Occupation Of Active Sites Research Articles

In Vivo Inhibition of Serine Protease Processing Requires a High Fractional Inhibition of Cathepsin C

Inhibition of cathepsin C, a dipeptidyl peptidase that activates many serine proteases, represents an attractive therapeutic strategy for inflammatory diseases with a high neutrophil burden. We recently showed the feasibility of blocking the activation of neutrophil elastase, cathepsin G, and proteinase-3 with a single cathepsin C selective inhibitor in cultured cells. Here we measured the fractional inhibition of cathepsin C that is required for blockade of downstream serine protease processing, in cell-based assays and in vivo. Using a radiolabeled active site probe and U937 cells, a 50% reduction of cathepsin G processing required approximately 50% of cathepsin C active sites to be occupied by an inhibitor. In EcoM-G cells, inhibition of 50% of neutrophil elastase activity required approximately 80% occupancy. Both of these serine proteases were fully inhibited at full cathepsin C active site occupancy, whereas granzyme B processing in TALL-104 cells was partially inhibited, despite complete occupancy. In vivo, leukocytes from cathepsin C(+/-) mice exhibited comparable levels of neutrophil elastase activity to wild-type animals, even though their cathepsin C activity was reduced by half. The long-term administration of a cathepsin C inhibitor to rats, at doses that resulted in the nearly complete blockade of cathepsin C active sites in bone marrow, caused significant reductions of neutrophil elastase, cathepsin G and proteinase-3 activities. Our results demonstrate that the inhibition of cathepsin C leads to a decrease of activity of multiple serine proteases involved in inflammation but also suggest that high fractional inhibition is necessary to reach therapeutically significant effects.

Cardiovascular Risk in Hypertension – Can We Ask for More?

The burden of cardiovascular (CV) disease remains high nowadays, despite the tremendous development in the therapeutic strategies that has occurred during the last 30 years. The growing focus on pharmacological strategies capable of interacting with key pathophysiological mechanisms has resulted in a better control of CV disease. The renin-angiotensin system (RAS) is involved in many pathophysiological processes underlying the development of major CV and renal diseases and, thus, it represents an 'ideal' target for the pharmacological treatment of these clinical conditions. Recently, in addition to the traditional therapeutic approaches, mostly based on the use of ACE inhibitors and/or angiotensin II type 1 receptor antagonists (angiotensin receptor blockers [ARBs]), the scientific and medical community have focused on a new therapeutic possibility to interfere with RAS activity. Indeed, the first compound of the new drug class of direct renin inhibitors, aliskiren, has been made available for clinical use. The therapeutic properties of aliskiren are of particular interest, since it interferes with the enzymatic activities of renin, the key rate-limiting step of the RAS cascade. This unique mechanism of action, e.g. occupation of the renin active site, provides an 'upstream' modulation in the RAS enzymatic-proteic cascade. This results in an inhibition of the biological properties of renin to promote the cleavage of the angiotensin I peptide from the angiotensinogen substrate.Clinical studies have demonstrated that the use of aliskiren provides antihypertensive efficacy comparable with, or even superior to, that observed with other classes of antihypertensive drugs. Also, the addition of aliskiren to different antihypertensive strategies results in a further significant increase in blood pressure (BP) reduction than those achieved with monotherapy based on diuretics, calcium-channel antagonists, ACE inhibitors and ARBs. The overall safety and tolerability of aliskiren are comparable with other classes of antihypertensive drugs and almost overlap with placebo. Recent evidence also demonstrates that aliskiren is effective in promoting the regression of organ damage (e.g. microalbuminuria and left ventricular hypertrophy), beyond its BP-lowering properties and in addition to standard therapies, including ARBs. An ambitious programme of clinical development of this modern antihypertensive drug will investigate whether aliskiren may further limit the incidence of major CV and renal events in hypertensive patients with co-morbidities, treated with the available therapeutic strategies, including ACE inhibitors and ARBs.

Stereospecific Proton Transfer by a Mobile Catalyst in Mammalian Fructose-1,6-bisphosphate Aldolase

Class I fructose-1,6-bisphosphate aldolases catalyze the interconversion between the enamine and iminium covalent enzymatic intermediates by stereospecific exchange of the pro(S) proton of the dihydroxyacetone-phosphate C3 carbon, an obligatory reaction step during substrate cleavage. To investigate the mechanism of stereospecific proton exchange, high resolution crystal structures of native and a mutant Lys(146) --> Met aldolase were solved in complex with dihydroxyacetone phosphate. The structural analysis revealed trapping of the enamine intermediate at Lys(229) in native aldolase. Mutation of conserved active site residue Lys(146) to Met drastically decreased activity and enabled trapping of the putative iminium intermediate in the crystal structure showing active site attachment by C-terminal residues 360-363. Attachment positions the conserved C-terminal Tyr(363) hydroxyl within 2.9A of the C3 carbon in the iminium in an orientation consistent with incipient re face proton transfer. We propose a catalytic mechanism by which the mobile C-terminal Tyr(363) is activated by the iminium phosphate via a structurally conserved water molecule to yield a transient phenate, whose developing negative charge is stabilized by a Lys(146) positive charge, and which abstracts the C3 pro(S) proton forming the enamine. An identical C-terminal binding mode observed in the presence of phosphate in the native structure corroborates Tyr(363) interaction with Lys(146) and is consistent with transient C terminus binding in the enamine. The absence of charge stabilization and of a mobile C-terminal catalyst explains the extraordinary stability of enamine intermediates in transaldolases.

Friedel–Crafts acylation of anisole and toluene with acetic anhydride over nano-sized Beta zeolites

Nano-sized Beta zeolites, with a crystal size of 80–100 nm, were synthesized via surface wet method. The nano-sized HBeta zeolites exhibit much higher activity and stability in the Friedel–Crafts acylation of anisole and toluene with acetic anhydride than the conventional zeolites of large particle size. The small crystal size of nano-sized zeolites may bring on more accessible active sites and then enhance the catalytic activity. The exposed pore openings in nano-sized zeolites allow a fast desorption of heavy products from the catalyst and can then reduce the occupancy of active sites by the adsorption of products; this can then alleviate the catalyst deactivation and improve the catalyst stability.

Water vapor sensitivity of nanosized La(Co, Mn, Fe)1−x(Cu, Pd)xO3 perovskites during NO reduction by C3H6 in the presence of oxygen

A series of La(Co, Mn, Fe)1−x(Cu, Pd)xO3 perovskites having high specific surface areas and nanosized crystal domains was prepared by reactive grinding. The solids were characterized by N2 adsorption, X-ray diffraction (XRD), scanning electron microscopy (SEM), temperature programmed desorption (TPD) of O2, NO+O2, C3H6, in the absence or presence of 5% H2O, Fourier transform infrared (FTIR) spectroscopy, as well as activity tests towards NO reduction by propene under the conditions of 3000ppm NO, 3000ppm C3H6, 1% O2, 0 or 10% H2O, and 50,000h−1 space velocity. The objective was to investigate the influence of H2O addition on catalytic behavior. A good performance (100% NO conversion, 77% N2 yield, and 90% C3H6 conversion) was achieved at 600°C over LaFe0.8Cu0.2O3 under a dry feed stream. With the exposure of LaFe0.8Cu0.2O3 to a humid atmosphere containing 10% water vapor, the catalytic activity was slightly decreased yielding 91% NO conversion, 51% N2 yield, and 86% C3H6 conversion. A competitive adsorption between H2O vapor with O2 and NO molecules at anion vacancies over LaFe0.8Cu0.2O3 was found by means of TPD studies here. A deactivation mechanism was therefore proposed involving the occupation of available active sites by water vapor, resulting in an inhibition of catalytic activity in C3H6+NO+O2 reaction. This H2O deactivation was also verified to be strictly reversible by removing steam from the feed.

The Influence of Impurities on the Crystal Growth Kinetics According to a Competitive Adsorption Model

A mathematical model describing the growth of crystals in impure solutions is presented. Evidence of competitive surface adsorption involving the crystallizing solute and impurities is discussed in terms of the adsorption isotherm in equilibrium and of the mechanism of occupation of active sites for growth. The impurity effect on crystal growth rates is characterized by the Langmuir adsorption constants and by the parameter β measuring the ability of the foreign species to move across the surface and occupy a stable position at the surface steps. Experimental growth rate data taken from the literature is used to test the proposed competitive adsorption model (CAM). The reported effects of the impurity concentration on the crystal growth rate at constant supersaturation are quantitatively described. Additionally, growth rate curves obtained in pure and impure solutions are used to investigate the influence of supersaturation on the relative growth rates. In all the examples considered, the CAM adequately describes the experimental data. An application example is given using the crystal growth rates of sucrose measured in a pilot evaporative crystallizer at different impurity concentrations. The kinetic effect of nonsucrose compounds existing in cane sugar solutions is characterized by estimating the average CAM parameters.

Adsorption of Cr(III), Ni(II), Zn(II), Co(II) ions onto phenolated wood resin

AbstractIn this study, phenolated wood resin was used an adsorbent for the removal of Cr(III), Ni(II), Zn(II), Co(II) ions by adsorption from aqueous solution. The adsorption of metal ions from solution was carried at different contact times, concentrations and pHs at room temperature (25°C). For individual metal ion, the amount of metal ions adsorbed per unit weight of phenolated wood resin at equilibrium time increased with increasing concentration and pH. Also, when the amounts of metal ions adsorbed are compared to each other, it was seen that this increase was order of Cr(III) > Ni(II) > Zn(II) > Co(II). This increase was order of Cr(III) > Ni(II) > Co(II) > Zn(II) for commercial phenol–formaldehyde resin. Kinetic studies showed that the adsorption process obeyed the intraparticle diffusion model. It was also determined that adsorption isotherm followed Langmuir and Freundlich models. Adsorption isotherm obtained for commercial phenol–formaldehyde resin was consistent with Freundlich model well. Adsorption capacities from Langmuir isotherm for commercial phenol–formaldehyde resin were higher than those of phenolated wood resin, in the case of individual metal ions. Original adsorption isotherm demonstrated the monolayer coverage of the surface of phenolated wood resin. Adsorption kinetic followed the intraparticle diffusion model. The positive values of ΔG° determined using the equilibrium constants showed that the adsorption was not of spontaneous nature. It was seen that values of distribution coefficient (KD) decreasing with metal ion concentration in solution at equilibrium (Ce) indicated that the occupation of active surface sites of adsorbent increased with metal ions. © 2006 Wiley Periodicals, Inc. J Appl Polym Sci 101: 2838–2846, 2006

Trapping of hydride forming elements within miniature electrothermal devices. Part 3. Investigation of collection of antimony and bismuth on a molybdenum foil strip following hydride generation

The interaction of stibine and bismuthine with a surface of bare and modified (Pt, Ir and Rh) molybdenum foil strips was investigated by atomic absorption spectrometry with a miniature hydrogen flame atomizer. Different trapping behavior of both analytes was observed contrary to selenium and arsenic hydrides. Maximum trapping efficiency of Sb and Bi is achieved at lower temperatures of 650–750 °C and 500–600 °C, respectively. Absence of hydrogen in argon does not affect trapping of these elements, whereas trapping of As and Se is feasible only in the presence of hydrogen. All used modifiers inhibit trapping of Bi and Sb when their amount exceeds 30 μg. Collected Sb is completely released at temperatures above 2200 °C, whereas a temperature of 1 200 °C is sufficient for vaporization of Bi. Aerodynamic conditions of the injected gas near the molybdenum surface play the same role in trapping both analytes as for As and Se. Maximum trapping efficiencies are achieved at total gas flow rates of 60–70 ml min − 1 and with a capillary distance of 2 mm. Trapping of Sb is not influenced within the whole interferent mass range of 0–30 μg of As and Se, and 0–300 ng of Bi. Trapping of other analytes is affected by 1 to 2 orders of magnitude higher amounts of concomitant hydrides. According to thermodynamic calculations, the competitive occupation of active sites on the molybdenum surface by the AsO (g) and SbO (g) species and the formation of the inactive analyte–interferent diatomic BiSe (g) and AsSe (g) species can probably be the reason for these interference effects.

The Crystal Structure of (S)-3-O-Geranylgeranylglyceryl Phosphate Synthase Reveals an Ancient Fold for an Ancient Enzyme

We report crystal structures of the citrate and sn-glycerol-1-phosphate (G1P) complexes of (S)-3-O-geranylgeranylglyceryl phosphate synthase from Archaeoglobus fulgidus (AfGGGPS) at 1.55 and 2.0 A resolution, respectively. AfGGGPS is an enzyme that performs the committed step in archaeal lipid biosynthesis, and it presents the first triose phosphate isomerase (TIM)-barrel structure with a prenyltransferase function. Our studies provide insight into the catalytic mechanism of AfGGGPS and demonstrate how it selects for the sn-G1P isomer. The replacement of "Helix 3" by a "strand" in AfGGGPS, a novel modification to the canonical TIM-barrel fold, suggests a model of enzyme adaptation that involves a "greasy slide" and a "swinging door." We propose functions for the homologous PcrB proteins, which are conserved in a subset of pathogenic bacteria, as either prenyltransferases or being involved in lipoteichoic acid biosynthesis. Sequence and structural comparisons lead us to postulate an early evolutionary history for AfGGGPS, which may highlight its role in the emergence of Archaea.

Adaptation of the behaviour of an aspartic proteinase inhibitor by relocation of a lysine residue by one helical turn

In addition to self-inhibition of aspartic proteinase zymogens by their intrinsic proparts, the activity of certain members of this enzyme family can be modulated through active-site occupation by extrinsic polypeptides such as the small IA3 protein from Saccharomyces cerevisiae. The unprecedented mechanism by which IA3 helicates to inhibit its sole target aspartic proteinase locates an i, i+4 pair of charged residues (Lys18+Asp22) on an otherwise-hydrophobic face of the amphipathic helix. The nature of these residues is not crucial for effective inhibition, but re-location of the lysine residue by one turn (+4 residues) in the helical IA3 positions its side chain in the mutant IA3-proteinase complex in an orientation essentially identical to that of the key lysine residue in zymogen proparts. The binding of the extrinsic mutant IA3 shows pH dependence reminiscent of that required for the release of intrinsic zymogen proparts so that activation can occur.

Insight into Factor IXa Selectivity and Reactivity Using Kunitz-Type Inhibitors: Importance of Factor IXa Residue K98.

Previous studies by us have shown that blood coagulation factor IXa is relatively resistant to inhibition by the Kunitz-type inhibitor bovine pancreatic trypsin inhibitor (BPTI; aprotinin), but that this resistance can be partially alleviated by the presence of low molecular weight heparin. In order to gain insight into potential mechansims of factor IXa selectivity and its modulation by heparin we undertook an examination of the reactivity of factor IXa with several other Kunitz-type inhibitors: the Kunitz-type inhibitor domain of protease nexin-2 (PN2KPI) and the first two Kunitz-type inhibitor domains of tissue factor pathway inhibitor (TFPI K1 and TFPI K2). As expected, factor IXa exhibited remarkable specificity towards these highly-homologous inhibitors and expressed the following order of reactivity: PN2KPI > TFPI K1/K2 > BPTI. Surprisingly, the enhancing effect of heparin (enoxaparin) was limited to factor IXa reactivity with BPTI and was not observed with PN2KPI or TFPI inhibitory domains. This effect of heparin was not due to a simple template or bridging mechanism between factor IXa and BPTI since progressively smaller oligosaccharides (H14, H10, H6) retained function. We performed molecular modeling and molecular dynamic simulation studies that suggested that BPTI residue R39 may sterically clash with the 99 loop region of factor IXa (specifically residue K98). In contrast, the corresponding position in PN2KPI (G39) does not appear to clash with factor IXa residue K98 in modeling studies. In addition, the 99 loop region of factor IXa is in close proximity to the heparin binding site. Thus, based on these observations we hypothesized that factor IXa residue K98 could be restricting occupation of the factor IXa active site, and that this steric hindrance could be partly alleviated by heparin binding. We therefore examined a mutant of factor IXa in which residue K98 was mutated to A. The fIXK98A mutant was expressed as a zymogen in human 293 cells and purified by a combination of ion exchange and heparin affinity chromatography. The fIXK98A mutant exhibited identical procoagulant activity compared to wild-type factor IXa in a standard clotting assay. In addition, upon activation with RVV-X the fIXaK98A enzyme showed similar activity as wild-type factor IXa towards the small peptide substrate CBS 31.39. In sharp contrast, however, examination of the inhibition of fIXaK98A by BPTI showed a dramatic enhancement in inhibition compared to the wild-type enzyme: Ki = 20 μM in the absence of heparin and Ki = 10 μM in the presence of heparin for fIXaK98A; and Ki > 500 μM in the absence of heparin and Ki = 40 μM in the presence of heparin for wild-type factor IXa. We conclude from this that factor IX residue K98 limits access of specific molecules into the active site of factor IXa and protects it from inhibition by BPTI. It would seem that some or all of this interference is alleviated upon heparin binding to factor IXa. The smaller effect of heparin on the mutant enzyme (2-fold) compared to the wild-type enzyme (>10-fold) further supports the supposition that heparin binding to factor IXa may in part be facilitating movement of the 99 loop of factor IXa to enhance access to the active site. This may have implications in the selectivity and reactivity of factor IXa with other inhibitors as well as certain substrates and provides some important insight into factor IXa function.

Poly‐l‐proline type II peptide mimics as probes of the active site occupancy requirements of cGMP‐dependent protein kinase

Based on the X-ray crystal structure of cAMP-dependent protein kinase (PKA) with the endogenous inhibitor PKI and the X-ray crystal structure of cyclin-dependent kinase 2 (CDK2) with a substrate peptide, a proposal is put forth that some protein kinases bind peptide substrates in their active sites in the poly-L-proline type II (PPII) conformation. In this work, PPII peptide mimics are evaluated as pseudosubstrate inhibitors of cGMP-dependent protein kinase (PKG) to explore if PKG also binds peptide substrates in the PPII conformation. Inhibition data of our PPII mimetics provide evidence that the P-1, P-2, and P-3 residues of substrate peptides bind in the PPII conformation (phi approximately -75 degrees, psi approximately 145 degrees). In addition, the inhibition data also suggest that the P-1, P-2, and P-3 residues in substrate peptides bind with a gauche(-) chi1 angle.

Active Site Occupancy Required for Catalytic Cooperativity by Escherichia coli Transcription Termination Factor Rho

Escherichia coli transcription termination factor Rho exhibits the phenomenon of catalytic cooperativity. The catalytic rate per site is 30-fold faster when all three sites are filled with substrate ATP than when only a single site is occupied (Stitt, B. L., and Xu, Y. (1998) J. Biol. Chem. 273, 26477-26486). Experiments presented here investigate whether all three active sites must be filled or whether only two occupied sites are required for catalytic cooperativity. The results indicate that all three Rho catalytic sites must be filled with substrate to achieve the enhanced catalytic rate, both in pre-steady-state and in steady-state hydrolysis. They further suggest that, once the enzyme is saturated with ATP, a V(max) enzyme conformation is achieved that is stable for at least three catalytic cycles.

Evidence for Two Different Mechanisms Triggering the Change in Quaternary Structure of the Allosteric Enzyme, Glucosamine-6-Phosphate Deaminase

The generation and propagation of conformational changes associated with ligand binding in the allosteric enzyme glucosamine-6-phosphate deaminase (GlcN6P deaminase, EC 3.5.99.6) from Escherichia coli were analyzed by fluorescence measurements. Single-tryptophan mutant forms of the enzyme were constructed on the basis of previous structural and functional evidence and used as structural-change probes. The reporter residues were placed in the active-site lid (position 174) and in the allosteric site (254 and 234); in addition, signals from the natural Trp residues (15 and 224) were also studied as structural probes. The structural changes produced by the occupation of either the allosteric or the active site by site-specific ligands were monitored through changes in the spectral center of mass (SCM) of their steady-state emission fluorescence spectra. Binding of the allosteric activator produces only minimal signals in titration experiments. In contrast, measurable spectral signals were found when the active site was occupied by a dead-end inhibitor. The results reveal that the two binary complexes, enzyme-activator (R(A)) and enzyme-inhibitor (R(S)) complexes, have structural differences and that they also differ from the ternary complex (R(AS)). The mobility of the active-site lid motif is shown to be independent of the allosteric transition. The active-site ligand induces cooperative SCM changes even in the enzyme-activator complex, indicating that the propagation pathway of the conformational relaxation triggered from the active site is different from that involved in the heterotropic activation. Analysis of the complete set of mutants shows that the occupation of the active site generates structural perturbations, which are propagated to the whole of the monomer and extend to the other subunits. The accumulative effect of these propagated changes should be responsible for the change in the sign of the DeltaG degrees ' of the T to R transition associated with the progression of the active-site occupation, resulting in the predominance of the R over the T forms in the population of deaminase hexamers.

Kinetic and regiospecific interrogation of covalent intermediates in the nonribosomal peptide synthesis of yersiniabactin.

For interrogation of enzyme-bound intermediates in nonribosomal peptide synthetases (NRPSs), mass spectrometry is used to read out the kinetics and substrate specificity of this medicinally important class of enzymes. The protein HMWP2 (230 kDa) catalyzes 11 chemical reactions, four of which could be resolved by fast quench approaches combined with mass spectrometry. The rate of complex intermediate accumulation at the PCP1 active site was observed to occur with a rate of 19 s(-1), with the rate of cysteine acylation faster than that of intermediate translocation. Use of alternative substrates for salicylic acid (at the ArCP carrier domain) and l-cysteine (at the PCP1 carrier domain) revealed a high penalty for omission of the salicyl alcohol. For some substrates, large discrepancies were found between prior adenylation assays and the current MS-based readouts. Indirect evidence for condensation via a thiolate attack (vs an amino group) was also accumulated. This is the first report to correlate the percent occupancy of multiple active sites in parallel with kinetic and structural resolution of intermediates and provides new evidence of interdomain and intermodule communication within thiotemplate assembly lines.

A Caspase Active Site Probe Reveals High Fractional Inhibition Needed to Block DNA Fragmentation

Apoptotic markers consist of either caspase substrate cleavage products or phenotypic changes that manifest themselves as a consequence of caspase-mediated substrate cleavage. We have shown recently that pharmacological inhibitors of caspase activity prevent the appearance of two such apoptotic manifestations, alphaII-spectrin cleavage and DNA fragmentation, but that blockade of the latter required a significantly higher concentration of inhibitor. We investigated this phenomenon through the use of a novel radiolabeled caspase inhibitor, [(125)I]M808, which acts as a caspase active site probe. [(125)I]M808 bound to active caspases irreversibly and with high sensitivity in apoptotic cell extracts, in tissue extracts from several commonly used animal models of cellular injury, and in living cells. Moreover, [(125)I]M808 detected active caspases in septic mice when injected intravenously. Using this caspase probe, an active site occupancy assay was developed and used to measure the fractional inhibition required to block apoptosis-induced DNA fragmentation. In thymocytes, occupancy of up to 40% of caspase active sites had no effect on DNA fragmentation, whereas inhibition of half of the DNA cleaving activity required between 65 and 75% of active site occupancy. These results suggest that a high and persistent fractional inhibition will be required for successful caspase inhibition-based therapies.

Investigation of manganese-doped iron ammonia synthesis catalysts

The iron catalysts for ammonia synthesis doped with manganese in amounts up to 3 wt.% has been investigated. The addition of manganese oxide did not influence the distribution of other promoters in the catalyst precursors, manganese oxide itself was entirely incorporated into phases of iron oxides, its concentration in the wustite phase was about twice that in the magnetite phase. At concentrations of manganese below 0.4 wt.% the activity of manganese-doped catalyst was about 10% higher than that of standard industrial catalyst in the temperatures 500 and 450 °C after reduction, and in 400 °C after overheating. At higher concentrations the manganese acted as a catalyst’s poison. The cause for the higher activity of catalysts of low manganese content is surface area development, poisoning effect is due to occupation of active sites by manganese atoms at manganese content exceeding 0.4 wt.%. The corresponding surface coverage calculated according to Langmuir–Maclean’s model of segregation amounted to θ=0.25. The estimated value of free energy of segregation of manganese in iron in the overheated catalysts amounted to approximately −15 kJ/mol. Before overheating no segregation process was observed. The surface coverage exceeding θ≈0.3 caused formation of a new phase of manganese oxide on the surface of catalyst. Little effect of manganese addition on specific surface area and mean iron crystallite size is observed after reduction. After overheating manganese caused the rise of specific surface area due to higher energy of manganeseoxygen bond than that of ironoxygen bond. Together with increasing surface area the mean diameter of iron crystallites also increased.

Avian sulfhydryl oxidase is not a metalloenzyme: adventitious binding of divalent metal ions to the enzyme.

Metal- and flavin-dependent sulfhydryl oxidases catalyze the generation of disulfide bonds with reduction of oxygen to hydrogen peroxide. The mammalian skin enzyme has been reported to be copper-dependent, but a recent protein sequence shows it belongs to the Quiescin/sulfhydryl oxidase (QSOX) flavoprotein family. This work demonstrates that avian QSOX is not a metalloenzyme, and that copper and zinc ions inhibit the oxidation of reduced pancreatic ribonuclease by the enzyme. Studies with Zn(2+), as a redox inactive surrogate for copper, show that one Zn(2+) binds to four-electron-reduced QSOX by diverting electrons away from the flavin and into two of the three redox active disulfide bridges in the enzyme. The resulting zinc complex is modestly air-stable, reverting to a spectrum of the native protein with a t(1/2) of 40 min, whereas the four-electron-reduced native QSOX is reoxidized in less than a second under comparable conditions. Using tris(2-carboxyethyl)phosphine hydrochloride (TCEP), an alternate substrate of QSOX that binds Zn(2+) relatively weakly (unlike dithiothreitol), allows rapid inhibition of oxidase activity to be demonstrated at low micromolar metal levels. Zinc binding was followed by rapid-scanning spectrophotometry. Copper also binds the four-electron-reduced form of QSOX with a visible spectrum suggestive of active site occupancy. In addition to interactions with the reduced enzyme, dialysis experiments show that multiple copper and zinc ions can bind to the oxidized enzyme without the perturbation of the flavin spectrum seen earlier. These data suggest that a reinvestigation of the metal content of skin sulfhydryl oxidases is warranted. The redox-modulated binding of zinc to QSOX is considered in light of evidence for a role of zinc-thiolate interactions in redox signaling and zinc mobilization.

Inhibition of Corrosion-Related Reduction Processes via Chromium Monolayer Formation

The oxygen reduction reaction (ORR) was examined on copper, platinum, and glassy carbon electrodes, with regard to its inhibition by exposure of the electrode to chromate ion in NaCl solution. All three electrode materials exhibited a mass transport limited current for the ORR at sufficiently negative potentials, but this current was strongly inhibited in the presence of Inhibition persisted in -free solution after the electrode was rinsed thoroughly, indicating that formed an irreversibly adsorbed inhibiting layer. A reduction peak observed in solution had an area of and the area varied little with concentration, electrode material, and potential in the range of to vs. Ag/AgCl. This reduction peak is attributed to formation, and corresponds to formation of approximately a monolayer of oxyhydroxide. Once formed, this monolayer inhibits both reduction and further reduction of The onset of monolayer formation at about vs. Ag/AgCl is the same as the potential of the onset of ORR inhbition in dilute The monolayer also decreases the electron transfer rate to ferrocene and which are known to be outer sphere redox systems that do not require adsorption to the electrode surface. The results indicate that the adsorbed film formed by reduction is a powerful inhibitor of oxygen reduction, due both to occupation of active chemisorption sites and to inhibition of electron transfer. In the context of corrosion protection, acts as a “site-directed” irreversible inhibitor which migrates to active sites for the ORR, then is reduced to and forms a permanent inhibiting monolayer. © 2002 The Electrochemical Society. All rights reserved.

Angiotensin peptides modulate bradykinin levels in the interstitium of the dog heart in vivo.

We previously demonstrated the substantial capacity for angiotensin (ANG) II formation in the interstitium of the dog heart in vivo. The current study tested the hypothesis that interstitial fluid (ISF) bradykinin (BK) is influenced by ANG II formation. Four microdialysis probes were inserted into the left ventricular myocardium of eight open-chest anesthetized dogs. The probe effluent was collected during four stages in each dog. Probes 1 and 3 sequentially delivered: 1) buffer; 2) ANG I (15 microM); 3) ANG II type 1 receptor antagonist (AT(1)-ant; irbesartan, 50 microM) or AT(2)-ant (PD123319, 50 microM); and 4) ANG I + AT(1)-ant or ANG I + AT(2)-ant. Probes 2 and 4 used the same protocol, substituting ANG II for ANG I in a concentration (0.5 microM) equivalent to that achieved during ANG I infusion. ISF BK levels increased 15-fold during ANG I (p < 0.001) but not during ANG II infusion. Co-infusion of selective AT(1)- and AT(2)-ants or nonselective AT-ant did not block the increase in ISF BK. ISF infusions of ANG I also produced a greater than 400-fold rise in ISF ANG-(1-7) over baseline. ISF infusion of ANG-(1-7) (10 microM) produced a 15-fold increase in ISF BK (p < 0.001). The metabolic machinery exists for the formation of BK and ANG-(1-7) in the cardiac ISF space that is not blocked by an AT receptor antagonist. The differential increase in ISF BK during ANG I and ANG-(1-7) but not during ANG II infusions suggests the possibility of decreased catabolism of ISF BK by an angiotensin-converting enzyme due to active site occupation by ANG I and ANG-(1-7).