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In many pro- and eukaryotes, a retinal-based proton pump equips the cell to drive ATP synthesis with (sun)light. Such pumps, therefore, have been proposed as a plug-in for cyanobacteria to artificially increase the efficiency of oxygenic photosynthesis. However, little information on the metabolism of retinal, their chromophore, is available for these organisms. We have studied the in vivo roles of five genes ( sll1541 , slr1648 , slr0091 , slr1192 , and slr0574 ) potentially involved in retinal metabolism in Synechocystis sp. strain PCC 6803. With a gene deletion approach, we have shown that Synechocystis apo- carotenoid-15,15-oxygenase (SynACO), encoded by gene sll1541 , is an indispensable enzyme for retinal synthesis in Synechocystis , presumably via asymmetric cleavage of β- apo -carotenal. The second carotenoid oxygenase (SynDiox2), encoded by gene slr1648 , competes with SynACO for substrate(s) but only measurably contributes to retinal biosynthesis in stationary phase via an as-yet-unknown mechanism. In vivo degradation of retinal may proceed through spontaneous chemical oxidation and via enzyme-catalyzed processes. Deletion of gene slr0574 (encoding CYP120A1), but not of slr0091 or of slr1192 , causes an increase (relative to the level in wild-type Synechocystis ) in the retinal content in both the linear and stationary growth phases. These results suggest that CYP120A1 does contribute to retinal degradation. Preliminary data obtained using 13 C-labeled retinal suggest that conversion to retinol and retinoic acid and subsequent further oxidation also play a role. Deletion of sll1541 leads to deficiency in retinal synthesis and allows the in vivo reconstitution of far-red-absorbing holo -proteorhodopsin with exogenous retinal analogues, as demonstrated here for all- trans 3,4-dehydroretinal and 3-methylamino-16-nor-1,2,3,4-didehydroretinal. IMPORTANCE Retinal is formed by many cyanobacteria and has a critical role in most forms of life for processes such as photoreception, growth, and stress survival. However, the metabolic pathways in cyanobacteria for synthesis and degradation of retinal are poorly understood. In this paper we identify genes involved in its synthesis, characterize their role, and provide an initial characterization of the pathway of its degradation. This led to the identification of sll1541 (encoding SynACO) as the essential gene for retinal synthesis. Multiple pathways for retinal degradation presumably exist. These results have allowed us to construct a strain that expresses a light-dependent proton pump with an action spectrum extending beyond 700 nm. The availability of this strain will be important for further work aimed at increasing the overall efficiency of oxygenic photosynthesis., (Copyright © 2018 American Society for Microbiology.)

We introduce the concept of using site-specific sulfation of various lignins for increasing their aqueous solubility and thereby their processability. Using p-nitrophenylsulfate as a sulfate source and an aryl sulfotransferase enzyme as catalyst, lignins are easily sulfated at ambient conditions. We demonstrate the specific sulfation of phenolic hydroxyl groups on five different lignins: Indulin AT (Kraft softwood), Protobind 1000 (mixed wheat straw/Sarkanda grass soda) and three organosolv lignins. The reaction proceeds smoothly and the increase in solubility is visible to the naked eye. We then examine the reaction kinetics, and show that these are easily monitored qualitatively and quantitatively using UV/Vis spectroscopy. The UV/Vis results are validated with 31 P NMR spectroscopy of the lignin phenol groups after derivatization with phosphorylation reagent II. In general, the results are more significant with organosolv lignins, as Kraft and soda lignins are produced from aqueous lignocellulose extraction processes., (© 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Objective: Inhibition of the mammalian target of rapamycin (mTOR) pathway has been suggested as a possible antiepileptogenic strategy in temporal lobe epilepsy (TLE). Here we aim to elucidate whether mTOR inhibition has antiepileptogenic and/or antiseizure effects using different treatment strategies in the electrogenic post-status epilepticus (SE) rat model., Methods: Effects of mTOR inhibitor rapamycin were tested using the following three treatment protocols: (1) "stop-treatment"-post-SE treatment (6 mg/kg/day) was discontinued after 3 weeks; rats were monitored for 5 more weeks thereafter, (2) "pretreatment"-rapamycin (3 mg/kg/day) was applied during 3 days preceding SE; and (3) "chronic phase-treatment"-5 days rapamycin treatment (3 mg/kg/day) in the chronic phase. We also tested curcumin, an alternative mTOR inhibitor with antiinflammatory and antioxidant effects, using chronic phase treatment. Seizures were continuously monitored using video-electroencephalography (EEG) recordings; mossy fiber sprouting, cell death, and inflammation were studied using immunohistochemistry. Blood was withdrawn regularly to assess rapamycin and curcumin levels with high performance liquid chromatography (HPLC)., Results: Stop-treatment led to a strong reduction of seizures during the 3-week treatment and a gradual reappearance of seizures during the following 5 weeks. Three days pretreatment did not prevent seizure development, whereas 5-day rapamycin treatment in the chronic phase reduced seizure frequency. Washout of rapamycin was slow and associated with a gradual reappearance of seizures. Rapamycin treatment (both 3 and 6 mg/kg) led to body growth reduction. Curcumin treatment did not reduce seizure frequency or lead to a decrease in body weight., Significance: The present study indicates that rapamycin cannot prevent epilepsy in the electrical stimulation post-SE rat model but has seizure-suppressing properties as long as rapamycin blood levels are sufficiently high. Oral curcumin treatment had no effect on chronic seizures, possibly because it did not reach the brain at adequate levels., (Wiley Periodicals, Inc. © 2016 International League Against Epilepsy.)

Introduction: Previous research showed an antimicrobial effect of vanadium chloroperoxidase (VCPO) on in vitro Enterococcus faecalis biofilms. The current study aimed to optimize the use of this enzyme at the root canal pH using a modified VCPO (mVCPO) that was adapted to function at a higher pH and to explore the biocompatibility of mVCPO., Methods: The activity of the original and modified VCPO was assessed using the monochlorodimedone assay. For antimicrobial assessment, 48-hour biofilms of E. faecalis OS-16 were incubated 5 or 30 minutes with mVCPO, bromide, and hydrogen peroxide, and colony-forming units were determined. A metabolic activity assay was used to evaluate the cytotoxic effect of mVCPO on oral fibroblasts., Results: Reaction products generated by mVCPO at a root canal pH of 7.7 significantly inactivated the biofilm after 5 minutes and even more after 30 minutes (Mann-Whitney U test, P < .05). The mVCPO reaction products showed less cytotoxic effects than control solutions and 0.5% sodium hypochlorite (Kruskal-Wallis test, P < .05)., Conclusions: The incubation of mVCPO in the presence of its substrates with in vitro E. faecalis biofilms showed a significant antimicrobial effect at the root canal pH. Also, cytotoxicity tests showed preliminary biocompatibility. Therefore, an interappointment dressing containing mVCPO could aid in improving current endodontic treatment through continuous and local generation of antimicrobials., (Copyright © 2013 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.)

Herein, we report a new flow process with immobilized enzymes to synthesize complex chiral carbohydrate analogues from achiral inexpensive building blocks in a three-step cascade reaction. The first reactor contained immobilized acid phosphatase, which phosphorylated dihydroxyacetone to dihydroxyacetone phosphate using pyrophosphate as the phosphate donor. The second flow reactor contained fructose-1,6-diphosphate aldolase (RAMA, rabbit muscle aldolase) or rhamnulose-1-phosphate aldolase (RhuA from Thermotoga maritima) and acid phosphatase. The immobilized aldolases coupled the formed dihydroxyacetone phosphate to aldehydes, resulting in phosphorylated carbohydrates. A final reactor containing acid phosphatase that dephosphorylated the phosphorylated product yielded the final product. Different aldehydes were used to synthesize carbohydrates on a gram scale. To demonstrate the feasibility of the flow systems, we synthesized 0.6 g of the D-fagomine precursor. By using immobilized aldolase RhuA we were also able to obtain other stereoisomers of the D-fagomine precursor., (Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Marine biofouling--the colonization of small marine microorganisms on surfaces that are directly exposed to seawater, such as ships' hulls--is an expensive problem that is currently without an environmentally compatible solution. Biofouling leads to increased hydrodynamic drag, which, in turn, causes increased fuel consumption and greenhouse gas emissions. Tributyltin-free antifouling coatings and paints based on metal complexes or biocides have been shown to efficiently prevent marine biofouling. However, these materials can damage the environment through metal leaching (for example, of copper and zinc) and bacteria resistance. Here, we show that vanadium pentoxide nanowires act like naturally occurring vanadium haloperoxidases to prevent marine biofouling. In the presence of bromide ions and hydrogen peroxide, the nanowires catalyse the oxidation of bromide ions to hypobromous acid (HOBr). Singlet molecular oxygen ((1)O(2)) is formed and this exerts strong antibacterial activity, which prevents marine biofouling without being toxic to marine biota. Vanadium pentoxide nanowires have the potential to be an alternative approach to conventional anti-biofouling agents.

Acid phosphatase, an enzyme that is able to catalyze the transfer of a phosphate group from cheap pyrophosphate to alcoholic substrates, was covalently immobilized on polymethacrylate beads with an epoxy linker (Immobeads-150 or Sepabeads EC-EP). After immobilization 70% of the activity was retained and the immobilized enzyme was stable for many months. With the immobilized enzyme we were able to produce and prepare D-glucose-6-phosphate, N-acetyl-D-glucosamine-6-phosphate, allyl phosphate, dihydroxyacetone phosphate, glycerol-1-phosphate, and inosine-5'-monophosphate from the corresponding primary alcohol on gram scale using either a fed-batch reactor or a continuous-flow packed-bed reactor., (Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Introduction: The aim of this study was to explore the antimicrobial effect of vanadium chloroperoxidase (VCPO) reaction products on Enterococcus faecalis biofilms of 4 different strains., Methods: Twenty-four-hour biofilms of E. faecalis strains V583, ER5/1, E2, and OS-16 were incubated in mixtures with VCPO, halide (either bromide or chloride), and hydrogen peroxide. The antibacterial efficacy was assessed by colony-forming unit counts., Results: The VCPO reaction products had a similar efficacy in reducing the viability of the 4 strains of E. faecalis (94%; range, 87%-100%). Bromide as the halogen of choice was more effective on E. faecalis strains E2 and OS-16, as compared with chloride (Mann-Whitney U test; P < .05). Despite different quantities of produced biofilms by the 4 strains, VCPO treatment was similarly effective toward all strains (Kruskal-Wallis test; P < .05)., Conclusions: VCPO treatment results in an antimicrobial effect toward in vitro E. faecalis biofilms and might provide an addition to current endodontic treatment, possibly as an antimicrobial dressing., (Copyright © 2012 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.)

To enhance the phosphorylating activity of the bacterial nonspecific acid phosphatase from Salmonella enterica ser. typhimurium LT2 towards dihydroxyacetone (DHA), a mutant library was generated from the native enzyme. Three different variants that showed enhanced activity were identified after one round of epPCR. The single mutant V78L was the most active and showed an increase in the maximal DHAP concentration to 25 % higher than that of the wild-type enzyme at pH 6.0. This variant is 17 times more active than the wild-type acid phosphatase from Salmonella enterica ser. typhimurium LT2 in the acid phosphatase/aldolase cascade reaction at pH 6.0 and is also six times more active than the phosphatase from Shigella flexneri that we previously used.

The aim of this study was to investigate the antimicrobial activity of vanadium chloroperoxidase (VCPO) reaction products on planktonic and biofilm cells of Streptococcus mutans C180-2. Planktonic and biofilm cells were incubated in a buffered reaction mixture containing VCPO, halide (either chloride or bromide) and hydrogen peroxide, and the killing efficacy was assessed by CFU counts. The enzymatic products formed by VCPO significantly reduced the viability of planktonic and biofilm cells compared to their negative controls and the effect on the biofilm cells was more effective than a 0.2% chlorhexidine digluconate treatment. We conclude that VCPO and its reaction products form a potent antimicrobial system against S. mutans.

The cytoskeletal proteins, FtsZ and tubulin, play a pivotal role in prokaryotic cell division and eukaryotic chromosome segregation, respectively. Selective inhibitors of the GTP-dependent polymerization of FtsZ could constitute a new class of antibiotics, while several inhibitors of tubulin are widely used in antiproliferative therapy. In this work, we set out to identify selective inhibitors of FtsZ based on the structure of its natural ligand, GTP. We found that GTP analogs with small hydrophobic substituents at C8 of the nucleobase efficiently inhibit FtsZ polymerization, whereas they have an opposite effect on the polymerization of tubulin. The inhibitory activity of the GTP analogs on FtsZ polymerization allowed us to crystallize FtsZ in complex with C8-morpholino-GTP, revealing the binding mode of a GTP derivative containing a nonmodified triphosphate chain.

Cross-links between amino acid residues in close proximity can provide distance constraints for the validation of models of the 3D structure proteins. The mapping of cross-links by the identification of linked peptides in proteolytic digests is facilitated by cleavable cross-linkers that enable isolation of the cleavage products while preserving information about the linkage. We present an amine-specific cross-linker, bis(succinimidyl)-3-azidomethyl glutarate (BAMG), that fulfils these requirements. Two parallel reaction pathways are induced by tris(carboxyethyl)phosphine (TCEP) in cross-linked peptides from BAMG-treated cytochrome c. One pathway leads to cleavage of the cross-linked species, while in the other the azido group of BAMG is reduced to an amino group without cleavage. Cross-linked peptides and peptides modified by partially hydrolysed BAMG yield distinct sets of TCEP-induced reaction products. These can be isolated by reversed-phase diagonal chromatography and identified by mass spectrometry to reveal the identity of the parent compounds. The ease with which cross-link-derived reaction products can be isolated and identified indicates that the mapping of cross-links in complex biological assemblies and mixtures of protein complexes might become feasible in the near future.

Ubiquinones (UQs) and menaquinones (MKs) perform distinct functions in Escherichia coli. Whereas, in general, UQs are primarily involved in aerobic respiration, the MKs serve as electron carriers in anaerobic respiration. Both UQs and MKs can accept electrons from various dehydrogenases, and may donate electrons to different oxidases. Hence, they play a role in maintaining metabolic flexibility in E. coli whenever this organism has to adapt to conditions with changing redox characteristics, such as oxygen availability. Here, the authors report on the changes in both the size and the redox state of the quinone pool when the environment changes from being well aerated to one with low oxygen availability. It is shown that such transitions are accompanied by a rapid increase in the demethylmenaquinone pool, and a slow increase in the MK pool. Moreover, in exponentially growing cultures in a well-shaken Erlenmeyer flask, it is observed that the assumption of a pseudo-steady state does not hold with respect to the redox state of the quinone pool.

A new enzymatic method for the generation of dihydroxyacetone phosphate (DHAP) using the acid phosphatase from Shigella flexneri (PhoN-Sf) and the cheap phosphate donor pyrophosphate (PPi) is described. The utility of this method was demonstrated in an aldolase-catalyzed condensation carried out in one pot in which DHAP was generated and coupled to propionaldehyde to give a yield of 53% of the isolated dephosphorylated end product.

Directed evolution was performed on vanadium chloroperoxidase from the fungus Curvularia inaequalis to increase its brominating activity at a mildly alkaline pH for industrial and synthetic applications and to further understand its mechanism. After successful expression of the enzyme in Escherichia coli, two rounds of screening and selection, saturation mutagenesis of a "hot spot," and rational recombination, a triple mutant (P395D/L241V/T343A) was obtained that showed a 100-fold increase in activity at pH 8 (k(cat) = 100 s(-1)). The increased K(m) values for Br(-) (3.1 mm) and H(2)O(2) (16 microm) are smaller than those found for vanadium bromoperoxidases that are reasonably active at this pH. In addition the brominating activity at pH 5 was increased by a factor of 6 (k(cat) = 575 s(-1)), and the chlorinating activity at pH 5 was increased by a factor of 2 (k(cat) = 36 s(-1)), yielding the "best" vanadium haloperoxidase known thus far. The mutations are in the first and second coordination sphere of the vanadate cofactor, and the catalytic effects suggest that fine tuning of residues Lys-353 and Phe-397, along with addition of negative charge or removal of positive charge near one of the vanadate oxygens, is very important. Lys-353 and Phe-397 were previously assigned to be essential in peroxide activation and halide binding. Analysis of the catalytic parameters of the mutant vanadium bromoperoxidase from the seaweed Ascophyllum nodosum also adds fuel to the discussion regarding factors governing the halide specificity of vanadium haloperoxidases. This study presents the first example of directed evolution of a vanadium enzyme.

The prokaryotic tubulin homologue FtsZ plays a key role in bacterial cell division. Selective inhibitors of the GTP-dependent polymerization of FtsZ are expected to result in a new class of antibacterial agents. One of the challenges is to identify compounds which do not affect the function of tubulin and various other GTPases in eukaryotic cells. We have designed a novel inhibitor of FtsZ polymerization based on the structure of the natural substrate GTP. The inhibitory activity of 8-bromoguanosine 5'-triphosphate (BrGTP) was characterized by a coupled assay, which allows simultaneous detection of the extent of polymerization (via light scattering) and GTPase activity (via release of inorganic phosphate). We found that BrGTP acts as a competitive inhibitor of both FtsZ polymerization and GTPase activity with a Ki for GTPase activity of 31.8 +/- 4.1 microM. The observation that BrGTP seems not to inhibit tubulin assembly suggests a structural difference of the GTP-binding pockets of FtsZ and tubulin.

The soluble [NiFe]-hydrogenase (SH) of the facultative lithoautotrophic proteobacterium Ralstonia eutropha H16 has up to now been described as a heterotetrameric enzyme. The purified protein consists of two functionally distinct heterodimeric moieties. The HoxHY dimer represents the hydrogenase module, and the HoxFU dimer constitutes an NADH-dehydrogenase. In the bimodular form, the SH mediates reduction of NAD(+) at the expense of H(2). We have purified a new high-molecular-weight form of the SH which contains an additional subunit. This extra subunit was identified as the product of hoxI, a member of the SH gene cluster (hoxFUYHWI). Edman degradation, in combination with protein sequencing of the SH high-molecular-weight complex, established a subunit stoichiometry of HoxFUYHI(2). Cross-linking experiments indicated that the two HoxI subunits are the closest neighbors. The stability of the hexameric SH depended on the pH and the ionic strength of the buffer. The tetrameric form of the SH can be instantaneously activated with small amounts of NADH but not with NADPH. The hexameric form, however, was also activated by adding small amounts of NADPH. This suggests that HoxI provides a binding domain for NADPH. A specific reaction site for NADPH adds to the list of similarities between the SH and mitochondrial NADH:ubiquinone oxidoreductase (Complex I).

Nonspecific acid phosphatases share a conserved active site with mammalian glucose-6-phosphatases (G6Pase). In this work we examined the kinetics of the phosphorylation of glucose and dephosphorylation of glucose-6-phosphate (G6P) catalysed by the acid phosphatases from Shigella flexneri (PhoN-Sf) and Salmonella enterica (PhoN-Se). PhoN-Sf is able to phosphorylate glucose regiospecifically to G6P, glucose-1-phosphate is not formed. The K(m) for glucose using pyrophosphate (PPi) as a phosphate donor is 5.3 mM at pH 6.0. This value is not significantly affected by pH in the pH region 4-6. The K(m) value for G6P by contrast is much lower (0.02 mM). Our experiments show these bacterial acid phosphatases form a good model for G6Pase. We also studied the phosphorylation of inosine to inosine monophosphate (IMP) using PPi as the phosphate donor. PhoN-Sf regiospecifically phosphorylates inosine to inosine-5'-monophosphate whereas PhoN-Se produces both 5'IMP and 3'IMP. The data show that during catalysis an activated phospho-enzyme intermediate is formed that is able to transfer its phosphate group to water, glucose or inosine. A general mechanism is presented of the phosphorylation and dephosphorylation reaction catalysed by the acid phosphatases. Considering the nature of the substrates that are phosphorylated it is likely that this class of enzyme is able to phosphorylate a wide range of hydroxy compounds.

Incubation of the isolated H(+)-ATPase from chloroplasts, CF(0)F(1), with 2-azido-[alpha-(32)P]ATP leads to the binding of this nucleotide to different sites. These sites were identified after removal of free nucleotides, UV-irradiation and trypsin treatment by separation of the tryptic peptides by ion exchange chromatography. The nitreno-AMP, nitreno-ADP and nitreno-ATP peptides were further separated on a reversed phase column, the main fractions were subjected to amino acid sequence analysis and the derivatized tyrosines were used to distinguish between catalytic (beta-Tyr362) and non-catalytic (beta-Tyr385) sites. Several incubation procedures were developed which allow a selective occupation of each of the three non-catalytic sites. The non-catalytic site with the highest dissociation constant (site 6) becomes half maximally filled at 50 microM 2-azido-[alpha-(32)P]ATP, that with the intermediate dissociation constant (site 5) at 2 microM. The ATP at the site with the lowest dissociation constant had to be hydrolyzed first to ADP before a replacement by 2-azido-[alpha-(32)P]ATP was possible. CF(0)F(1) with non-covalently bound 2-azido-[alpha-(32)P]ATP and after covalent derivatization was reconstituted into liposomes and the rates of ATP synthesis as well as ATP hydrolysis were measured after energization of the proteoliposomes by Delta pH/Delta phi. Non-covalent binding of 2-azido-ATP to any of the three non-catalytic sites does not influence ATP synthesis and ATP hydrolysis, whereas covalent derivatization of any of the three sites inhibits both, the degree being proportional to the degree of derivatization. Extrapolation to complete inhibition indicates that derivatization of one site (either 4 or 5 or 6) is sufficient to block completely multi-site catalysis. The rates of ATP synthesis and ATP hydrolysis were measured as a function of the ADP and ATP concentration from uni-site to multi-site conditions with covalently derivatized and non-derivatized CF(0)F(1). Uni-site ATP synthesis and ATP hydrolysis were not inhibited by covalent derivatization of any of the non-catalytic sites, whereas multi-site catalysis is inhibited. These results indicate that multi-site catalysis requires some flexibility between beta- and alpha-subunits which is abolished by covalent derivatization of beta-Tyr385 with a 2-nitreno-adenine nucleotide. Conformational changes connected with energy transduction between the F(0)-part and the F(1)-part are either not required for uni-site ATP synthesis or they are not impaired by the derivatization of any of the three beta-Tyr385.

Mass spectrometric structural analysis of crosslinked peptides is a powerful method to elucidate the spatial arrangement of polypeptides in protein complexes. Our aim is to develop bifunctional crosslinkers that, after crosslinking protein complexes followed by proteolytic digestion, give rise to crosslinked peptides that can be readily tracked down by mass spectrometry. To this end we synthesized the crosslinker N-benzyliminodiacetoyloxysuccinimid (BID), which yields stable benzyl cation marker ions upon low-energy collision-induced dissociation (CID) tandem mass spectrometry. Sensitive detection of the marker ion upon low-energy CID is demonstrated with different BID-crosslinked peptide preparations. With BID it becomes possible to retrieve crosslinked and crosslinker-adducted peptides, without the necessity of purifying crosslinked peptides prior to identification. The basic design of this crosslinker can be varied upon, in order to meet specific crosslinking needs.

After isolation and purification, the H(+)-ATPase from chloroplasts, CF(0)F(1), contains one endogenous ADP at a catalytic site, and two endogenous ATP at non-catalytic sites. Incubation with 2-azido-[alpha-(32)P]ADP leads to tight binding of azido-nucleotides. Free nucleotides were removed by three consecutive passages through centrifugation columns, and upon UV-irradiation most of the label was covalently bound. The labelled enzyme was digested by trypsin, the peptides were separated by ion exchange chromatography into nitreno-AMP, nitreno-ADP and nitreno-ATP labelled peptides, and these were then separated by reversed phase chromatography. Amino acid sequence analysis was used to identify the type of the nucleotide binding site. After incubation with 2-azido-[alpha-(32)P]ADP, the covalently bound label was found exclusively at beta-Tyr-362. Incubation conditions with 2-azido-[alpha-(32)P]ADP were varied, and conditions were found which allow selective binding of the label to different catalytic sites, designated as 1, 2 and 3 in order of decreasing affinity for ADP, and either catalytic site 1 or catalytic sites 1 and 2 together were labelled. For measurements of the degree of inhibition by covalent modification, CF(0)F(1) was reconstituted into phosphatidylcholine liposomes, and the membranes were energised by an acid-base transition in the presence of a K(+)/valinomycin diffusion potential. The rate of ATP synthesis was 50-80 s(-1), and the rate of ATP hydrolysis was 15 s(-1) measured under multi-site conditions. Covalent modification of either catalytic site 1 or catalytic sites 1 and 2 together inhibited ATP synthesis and ATP hydrolysis equally, the degree of inhibition being proportional to the degree of modification. Extrapolation to complete inhibition indicates that derivatisation of catalytic site 1 leads to complete inhibition when 1 mol 2-nitreno-ADP is bound per mol CF(0)F(1). Derivatisation of catalytic sites 1 and 2 together extrapolates to complete inhibition when 2 mol 2-nitreno-ADP are bound per CF(0)F(1). The rate of ATP synthesis and the rate of ATP hydrolysis were measured as a function of the substrate concentration from multi-site to uni-site conditions with derivatised CF(0)F(1) and with non-derivatised CF(0)F(1). ATP synthesis and ATP hydrolysis under uni-site and under multi-site condition were inhibited by covalent modification of either catalytic site 1 or catalytic sites 1 and 2 together. The results indicate that derivatisation of site 1 inhibits activation of the enzyme and that cooperative interactions occur at least between the catalytic sites 2 and 3.

After isolation and purification, the H+-ATPase from chloroplasts, CF0F1, contains one endogenous ADP at a catalytic site, and two endogenous ATP at non-catalytic sites. Incubation with 2-azido-[alpha-32P]ADP leads to tight binding of azidonucleotides. Free nucleotides were removed by three consecutive passages through centrifugation columns, and upon UV-irradiation most of the label was covalently bound. The labelled enzyme was digested by trypsin, the peptides were separated by ion exchange chromatography into nitreno-AMP, nitreno-ADP and nitreno-ATP labelled peptides, and these were then separated by reversed phase chromatography. Amino acid sequence analysis was used to identify the type of the nucleotide binding site. After incubation with 2-azido-[alpha-32P]ADP, the covalently bound label was found exclusively at beta-Tyr-362. Incubation conditions with 2-azido-[alpha-32P]ADP were varied, and conditions were found which allow selective binding of the label to different catalytic sites, designated as 1, 2 and 3 in order of decreasing affinity for ADP, and either catalytic site 1 or catalytic sites 1 and 2 together were labelled. For measurements of the degree of inhibition by covalent modification, CF0F1 was reconstituted into phosphatidylcholine liposomes, and the membranes were energised by an acid-base transition in the presence of a K+/valinomycin diffusion potential. The rate of ATP synthesis was 50-80 s(-1), and the rate of ATP hydrolysis was 15 s(-1) measured under multi-site conditions. Covalent modification of either catalytic site 1 or catalytic sites 1 and 2 together inhibited ATP synthesis and ATP hydrolysis equally, the degree of inhibition being proportional to the degree of modification. Extrapolation to complete inhibition indicates that derivatisation of catalytic site 1 leads to complete inhibition when 1 mol 2-nitreno-ADP is bound per mol CF0F1. Derivatisation of catalytic sites 1 and 2 together extrapolates to complete inhibition when 2 mol 2-nitreno-ADP are bound per CF0F1. The rate of ATP synthesis and the rate of ATP hydrolysis were measured as a function of the substrate concentration from multi-site to uni-site conditions with derivatised CF0F1 and with non-derivatised CF0F1. ATP synthesis and ATP hydrolysis under uni-site and under multi-site condition were inhibited by covalent modification of either catalytic site 1 or catalytic sites 1 and 2 together. The results indicate that derivatisation of site 1 inhibits activation of the enzyme and that cooperative interactions occur at least between the catalytic sites 2 and 3.

The H(+)-ATPase from chloroplasts, CF(0)F(1), was isolated and purified. The enzyme contained one endogenous ADP at a catalytic site, and two endogenous ATP at non-catalytic sites. Incubation with 2-azido-[alpha-(32)P]AD(T)P leads to a tight binding of the azido-nucleotides. Free nucleotides were removed by three consecutive passages through centrifugation columns, and after UV-irradiation, the label was covalently bound. The labelled enzyme was digested by trypsin, the peptides were separated by ion exchange chromatography into nitreno-AMP, nitreno-ADP and nitreno-ATP labelled peptides, and these were then separated by reversed phase chromatography. Amino acid sequence analysis was used to identify the type of the nucleotide binding site. After incubation with 2-azido-[alpha-(32)P]ADP, the covalently bound label was found exclusively at beta-Tyr-362, i.e. binding occurs only to catalytic sites. Incubation conditions with 2-azido-[alpha-(32)P]ADP were varied, and conditions were found which allow selective binding of the label to different catalytic sites, either to catalytic site 2 or to catalytic site 3. For measurements of the degree of inhibition by covalent modification, CF(0)F(1) was reconstituted into phosphatidylcholine liposomes, and the membranes were energised by an acid-base transition in the presence of a K(+)/valinomycin diffusion potential. The rate of ATP synthesis was 120 s(-1), and the rate of ATP hydrolysis was 20 s(-1), both measured under multi-site conditions. Covalent modification of either catalytic site 2 or catalytic site 3 inhibited both ATP synthesis and ATP hydrolysis, the degree of inhibition being proportional to the degree of modification. Extrapolation to complete inhibition indicates that modification of one catalytic site, either site 2 or site 3, is sufficient to completely block multi-site ATP synthesis and ATP hydrolysis. The rate of ATP synthesis and the rate of ATP hydrolysis were measured as a function of the substrate concentration from multi-site to uni-site conditions with covalently modified CF(0)F(1) and with non-modified CF(0)F(1). The result was that uni-site ATP synthesis and ATP hydrolysis were not inhibited by covalent modification of either catalytic site 2 or site 3. The results indicate cooperative interactions between catalytic nucleotide binding sites during multi-site catalysis, whereas neither uni-site ATP synthesis nor uni-site ATP hydrolysis require interaction with other sites.

In active MF1, one of the two non-exchangeable tightly bound adenine nucleotides is an ATP, while the other is an ADP. The respective sites are called the T-site and the D-site. The activity of the enzyme correlates linearly with the amount of bound ATP, ADP at the T-site being inhibitory. When MF1 is stored at room temperature in 50% glycerol and 100 mM Tris-HCl (pH 7.3) after slow passage through a Sephadex column, the tightly bound ATP is slowly dephosphorylated to ADP which is subsequently released, without effect on activity. When enzyme with about one residual ADP left (at the D-site) was incubated at pH 7.3, after dilution of the glycerol, with 400 &mgr;M [14C]ATP under varying conditions, the amount of tightly bound nucleotide triphosphate again correlated well with activity, the residual ADP being bound at the D-site. Optimal results were obtained when the incubation was performed in the presence of a regenerating system. Binding of 2-azido-ATP instead of ATP to the T-site as a triphosphate, as indicated by the specific activity of the enzyme, appeared to be optimal when the binding was performed at pH 6.4 in the absence of Mg2+ and with high concentrations of the nucleotide. Under such conditions, 3 mol 2-azido-AXP per mol F1 remained tightly bound after ammonium sulfate precipitation and column centrifugation, in addition to about one residual ADP at the D-site. After a 2-min period of turnover with ATP/Mg2+ as substrate two mol 2-azido-AXP were left on the enzyme, of which one was bound at a beta-site. These results show that one of the non-catalytic nucleotide binding sites that contain tightly bound nucleotides, is a beta-site, in conflict with the requirements for a rotatory tri-site mechanism for ATP hydrolysis. This beta-site can further be identified with the T-site. The validity of these conclusions for F1 from other sources and for catalysis by membrane-bound enzyme is discussed.

Mitochondrial mRNAs encoding subunits of respiratory-chain complexes in kinetoplastids are post-transcriptionally edited by uridine insertion and deletion. In order to identify the proteins encoded by these mRNAs, we have analyzed respiratory-chain complexes from cultured cells of Crithidia fasciculata with the aid of 2D polyacrylamide gel electrophoresis (PAGE). The subunit composition of F0F1-ATPase (complex V), identified on the basis of its activity as an oligomycin-sensitive ATPase, is similar to that of bovine mitochondrial F0F1-ATPase. Amino acid sequence analysis, combined with binding studies using dicyclohexyldiimide and azido ATP allowed the identification of two F0 subunits (b and c) and all of the F1 subunits. The F0 b subunit has a low degree of similarity to subunit b from other organisms. The F1 alpha subunit is extremely small making the beta subunit the largest F1 subunit. Other respiratory-chain complexes were also analyzed. Interestingly, an NADH: ubiquinone oxidoreductase (complex I) appeared to be absent, as judged by electron paramagnetic resonance (EPR), enzyme activity and 2D PAGE analysis. Cytochrome c oxidase (complex IV) displayed a subunit pattern identical to that reported for the purified enzyme, whereas cytochrome c reductase (complex III) appeared to contain two extra subunits. A putative complex II was also identified. The amino acid sequences of the subunits of these complexes also show a very low degree of similarity (if any) to the corresponding sequences in other organisms. Remarkably, peptide sequences derived from mitochondrially encoded subunits were not found in spite of the fact that sequences were obtained of virtually all subunits of complex III, IV and V.

Binding of 1 mole 5'-fluorosulfonylbenzoyladenosine (FSBA) per mol F1 induces about 50% inhibition of ATPase activity and 80% inhibition of ITPase activity. The binding of additional ligand results in a further inhibition of both activities. Maximally 5 mol/mol F1, causing complete inhibition of activity, can be bound. Using radioactive FSBA more label is found on alpha-subunits than on beta-subunits under the usual buffer conditions. The modified amino acids are alpha-Tyr300, alpha-Tyr244 and beta-Tyr368. Binding of FSBA, at least up to 3 mol/mol F1, does not result in loss of bound ADP, whether the starting enzyme contains 2, 3 or 4 bound nucleotides. Added adenine nucleotides compete with FSBA only for binding that results in modification of beta-subunits, shifting the alpha/beta ratio of bound label to higher values. It is concluded that the alpha-subunits contain two hydrophobic pockets for the binding of nucleoside moieties, with a different orientation relative to the P-loop. One pocket contains alpha-Tyr244 and alpha-Tyr300, the other beta-Tyr368. Since, however, in the binding of adenine nucleotide di- or triphosphates the P-loop is involved, only one of these ligands can bind per subunit. The previously not understood binding characteristics of several substrate analogues have now become interpretable on the assumption that also the structurally homologous beta-subunits contain 2 pockets where nucleoside moieties can bind. The kinetic effects of FSBA binding indicate that the first FSBA binds at the regulatory site that has a high affinity for ADP and pyrophosphate. Binding of pyrophosphate at this high-affinity regulatory site increases the Vmax of the enzyme, while binding at a second regulatory site, a low-affinity site, increases the rate of binding of FSBA with a factor of about 3. Binding of bicarbonate at this latter site is responsible for the disappearance of the apparent negative cooperativity of the ATPase activity.

Bovine heart submitochondrial particles (smp) were incubated with p-fluorosulfonylbenzoyl-5'-adenosine (FSBA) in order to study the binding of this ligand and its effect on ATP synthesis and ATP hydrolysis in smp and to compare the results with those obtained with isolated F1. The binding was measured with the 14C-labeled compound. ATP hydrolysis was in all cases as much inhibited as succinate-driven ATP synthesis and ITP hydrolysis was more inhibited than ATP hydrolysis. The binding experiments show that modification of three nucleotide binding sites results in nearly complete inhibition of ATPase activity. In the presence of pyrophosphate up to 6 mol [14C]SBA/mol F1 can be bound. FSBA preferentially modifies amino acids of the alpha-subunits but also beta-subunits are modified. It is concluded that modification of both subunits results in inhibition of activity. The results are very well comparable with the results obtained with isolated F1, which indicates that our preparation of F1 is a good model for F1 in the intact system. Furthermore it is concluded that each alpha-subunit of F1 in smp, just like in the isolated form, contains two pockets where adenosine moieties can bind, one located above the P-loop, modifying alpha-Tyr-244 and alpha-Tyr-300 and the other one located below the P-loop where also the adenosine moiety of AD(T)P binds, modifying beta-Tyr-368.

We have studied the secondary structure of mitochondrial F1 using infrared spectroscopy. Our results show that in the absence of added nucleotides this complex contains similar percentages of alpha-helices, beta-structures and reverse turns (30%, 28% and 31%, respectively). The influence of ADP and ATP on the different types of secondary structure was determined; when all the nucleotide-binding sites were occupied, small but reproducible changes were observed, corresponding to a decrease in beta-structure and an increase in alpha-helix and reverse turns. The effect of nucleotide binding on the thermal stability of F1 was also studied; the thermal denaturation temperature, 55 degrees C, was increased by 11 degrees C and 7 degrees C by ATP and ADP, respectively. These results indicate that nucleotide binding affects the secondary structure of F1, stabilizing the complex.

Labeling of mitochondrial F1-ATPase with 8-azido-ATP or 8-azido-ADP under turnover conditions with Mg(2+)-ATP resulted in the identification of one exchangeable non-catalytic site whose occupation with a ligand does not influence the ATPase activity of F1 when measured at Vmax. With 8-azido-ADP two exchangeable non-catalytic sites could be labeled, but at one of them the bound ligand exchanges, at least partly, during the illumination under turnover conditions. After labeling an exchangeable non-catalytic site under turnover conditions with 8-azido-ATP or with 8-azido-ADP, F1-ATPase kept the ability to bind NAP3-2N3ADP at the slowly exchangeable noncatalytic site, thereby inhibiting the ATPase activity by 45%, as recently described (Edel et al. (1992) Biochim. Biophys. Acta 1101, 329-338). Covalent modification of the low-affinity non-catalytic site with 8-nitreno-AT(D)P increased the Km of ATP and abolished the negative cooperativity of ATP hydrolysis. This site can therefore be marked as a regulatory site, whose occupation with a nucleotide decreases the affinity of the catalytic sites for ATP.

Purified nicotinamide nucleotide transhydrogenase from beef heart was investigated with respect to labeling and subsequent sequence analysis of a nicotinamide nucleotide-binding site. A photo-activated azide derivative, 8-azidoadenosine 5'-monophosphate, was used as an active-site-directed photoaffinity label, which was shown to be specific for the NAD(H)-binding site in the dark. Light-activated incorporation of the label in transhydrogenase was accompanied by an inactivation, which approached 100% at the incorporation of about 1 mol label/mol transhydrogenase monomer. As expected from the assumed site-specificity of the label. NADH prevented both labeling and inactivation to some extent. However, NADPH also prevented labeling and inactivation marginally. The oxidized substrates NAD+ and NADP+ were inhibitory by themselves under these conditions, and the substrate analogs 5'-AMP and 2'-AMP were also poor protectors. The NAD(H)-site specificity of the azido compound was thus largely lost upon illumination and covalent modification. Radioactive labeling of transhydrogenase with 8-azido-[2-3H]-adenosine 5'-monophosphate followed by protease digestion, isolation of labeled peptides and amino-acid sequence analysis showed that Tyr 1006 in the sequence 1001-1027 close to the C-terminus was labeled. This sequence shows homologies with nucleotide-binding sequences in, e.g., F1-ATPase. On the basis of sequence homologies with other NAD(P)-dependent enzymes it is proposed that transhydrogenase contains 4 nucleotide-binding sites, of which 2 constitute the adenine nucleotide-binding domains of the catalytic sites for NAD(H) and NADP(H) close to the N- and C-terminals, respectively. Each of these domains has an additional vicinal nucleotide-binding sequence which may constitute a non-catalytic nucleotide-binding site or the nicotinamide nucleotide-binding domain of the catalytic site. The present results indicate that 8-azidoadenosine 5'-monophosphate is kinetically specific for the catalytic NAD(H)-binding site, but reacts covalently with Tyr 1006 of the putative non-catalytic site or nicotinamide nucleotide-binding domain formed by the 1001-1027 amino acid sequence of the catalytic NADP(H)-binding site. Interactions between the catalytic NAD(H) and NADP(H) binding sites, and the assumed non-catalytic sites, may be facilitated by a ligand-triggered formation of a narrow pocket, which normally allows an efficient hydride ion transfer between the natural substrates.

F1-ATPase was treated so that it contained three tightly bound nucleotides per molecule. One of these was bound at a catalytic site and was rapidly exchangeable, the two remaining nucleotides were nonexchangeable. Incubation of this preparation with ADP in the presence of Mg2+ results in 40-45% inhibition of the ATPase activity. With 2-azido-ADP instead of ADP, the ligand was covalently bound to F1 by illumination, in the presence or absence of turnover of the enzyme, and the site of binding was determined. In this way, one site could be identified, which induces the inhibition. The attachment of the covalently bound 2-nitreno-ADP is at Tyr-368 of a beta-subunit, characterized in the literature as a non-catalytic site. A second, non-catalytic site also binds 2-azido-ADP, but this binding is partially reversed by the addition of ATP and does not cause further inhibition of the ATPase activity. It is concluded that the slowly exchangeable non-catalytic site is the site of inhibition by ADP.

The dissociation of mitochondrial F1-ATPase with 3 M LiCl at 0 degrees C, followed by reconstitution, has been analysed. FPLC over a gel filtration column in the dissociation buffer revealed the presence of two protein moieties, an alpha 3 gamma delta epsilon complex and single beta-subunits. When the dissociation and chromatography is performed at pH 6.2, the former protein moiety still contains some adenine nucleotides. Reconstitution of the dissociated complex is not possible any more after FPLC, probably due to the loss of residual adenine nucleotides. After a single column centrifugation step one nucleotide per F1 still remains bound. For reconstitution, additional ATP, or a suitable analog, is required. 2-Azido-ATP, but not 8-azido-ATP or ITP, can replace ATP during the reconstitution. F1, reconstituted in the presence of 2-azido-ATP, contains three tightly bound nucleotides, similar to freshly isolated F1, of which in this case one is an adenine nucleotide and two are azido-adenine nucleotides. One of the latter can be rapidly exchanged and is bound to a catalytic site. Covalent binding (at a beta-subunit) of the other tightly bound 2-azido-ATP by ultraviolet illumination does not result in inhibition of the enzyme. Digestion of F1 with trypsin, followed by HPLC, showed that the label is not bound to the fragment containing Tyr-368, nor to the fragment containing Tyr-345. This result was confirmed by CNBr digestion, followed by SDS-urea PAGE. We conclude that during dissociation of F1 one tightly bound nucleotide (ADP) remains bound at an alpha/beta interface site and that for reconstitution binding of ATP to a (non-catalytic) beta-site is required. The conformation of this site differs from that of the two catalytic beta-sites.

Mitochondrial F1, inactivated to various extents with 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl), was dissociated with LiCl and reconstituted after removal of the salt. This procedure resulted in a reactivation that corresponded with a reactivation theoretically expected on the basis of the assumption that the reassociation of beta-subunits into native F1 molecules is random and that two out of the three beta-subunits are directly involved in catalysis. Repeated inactivation of such reactivated F1, followed by the same dissociation-association procedure, resulted in similar data. After inactivation of F1 by covalent binding of 2-N-AT(D)P to one catalytic site, no reactivation upon dissociation-reassociation was obtained due to the fact that such modified F1 did not dissociate under the experimental conditions used.

The synthesis of the photoaffinity label 5-azidopyridine-2-carboxylic acid is described. The 2-oxoglutarate analogue photoaffinity label is a competitive inhibitor with respect to 2-oxoglutarate with a Ki value of 9 X 10(-3) M. Upon ultraviolet irradiation, 5-azidopyridine-2-carboxylic acid inactivated prolyl 4-hydroxylase irreversibly by up to 50%. The extent of inactivation depended on the 5-azidopyridine-2-carboxylic acid concentration and the irradiation time. Inactivation was prevented in the presence of an excess of 2-oxoglutarate. It is concluded that the 5-azidopyridine-2-carboxylic acid became covalently bound to the alpha subunit of prolyl 4-hydroxylase, as the alpha subunit of the photoaffinity labelled enzyme had a decreased electrophoretic mobility in polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate.

The kinetic parameters for the hydrolysis by F1 of the photoreactive nucleotide analogue 2-azido-ATP were determined (Vmax, 105 U/mg F1; Km, 250 microM, in the presence of 1.0 mM SO2-3). In the absence of an activating anion, a non-linear relationship in a Lineweaver-Burk plot was found for the hydrolysis of 2-azido-ATP. The 2-azido-analogues of ATP and ADP proved to be good photoaffinity labels causing notable inactivation of the F1-ATPase activity upon irradiation at 360 nm. This inhibition was also used to demonstrate high-affinity binding of these analogues to a catalytic binding site on the F1. High-affinity binding proved to be an Mg2+-requiring process, occurring with both 2-azido-ATP and 2-azido-ADP but hardly or not occurring with 8-azido-AT(D)P. Covalent binding of 2-nitreno-ATP upon irradiation of F1 containing tightly bound [beta-32P]2-azido-ATP results in a proportional inhibition of ATPase activity, extrapolating to 0.92 mol of covalently bound label per mol of F1 needed for the complete inactivation of the enzyme. When the F1 was irradiated in the presence of excess [beta-32P]2-azido-AT(D)P, 3-4 mol of label were bound when the enzyme was fully inactivated. In all cases, all or most of the radioactivity was found on the beta subunits.

Modification of mitochondrial nicotinamide nucleotide transhydrogenase (NADPH: NAD+ oxidoreductase, EC 1.6.1.1) with 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl), followed by measurement of the absorption or fluorescence of the transhydrogenase-NBD adducts, resulted in a biphasic labelling of approx. 4-6 sulfhydryls, presumably cysteine residues. Of these 1-2 (27%) were fast-reacting and 3-4 (73%) slow-reacting sulfhydryls. In the presence of substrates, e.g., NADPH, the labelling was monophasic and all sulfhydryls were fast-reacting, suggesting that the modified sulfhydryls are predominantly localized peripheral to the NAD(P)(H)-binding sites. The rates of modification allowed the calculation of the rate constants for each phase of the labelling. Both in the absence and in the presence of a substrate, e.g., NADPH, the extent of labelling essentially parallelled the inhibition of transhydrogenase activity. Attempts to reactivate transhydrogenase by reduction of labelled sulfhydryls were not successful. Photo-induced transfer of the NBD adduct in partially inhibited transhydrogenase, from the sulfhydryls to reactive NH2 groups of amino-acid residue(s), identified as lysine residue(s), was parallelled by an inhibition of the residual transhydrogenase activity. It is suggested that a lysine localized close to the fast-reacting NBD-Cl-reactive sulfhydryl groups is essential for activity.

The synthesis is described of the photoaffinity label N-(4-azido-2-nitrophenyl)glycyl-(Pro-Pro-Gly)5 for the peptide binding site of prolyl 4-hydroxylase. The photoaffinity label is a good substrate and is capable of light-induced inactivation of prolyl 4-hydroxylase activity. Inactivation depends on the concentration of photoaffinity label and is prevented by competition with excess (Pro-Pro-Gly)5. Two moles of photoaffinity label per mole of enzyme is needed for 100% inactivation of enzymic activity. Oxidative decarboxylation of 2-oxoglutarate measured in the absence of added peptide substrate is not affected by labeling. We conclude that the covalently bound nitreno derivative of N-(4-azido-2-nitrophenyl)glycyl-(Pro-Pro-Gly)5 acts by preventing the binding of peptide substrate to the catalytic site without interfering with the binding of the other substrates and cofactors 2-oxoglutarate, O2, Fe2+, and ascorbate. Labeling is specific for the alpha subunit of the tetrameric alpha 2 beta 2 enzyme. In addition to two catalytic binding sites that are blocked by the photoaffinity label, the enzyme contains binding subsites for peptide substrates, as judged from the capability of photoinactivated enzyme to bind to a poly(L-proline) affinity column. These binding subsites may account for the rapidly increasing affinity for peptide substrates with increasing chain length.