the current study, we developed a method employing isothermal titration calorimetry (ITC) to characterize the binding of several distinct classes of cPLA₂α inhibitors. Thermodynamic parameters and the binding constants were obtained following titration of the inhibitor to the protein at 30 °C and pH 7.4. The compounds tested bound cPLA₂α with a 1:1 stoichiometry, and the dissociation constant K_d of the inhibitors calculated from the ITC experiments correlated well with the IC₅₀ values obtained from enzymatic assays. Interestingly, binding was observed only in the presence of a micellar surface, even for soluble compounds. The site of binding of these inhibitors within cPLA₂α was analyzed by testing for binding in the presence of methyl arachidonyl fluorophosphonate (MAFP), an irreversible active site inhibitor of cPLA₂α. Lack of binding of inhibitors in the presence of MAFP suggested that the compounds tested bound specifically at or near the active site of the protein. Furthermore, the effect of various detergents on the binding of certain inhibitors to cPLA₂α was also tested. The results are discussed with reference to thermodynamic parameters such as changes in enthalpy (ΔH), entropy (ΔS), and free energy (ΔG). The data obtained from these studies provide not only structure-activity relationships for compounds but also important information regarding mechanism of binding. This is the first example of ITC used for studying inhibitors of enzymes with interfacial kinetics.     

New potent inhibitors of tyrosinase: Novel clues to binding of 1,3,4-thiadiazole-2(3H)-thiones, 1,3,4-oxadiazole-2(3H)-thiones, 4-amino-1,2,4-triazole-5(4H)-thiones,and substituted hydrazides to the dicopper active site

A series of 1,3,4-thiadiazole-2(3H)-thiones, 1,3,4-oxadiazole-2(3H)-thiones, 4-amino-1,2,4-triazole-5(4H)-thiones, and substituted hydrazides were tailored and synthesized as new potent inhibitors of tyrosinase. The rationale for inhibitor design was based on the active site structural evidence from the crystal structures of bacterial tyrosinase and potato catechol oxidase enzymes. Kinetic and active site binding studies suggested mono-dentate binding of thiadiazole, oxadiazole, and triazole rings to the active site dicopper center of tyrosinase including hydrophobicity contributing to the potent inhibition. Kinetic plots showed mixed-type of inhibition by all 25 compounds. Substitutions at C3 of the triazole ring and C5 of the thiadiazole/oxadiazole rings were found to be playing a major role in the high binding affinity to tyrosinase. The current work may help develop new potent tyrosinase inhibitors against hyperpigmentation including potential insecticides.     

Acceptor and donor-side interactions of phenolic inhibitors in Photosystem II

Certain phenolic compounds represent a distinct class of Photosystem (PS) II Q_B site inhibitors. In this paper, we report a detailed study of the effects of 2,4,6-trinitrophenol (TNP) and other phenolic inhibitors, bromoxynil and dinoseb, on PS II energetics. In intact PS II, phenolic inhibitors bound to only 90-95% of Q_B sites even at saturating concentrations. The remaining PS II reaction centers (5-10%) showed modified Q_A to Q_B electron transfer but were sensitive to urea/triazine inhibitors. The binding of phenolic inhibitors was 30- to 300-fold slower than the urea/triazine class of Q_B site inhibitors, DCMU and atrazine. In the sensitive centers, the S₂Q_A⁻ state was 10-fold less stable in the presence of phenolic inhibitors than the urea/triazine herbicides. In addition, the binding affinity of phenolic herbicides was decreased 10-fold in the S₂Q_A⁻ state than the S₁Q_A state. However, removal of the oxygen-evolving complex (OEC) and associated extrinsic polypeptides by hydroxylamine (HA) washing abolished the slow binding kinetics as well as the destabilizing effects on the charge-separated state. The S₂-multiline electron paramagnetic resonance (EPR) signal and the ‘split’ EPR signal, originating from the S₂Y_Z state showed no significant changes upon binding of phenolic inhibitors at the Q_B site. We thus propose a working model where Q_A redox potential is lowered by short-range conformational changes induced by phenolic inhibitor binding at the Q_B niche. Long-range effects of HA-washing eliminate this interaction, possibly by allowing more flexibility in the Q_B site.     

Secondary mutations M36I and A71V in the human immunodeficiency virus type 1 protease can provide an advantage for the emergence of the primary mutation D30N

Development of resistance mutations in enzymatic targets of human immunodeficiency virus 1 (HIV-1) hampers the ability to provide adequate therapy. Of special interest is the effect mutations outside the active site of HIV-1 protease have on inhibitor binding and virus viability. We engineered protease mutants containing the active site mutation D30N alone and with the nonactive site polymorphisms M36I and/or A71V. We determined the K(i) values for the inhibitors nelfinavir, ritonavir, indinavir, KNI272, and AG1776 as well as the catalytic efficiency of the mutants. Single and double mutation combinations exhibited a decrease in catalytic efficiency, while the triple mutant displayed catalytic efficiency greater than that of the wild type. Variants containing M36I or A71V alone did not display a significant change in binding affinities to the inhibitors tested. The variant containing mutation D30N displayed a 2-6-fold increase in K(i) for all inhibitors tested, with nelfinavir showing the greatest increase. The double mutants containing a combination of mutations D30N, M36I, and A71V displayed -0.5-fold to +6-fold changes in the K(i) of all inhibitors tested, with ritonavir and nelfinavir most affected. Only the triple mutant showed a significant increase (>10-fold) in K(i) for inhibitor nelfinavir, ritonavir, or AG-1776 displaying 22-, 19-, or 15-fold increases, respectively. Our study shows that the M36I and A71V mutations provide a greater level of inhibitor cross-resistance combined with active site mutation D30N. M36I and A71V, when present as natural polymorphisms, could aid the virus in developing active site mutations to escape inhibitor binding while maintaining catalytic efficiency.     

Evidence for an association between a 33 kDa extrinsic membrane protein,manganese and photosynthetic oxygen evolution. I. Correlation with the S2 multiline EPR signal

The removal of peripheral membrane proteins of a molecular mass of 17 and 23 kDa by washing of spinach Photosystem-II (PS II) membranes in 1 M salt between pH 4.5 and 6.5 produces a minimal loss of the S₁ → S₂ reaction, as seen by the multiline EPR signal for the S₂ state of the water-oxidizing complex, while reversibly inhibiting O₂ evolution. The multiline EPR signal simplifies from a ‘19-line’ spectrum to a ‘16-line’ spectrum, suggestive of partial uncoupling of a cluster of 3 or 4 to yield photo-oxidation of a binuclear Mn site. Alkaline salt washing progressively releases a 33 kDa peripheral protein between pH 6.5 and 9.5, in direct parallel with the loss of O₂ evolution and the S₂ multiline EPR signal. The 33 kDa protein can be partially removed (20%) at pH 8.0 prior to managanese release. Salt treatment releases four Mn ions between pH 8.0 and 9.5 with the first 2 or 3 Mn ions released cooperatively. A common binding site is thus suggested in agreement with earlier EPR spectroscopic data establishing a tetranuclear Mn site. At least two of these Mn ions bind directly at a site in the PS II complex for which photooxidation by the reaction center is controlled by the 33 kDa protein. The washing of PS II membranes with 1 M CaCl₂ to affect the release of the 33 kDa protein, while preserving Mn binding to the membrane (Ono, T.-A. and Inoue, Y. (1983) FEBS Lett. 164, 255–260), is found to leave some 33 kDa protein undissociated in proportion to the extent of O₂ evolution and S₂ multiline yield. These depleted membranes do not oxidize water or produce the normal S₂ state without the binding of the 33 kDa protein. A method for the accurate determination of relative concentrations of the peripheral membrane proteins using gel electrophoresis is presented.     

Novel monoamine oxidase inhibitors based on the privileged 2-imidazoline molecular framework

Series of structurally diverse 2-imidazoline derivatives have been synthesized by condensation of substituted aldehydes with ethylenediamine, Pd-catalyzed N-arylation of 2-imidazolines and by the formation of 1,2,4-oxadiazoles and benzoxazepines from 2-imidazoline-containing precursors. The 2-imidazoline derivatives were evaluated as potential inhibitors of human monoamine oxidase (MAO) A and B. Among the 2-imidazolines, good potency inhibitors were discovered with compound 9p (IC₅₀?=?0.012?µM) being the most potent MAO-B inhibitor, while compound 9d (IC₅₀?=?0.751?µM) was the most potent MAO-A inhibitor of the series. These potencies are in the same range as those of reference MAO inhibitors used in the clinic. Among 33 compounds evaluated, 13 exhibited IC₅₀ values in the submicromolar range for the inhibition of an MAO isoform. It is postulated that the imidazoline moieties of some of these inhibitors may be recognized by the imidazoline I₂-binding site of MAO. Good potency MAO inhibitors may be useful for the treatment of neuropsychiatric and neurodegenerative disorders such as depression and Parkinson’s disease, and future application for the treatment of prostate cancer, congestive heart failure and Alzheimer’s disease. In addition, high potency 2-imidazoline-derived MAO inhibitors may be used as potential probes for the imidazoline binding sites of the MAOs, as well as to determine alternative binding regions of imidazoline within the MAO active site.     

X-ray and model-building studies on the specificity of the active site of proteinase K

Proteinase K, the extracellular serine endopeptidase (E.C. 3.4.21.14) from the fungus Tritirachium album limber, is homologous to the bacterial subtilisin proteases. The binding geometry of the synthetic inhibitor carbobenzoxy-Ala-Phechloromethyl Ketone to the active site of proteinase K was the first determined from a Fourier synthesis based on synchrotron X-ray diffraction data between 1.8 Å and 5.0 Å resolution. The protein inhibitor complexes was refined by restrained least-squares minimization with the data between 10.0 and 1.8 Å. The final R factor was 19.1% and the model contained 2,018 protein atoms, 28 inhibitors atoms, 125 water molecules, and two Ca²⁺ ions. The peptides portion of the inhibitor is bound to the active center of proteinase K by means of a three-stranded antiparallel pleated sheet, with the side chain of the phenylalanine located in the P1 site. Model building studies, with lysine replacing phenylalanine in the inhibitor, explain the relatively unspecific catalytic activity of the enzyme.     

Binding of the potential antitumour agent indirubin-5-sulphonate at the inhibitor site of rabbit muscle glycogen phosphorylase b. Comparison with ligand binding to pCDK2-cyclin A complex.

The binding of indirubin-5-sulphonate (E226), a potential anti-tumour agent and a potent inhibitor (IC(50) = 35 nm) of cyclin-dependent kinase 2 (CDK2) and glycogen phosphorylase (GP) has been studied by kinetic and crystallographic methods. Kinetic analysis revealed that E226 is a moderate inhibitor of GPb (K(i) = 13.8 +/- 0.2 micro m) and GPa (K(i) = 57.8 +/- 7.1 micro m) and acts synergistically with glucose. To explore the molecular basis of E226 binding we have determined the crystal structure of the GPb/E226 complex at 2.3 A resolution. Structure analysis shows clearly that E226 binds at the purine inhibitor site, where caffeine and flavopiridol also bind [Oikonomakos, N.G., Schnier, J.B., Zographos, S.E., Skamnaki, V.T., Tsitsanou, K.E. & Johnson, L.N. (2000) J. Biol. Chem.275, 34566-34573], by intercalating between the two aromatic rings of Phe285 and Tyr613. The mode of binding of E226 to GPb is similar, but not identical, to that of caffeine and flavopiridol. Comparative structural analyses of the GPb-E226, GPb-caffeine and GPb-flavopiridol complex structures reveal the structural basis of the differences in the potencies of the three inhibitors and indicate binding residues in the inhibitor site that can be exploited to obtain more potent inhibitors. Structural comparison of the GPb-E226 complex structure with the active pCDK2-cyclin A-E226 complex structure clearly shows the different binding modes of the ligand to GPb and CDK2; the more extensive interactions of E226 with the active site of CDK2 may explain its higher affinity towards the latter enzyme.     

Design,synthesis and biological evaluation of 2-aminopyrimidinones and their 6-aza-analogs as a new class of CK2 inhibitors

Abstract

In order to find the new potent CK2 inhibitors the 60 derivatives of 2-aminopyrimidinone and their 6-aza-substituted analogs were synthesized and tested in vitro. Among them, the most efficient inhibitor 2-hydroxy-5-[4-(4-methoxyphehyl)-6-oxo-1,6-dihydropyrimidin-2-ylamino] benzoic acid was identified (IC₅₀?=?1.1?μM). The structure--activity relationship study of newly synthesized derivatives was carried out and their binding mode with adenosine triphosphate-acceptor site of CK2 was proposed.     

Structure-activity relationships for the interaction of bovine pancreatic trypsin inhibitor with an intracellular site on a large conductance Ca(2+)-activated K(+) channel

Large conductance Ca(2+)-activated K(+) channels (BK(Ca)) contain an intracellular binding site for bovine pancreatic trypsin inhibitor (BPTI), a well-known inhibitor of various serine proteinase (SerP) enzymes. To investigate the structural basis of this interaction, we examined the activity of 11 BPTI mutants using single BK(Ca) channels from rat skeletal muscle incorporated into planar lipid bilayers. All of the mutants induced discrete substate events at the single-channel level. The dwell time of the substate, which is inversely related to the dissociation rate constant of BPTI, exhibited relatively small changes (<9-fold) for the various mutants. However, the apparent association rate constant varied up to 190-fold and exhibited a positive correlation with the net charge of the molecule, suggesting the presence of a negative electrostatic surface potential in the vicinity of the binding site. The substate current level was unaffected by most of the mutations except for substitutions of Lys15. Different residues at this position were found to modulate the apparent conductance of the BPTI-induced substate to 0% (K15G), 10% (K15F), 30% (K15 wild-type), and 55% (K15V) of the open state at +20 mV. Lys15 is located on a loop of BPTI that forms the primary contact region for binding to many SerPs such as trypsin, chymotrypsin, and elastase. The finding that Lys15 is a determinant of the conductance behavior of the BK(Ca) channel when BPTI is bound implies that the same inhibitory loop that contacts SerP's is located close to the protein interface in the BK(Ca) channel complex. This supports the hypothesis that the C-terminal region of the BK(Ca) channel protein contains a domain homologous to SerP's. We propose a domain interaction model for the mechanism of substate production by Kunitz inhibitors based on current ideas for allosteric activation of BK(Ca) channels by voltage and Ca(2+).     

Thermodynamic and NMR analysis of inhibitor binding to dihydrofolate reductase

Isothermal titration calorimetry (ITC) was used to determine the thermodynamic driving force for inhibitor binding to the enzyme dihydrofolate reductase (DHFR) from Escherichia coli. 1,4-Bis-{[N-(1-imino-1-guanidino-methyl)]sulfanylmethyl}-3,6-dimethyl-benzene (1) binds DHFR:NADPH with a K(d) of 13±5 nM while the related inhibitor 1-{[N-(1-imino-guanidino-methyl)]sulfanylmethyl}-3-trifluoromethyl-benzene (2) binds DHFR:NADPH with a K(d) of 3.2±2.2 μM. The binding of these inhibitors has both a favorable entropy and enthalpy of binding. Additionally, we observe positive binding cooperativity between both 1 and 2 and the cofactor NADPH. Binding of compound 1 to DHFR is 285-fold tighter in the presence of the NADPH cofactor than in its absence. We did not detect binding of 2 to DHFR in the absence of NADPH. The backbone amide (1)H and (15)N NMR resonances of DHFR:NADPH and both DHFR:NADPH inhibitor complexes were assigned in order to better understand the binding of these inhibitors in solution. The chemical shift perturbations observed with the binding of 1 were greatest at residues closest to the binding site, but significant perturbations also occur away from the inhibitor location at amino acids in the vicinity of residue 58 and in the GH loop. The pattern of chemical shift changes observed with the binding of 2 is similar to that seen with 1. The main differences in chemical shift perturbation between the two inhibitors are in the Met20 loop and in residues at the interface between the inhibitor and NADPH.     

Structural Basis for Resistance of the Genotype 2b Hepatitis C Virus NS5B Polymerase to Site A Non-Nucleoside Inhibitors

Hepatitis C virus (HCV) exists in six major genotypes. Compared with the 1b enzyme, genotype 2b HCV polymerase exhibits a more than 100-fold reduction in sensitivity to the indole-N-acetamide class of non-nucleoside inhibitors. These compounds have been shown to bind in a pocket occupied by helix A of the mobile Λ1 loop in the apoenzyme. The three-dimensional structure of the HCV polymerase from genotype 2b was determined to 1.9-Å resolution and compared with the genotype 1b enzyme. This structural analysis suggests that genotypic variants result in a different shape of the inhibitor binding site. Mutants of the inhibitor binding pocket were generated in a 1b enzyme and evaluated for their binding affinity and sensitivity to inhibition by indole-N-acetamides. Most of the point mutants showed little variation in activity and IC₅₀, with the exception of 15- and 7-fold increases in IC₅₀ for Leu392Ile and Val494Ala mutants (1b→2b), respectively. Furthermore, a 1b replicon with 20-fold resistance to this class of inhibitors was selected and shown to contain the Leu392Ile mutation. Chimeric enzymes, where the 2b fingertip Λ1 loop, pocket or both replaced the corresponding regions of the 1b enzyme, were also generated. The fingertip chimera retained 1b-like inhibitor binding affinity, whereas the other two chimeric constructs and the 2b enzyme displayed between 50- and 100-fold reduction in binding affinity. Together, these data suggest that differences in the amino acid composition and shape of the indole-N-acetamide binding pocket are responsible for the resistance of the 2b polymerase to this class of inhibitors.     

Identity of the Photosystem II reaction center polypeptide

A Photosystem-II (PS-II)-enriched chloroplast submembrane fraction has been subjected to non-denaturing gel-electrophoresis. Two chlorophyll a (Chl a)-binding proteins associated with the core complex were isolated and spectrally characterized. The Chl protein with apparent apoprotein mass of 47 kDa (CP47) displayed a 695 nm fluorescence emission maximum (77 K) and light-induced absorption characteristics indicating the presence of the reaction center Chl, P-680, and its primary electron acceptor, pheophytin. A Chl protein of apparent apoprotein mass of 43 kDa (CP43) displayed a fluorescence emission maximum at 685 nm. We conclude that CP43 serves as an antenna Chl protein and the PS II reaction center is located in CP47.     

Differential efficacy of inhibition of mitochondrial and bacterial cytochrome bc1 complexes by center N inhibitors antimycin, ilicicolin H and funiculosin

We have compared the efficacy of inhibition of the cytochrome bc₁ complexes from yeast and bovine heart mitochondria and Paracoccus denitrificans by antimycin, ilicicolin H, and funiculosin, three inhibitors that act at the quinone reduction site at center N of the enzyme. Although the three inhibitors have some structural features in common, they differ significantly in their patterns of inhibition. Also, while the overall folding pattern of cytochrome b around center N is similar in the enzymes from the three species, amino acid sequence differences create sufficient structural differences so that there are striking differences in the inhibitors binding to the three enzymes. Antimycin is the most tightly bound of the three inhibitors, and binds stoichiometrically to the isolated enzymes from all three species under the cytochrome c reductase assay conditions. Ilicicolin H also binds stoichiometrically to the yeast enzyme, but binds approximately 2 orders of magnitude less tightly to the bovine enzyme and is essentially non-inhibitory to the Paracoccus enzyme. Funiculosin on the other hand inhibits the yeast and bovine enzymes similarly, with IC₅₀ ∼ 10 nM, while the IC₅₀ for the Paracoccus enzyme is more than 10-fold higher. Similar differences in inhibitor efficacy were noted in bc₁ complexes from yeast mutants with single amino acid substitutions at the center N site, although the binding affinity of quinone and quinol substrates were not perturbed to a degree that impaired catalytic function in the variant enzymes. These results reveal a high degree of specificity in the determinants of ligand-binding at center N, accompanied by sufficient structural plasticity for substrate binding as to not compromise center N function. The results also demonstrate that, in principle, it should be possible to design novel inhibitors targeted toward center N of the bc₁ complex with appropriate species selectivity to allow their use as drugs against pathogenic fungi and parasites.     

Synthesis,structure, and structure-activity relationships of divalent thrombin inhibitors containing an α-keto-amide transition-state mimetic

A new class of divalent thrombin inhibitors is described that contains an α-keto-amide transition-state mimetic linking an active site binding group and a group that binds to the fibrinogen-binding exosite. The X-ray crystallographic structure of the most potent member of this new class, CVS995, shows many features in common with other divalent thrombin inhibitors and clearly defines the transition-state-like binding of the α-keto-amide group. The structure of the active site part of the inhibitor shows a network of water molecules connecting both the side-chain and backbone atoms of thrombin and the inhibitor. Direct peptide analogues of the new transition-state-containing divalent thrombin inhibitors were compared using in vitro assays of thrombin inhibition. There was no direct correlation between the binding constants of the peptides and their α-keto-amide counterparts. The most potent cv-keto-amide inhibitor, CVS995, with a K_i = 1 pM, did not correspond to the most potent divalent peptide and contained a single amino acid deletion in the exosite binding region with respect to the equivalent region of the natural thrombin inhibitor hirudin. The interaction energies of the active site, transition state, and exosite binding regions of these new divalent thrombin inhibitors are not additive.     

Inhibitor and ion binding sites on the gastric H,K-ATPase

The gastric H,K-ATPase catalyzes electroneutral exchange of H(+) for K(+) as a function of enzyme phosphorylation and dephosphorylation during transition between E(1)/E(1)-P (ion site in) and E(2)-P/E(2) (ion site out) conformations. Here we present homology modeling of the H,K-ATPase in the E(2)-P conformation as a means of predicting the interaction of the enzyme with two known classes of specific inhibitors. All known proton pump inhibitors, PPIs, form a disulfide bond with cysteine 813 that is accessible from the luminal surface. This allows allocation of the binding site to a luminal vestibule adjacent to Cys813 enclosed by part of TM4 and the loop between TM5 and TM6. K(+) competitive imidazo-1,2alpha-pyridines also bind to the luminal surface of the E(2)-P conformation, and their binding excludes PPI reaction. This overlap of the binding sites of the two classes of inhibitors combined with the results of site-directed mutagenesis and cysteine cross-linking allowed preliminary assignment of a docking mode for these reversible compounds in a position close to Glu795 that accounts for the detailed structure/activity relationships known for these compounds. The new E(2)-P model is able to assign a possible mechanism for acid secretion by this P(2)-type ATPase. Several ion binding side chains identified in the sr Ca-ATPase by crystallography are conserved in the Na,K- and H,K-ATPases. Poised in the middle of these, the H,K-ATPase substitutes lysine in place of a serine implicated in K(+) binding in the Na,K-ATPase. Molecular models for hydronium binding to E(1) versus E(2)-P predict outward displacement of the hydronium bound between Asp824, Glu820, and Glu795 by the R-NH(3)(+) of Lys791 during the conformational transition from E(1)P and E(2)P. The site for luminal K(+) binding at low pH is proposed to be between carbonyl oxygens in the nonhelical part of the fourth membrane span and carboxyl oxygens of Glu795 and Glu820. This site of K(+) binding is predicted to destabilize hydrogen bonds between these carboxylates and the -NH(3)(+) group of Lys791, allowing the Lys791 side chain to return to its E(1) position.