Lack Of Catalytic Activity Research Articles

Materials for Symmetrical Solid Oxide Fuel Cells

A new concept of Solid Oxide Fuel Cell (SOFC), Symmetrical SOFC (SFC), has been investigated. In this new configuration, the same electrode material is used simultaneously as anode and cathode. Interconnector materials, such as chromites, could be potential symmetrical electrodes, at least, under pure hydrogen at relatively high temperatures. However, their lack of catalytic activity towards hydrocarbon oxidation led to the use of substituted-chromites to enhance the aforementioned catalytic activity. Thus, La0.75Sr0.25Cr0.5Mn0.5O3-δ (LSCM) is a good option for such symmetrical configuration, rendering fuel cell performances in excess of 500 and 300mW/cm2 using pure H2 and CH4 as fuel, at 950oC. Other potential materials could be typical n-type anode materials, such as strontium titanates, that could be activated for oxidising conditions by substituting some of Ti content for Fe to introduce p-type conductivity. Preliminary electrochemical experiments on La4Sr8Ti12-xFexO38-δ-based SFCs show that they perform reasonably well under humidified H2.

Ordered Cleavage of Prothrombin by Prothrombinase Results from Constrained Binding and Preferential Active Site Engagement by One of the Two Cleavage Sites in Prothrombin.

The conversion of prothrombin to thrombin is a paradigm for proteolytic activation reactions wherein product is produced following cleavage at more than one site in the substrate. Human prothrombinase catalyzes thrombin formation by sequential cleavage of prothrombin at Arg320 followed by cleavage at Arg271. The largely ordered activation arises because Arg320 in intact prothrombin is hydrolyzed by prothrombinase with a Vmax that is ~30-fold greater than that for cleavage at Arg271 while substrate affinity is unchanged. Paradoxically, this phenomenon has been proposed to arise from the constrained binding of prothrombin to prothrombinase. The alternate proposal is that two interconverting forms of the enzyme differentially recognize and cleave the two sites in prothrombin. We have investigated substrate binding using prothrombinase assembled with a catalytically inactive recombinant factor Xa (XaS195A) in which Ser195 was replaced with Ala and a series of recombinant variants of prothrombin. The prothrombin variants included wild type prothrombin (IIWT) containing both cleavage sites, IIQ271 with Arg271 replaced with Gln and a single cleavable site at Arg320, IIQ320 with Arg320 replaced with Gln and a single cleavable site at Arg271 and the uncleavable IIQQ containing both Gln substitutions. Titration of XaS195A assembled into prothrombinase with increasing concentrations of the fluorescent active site probe p-aminobenzamidine (PAB) produced a saturable increase in fluorescence confirming the expected ability of XaS195A to bind ligands, such as PAB, at the active site despite its lack of catalytic activity. The addition of a saturating concentration of IIWT to reaction mixtures containing PAB and prothrombinase assembled with XaS195A resulted in a decrease in PAB fluorescence arising from the engagement of the active site by the substrate and the associated displacement of PAB. Only a minor change in PAB fluorescence was observed with IIQQ even though this uncleavable derivative binds to prothrombinase with the same affinity as IIWT. Thus, features of the substrate-enzyme interaction that determine affinity are independent of active site engagement by the substrate. Saturating concentrations of IIQ271 displaced PAB from the active site of XaS195A within prothrombinase to the same extent observed with IIWT while essentially no fluorescence decrease was observed with IIQ320. It follows that although all prothrombin derivatives bind with similar affinity to prothrombinase, elements flanking Arg320 can readily engage the active site of XaS195A within the enzyme complex while those flanking the Arg271 site cannot. Our equilibrium binding measurements establish a primary role for active site binding in determining the perceived rate constant for catalysis without influencing substrate affinity. The differential action of prothrombinase on the two cleavage sites in prothrombin likely arises from constraints imposed by active site-independent interactions that drive substrate affinity and permit active site docking by the Arg320 site in the substrate but not the Arg271 site. These constraints in active site engagement explain asymmetry in the recognition of the two bonds in intact prothrombin and its ordered cleavage by prothrombinase.

The Crystal Structure of a Novel, Inactive, Lysine 49 PLA2 from Agkistrodon acutus Venom: AN ULTRAHIGH RESOLUTION, AB INITIO STRUCTURE DETERMINATION

The crystal structure of acutohaemolysin, a lysine 49 phospholipase A2 protein with 1010 non-hydrogen protein atoms and 232 water molecules, has been determined ab initio using the program SnB at an ultrahigh resolution of 0.8 A. The lack of catalytic activity appears to be related to the presence of Phe102, which prevents the access of substrate to the active site. The substitution of tryptophan for leucine at residue 10 interferes with dimer formation and may be responsible for the additional loss of hemolytic activity. The ultrahigh resolution of the experimental diffraction data permits alternative conformations to be modeled for disordered residues, many hydrogen atoms to be located, the protonation of the Nepsilon2 atom in the catalytic residue His48 to be observed experimentally, and the density of the bonding electrons to be analyzed in detail.

Insights into the Catalytic Mechanism of Cofactor-independent Phosphoglycerate Mutase from X-ray Crystallography, Simulated Dynamics and Molecular Modeling

Phosphoglycerate mutases catalyze the isomerization of 2 and 3-phosphoglycerates, and are essential for glucose metabolism in most organisms. Here, we further characterize the 2,3-bisphosphoglycerate-independent phosphoglycerate mutase (iPGM) from Bacillus stearothermophilus by determination of a high-resolution (1.4 Å) crystal structure of the wild-type enzyme and the crystal structure of its S62A mutant. The mutant structure surprisingly showed the replacement of one of the two catalytically essential manganese ions with a water molecule, offering an additional possible explanation for its lack of catalytic activity. Crystal structures invariably show substrate phosphoglycerate to be entirely buried in a deep cleft between the two iPGM domains. Flexibility analyses were therefore employed to reveal the likely route of substrate access to the catalytic site through an aperture created in the enzyme's surface during certain stages of the catalytic process. Several conserved residues lining this aperture may contribute to orientation of the substrate as it enters. Factors responsible for the retention of glycerate within the phosphoenzyme structure in the proposed mechanism are identified by molecular modeling of the glycerate complex of the phosphoenzyme. Taken together, these results allow for a better understanding of the mechanism of action of iPGMs. Many of the results are relevant to a series of evolutionarily related enzymes. These studies will facilitate the development of iPGM inhibitors which, due to the demonstrated importance of this enzyme in many bacteria, would be of great potential clinical significance.

Involvement of crk adapter proteins in regulation of lymphoid cell functions.

The Crk adapter proteins consist of Src homology 2 (SH2) SH2 and SH3 domains, which bind tyrosine-phosphorylated peptides and polyproline-rich motives, respectively. They are linked to multiple signaling pathways in different cell types, including lymphocytes, and because of their lack of catalytic activity, many studies on Crk were aimed at the identification of their binding partners and determination of the physiologic meaning of these interactions. Crk proteins were found to be involved in the early steps of lymphocyte activation through their SH2-mediated transient interaction with signal-transducing molecules, such as Cbl, ZAP-70, CasL, and STAT5. In addition, Crk proteins are constitutively associated with effector molecules that mediate cell adhesion and thereby regulate lymphocyte extravasation and recruitment to sites of inflammation. This article describes selected studies of Crk, performed predominantly in lymphocytes, and discusses their potential relevance to the role of Crk in the regulation of lymphocyte functions.

Resonant Raman-scattering measurements of trichloroethene in a thermal boundary layer.

Concentration profiles of trichloroethene were measured in a boundary-layer flow over a heated ceramic surface. Raman scattering was excited with the fifth harmonic of a Nd:YAG laser at 213 nm. This wavelength took advantage of a resonance in the trichloroethene molecule to significantly enhance the C2HCl3 scattering cross section. The resonant Raman system was calibrated in a heated flow. The optical system was optimized so that measurements could be obtained close to the solid surface, normally a significant challenge for a spontaneous Raman-scattering setup. Measured concentrations indicated the lack of catalytic activity on a bare alumina surface. However, the results showed that a surface that was coated with Cr2O3-based zeolite was catalytically active.

Domain organization of p130, PLC-related catalytically inactive protein, and structural basis for the lack of enzyme activity.

The 130-kDa protein (p130) was isolated as a novel inositol 1,4, 5-trisphosphate [Ins(1,4,5)P3]-binding protein similar to phospholipase C-delta1 (PLC-delta1), but lacking catalytic activity [Kanematsu, T., Takeya, H., Watanabe, Y., Ozaki, S., Yoshida, M., Koga, T., Iwanaga, S. & Hirata, M. (1992) J. Biol. Chem. 267, 6518-6525; Kanematsu, T., Misumi, Y., Watanabe, Y., Ozaki, S., Koga, T., Iwanaga, S., Ikehara, Y. & Hirata, M. (1996) Biochem. J. 313, 319-325]. To test experimentally the domain organization of p130 and structural basis for lack of PLC activity, we subjected p130 to limited proteolysis and also constructed a number of chimeras with PLC-delta1. Trypsin treatment of p130 produced four major polypeptides with molecular masses of 86 kDa, 55 kDa, 33 kDa and 25 kDa. Two polypeptides of 86 kDa and 55 kDa started at Lys93 and were calculated to end at Arg851 and Arg568, respectively. Using the same approach, it has been found that the polypeptides of 33 kDa and 25 kDa are likely to correspond to regions between Val569 and Arg851 and Lys869 and Leu1096, respectively. All the proteolytic sites were in interconnecting regions between the predicted domains, therefore supporting domain organization based on sequence similarity to PLC-delta1 and demonstrating that all domains of p130, including the unique region at the C-terminus, are stable, tightly folded structures. p130 truncated at either or both the N-terminus (94 amino acids) and C-terminus (851-1096 amino acids) expressed in COS-1 cells showed no catalytic activity, indicating that p130 has intrinsically no PLC activity. A number of chimeric molecules between p130 and PLC-delta1 were constructed and assayed for PLC activity. It was shown that structural differences in interdomain interactions exist between the two proteins, as only some domains of p130 could replace the corresponding structures in PLC-delta1 to form a functional enzyme. These results suggest that p130 and the related proteins could represent a new protein family that may play some distinct role in cells due to the capability of binding Ins(1,4,5)P3 but the lack of catalytic activity.

Novel rhodium(I) complexes with (2-hydroxyphenyl)diphenylphosphine ligand: catalytic properties and X-ray structures of Rh(OC6H4PPh2)(CO)(PPh3) and Rh(OC6H4PPh2){P(OPh)3}2 · 0.5C6H6

The novel rhodium complexes with the bidentate PO ligand (PO=OC6H4PPh2−) of the form Rh(PO)(CO)L (La=POH=HOC6H4PPh2 (1), PPh3 (2), P(NC4H4)3 (4), PPh2(NC4H4) (6)) and Rh(PO)L2 (Lb=P(OPh)3 (3), P(NC4H4)3 (5)) were obtained by ligand exchange in Rh(β-diketone)(CO)2, Rh(β-diketone)(CO)L and Rh(β-diketone)L2 complexes. All complexes of the Rh(PO)(CO)La type exist in solution as isomers with both phosphorus atoms in the trans position as was shown by 31P{1H}-NMR. The trans influence of the phosphorus atom of a bidentate PO ligand is stronger than that of oxygen atom, which is manifested by the differences of Rh–P bonds in (2) (2.283(1) and 2.327(1) Å) and of Rh–P (phosphite) bonds in (3) (2.233(2) and 2.139(2) Å). The complexes (1) and (2) used alone or with an excess of free phosphine (POH, PPh3, P(NC4H4)3) are not active in hexen-1-e hydroformylation at 1 MPa CO/H2=1 and at 353 K. The lack of catalytic activity is explained by the extremely high stability of the chelate (PO) ring which does not allow the formation of the active form of the catalyst. In contrast, the complex (3) used alone as the catalyst precursor produces 54 and 72.9% of aldehydes when used with a six-fold excess of P(OPh)3. Complex (1) modified with P(OPh)3 catalyses hexen-1-e hydroformylation with a 73.6–84.6% yield of aldehydes. Under hydroformylation reaction conditions, the PO ligand is removed from the coordination sphere of (1) and complexes of the form HRh(CO){P(OPh)3}3 and HRh{P(OPh)3}4 are formed.

Functionally Active Catalytic Domain Is Essential for Guanylyl Cyclase-Linked Receptor Mediated Inhibition of Human Aldosterone Synthesis

An aspartate-to-alanine point mutation in the catalytic domain (D853A) of guanylyl cyclase-C (GC-C), the heat-stable enterotoxin (STa) receptor, rendered the enzyme catalytically inactive. Mn2+/Triton X-100-stimulated guanylyl cyclase activity was detected in membranes from COS7 cells overexpressing GC-C but not GC-CD853A. STa treatment of paired cells resulted in cGMP production in those transiently expressing GC-C but not GC-CD853A. GC-C and GC-CD853A showed similar Bmax and Kd values for [125I]STa binding in these cells, indicating that the lack of catalytic activity in the latter was not due to differing expression levels or reduced binding affinity. The involvement of the catalytic domain in aldosteronogenesis was studied in human adrenocortical H295R cells. COS7 and H295R cells infected with vaccinia virus-expressing GC-C and GC-CD853A (VVGC-CD853A) had [125I]STa-binding characteristics akin to those in transfected cells. Immunoblot confirmed that both GC-C and GC-CD853A formed similar higher order oligomers in infected cells. Virus-mediated expression of GC-C in H295R cells revealed concentration-dependent STa-stimulated cGMP formation that was undetectable in VVGC-CD853A-infected cells. STa decreased angiotensin II-stimulated human aldosterone generation in a concentration-dependent manner in vaccinia virus-expressing GC-C-infected cells but not in those infected with VVGC-CD853A. These results demonstrate that a catalytically active guanylyl cyclase is required for the inhibition of aldosteronogenesis.

The Conserved Asparagine and Arginine Are Essential for Catalysis of Mammalian Adenylyl Cyclase

Mammalian adenylyl cyclases have two homologous cytoplasmic domains (C1 and C2), and both domains are required for the high enzymatic activity. Mutational and genetic analyses of type I and soluble adenylyl cyclases suggest that the C2 domain is catalytically active and the C1 domain is not; the role of the C1 domain is to promote the catalytic activity of the C2 domain. Two amino acid residues, Asn-1025 and Arg-1029 of type II adenylyl cyclase, are conserved among the C2 domains, but not among the C1 domains, of adenylyl cyclases with 12 putative transmembrane helices. Mutations at each amino acid residue alone result in a 30-100-fold reduction in Kcat of adenylyl cyclase. However, the same mutations do not affect the Km for ATP, the half-maximal concentration (EC50) for the C2 domain of type II adenylyl cyclase to associate with the C1 domain of type I adenylyl cyclase and achieve maximal enzyme activity, or the EC50 for forskolin to maximally activate enzyme activity with or without Gsalpha. This indicates that the mutations at these two residues do not cause gross structural alteration. Thus, these two conserved amino acid residues appear to be crucial for catalysis, and their absence from the C1 domains may account for its lack of catalytic activity. Mutations at both amino acid residues together result in a 3,000-fold reduction in Kcat of adenylyl cyclase, suggesting that these two residues have additive effects in catalysis. A second site suppressor of the Asn-1025 to Ser mutant protein has been isolated. This suppressor has 17-fold higher activity than the mutant and has a Pro-1015 to Ser mutation.

Identification of a region important for human monoamine oxidase b substrate and inhibitor selectivity

Monoamine oxidase (MAO) A and B are flavoenzymes that catalyze the oxidative deamination of biogenic and xenobiotic amines. To search for domains that confer substrate and inhibitor selectivities, two chimeric proteins were constructed and expressed in yeast. The kinetic constants and IC 50 values were determined for these chimeric enzymes using MAO-A/B selective substrates and inhibitors. Replacement of MAO-A amino acids 161–375 with the corresponding region of MAO-B, termed AB 161–375A, converted MAO-A catalytic properties to MAO-B like ones. The specificity constants ( k cat K m ) for the oxidation of β-phenylethylamine (PEA) (1.6 × 10 5s −1 M −1) and benzylamine (2.4 × 10 4s −1 M −1) by AB 161– 375A were similar to wild-type MAO-B (PEA, 8 × 10 5s −1 M −1; benzylamine, 4.9 × 10 4s −1 M −1)-Serotonin (5-HT), a preferred substrate for MAO-A, was not oxidized by AB 161– 375A or wild-type MAO-B. Furthermore, AB 161– 375A was more sensitive to the MAO-B specific inhibitor deprenyl (IC 50 2.7 ± 0.4 × 10 −8M) than to the MAO-A specific inhibitor clorgyline (IC 505.4 ± 0.8 × 10 −7M). However, the reciprocal chimera in which a MAO-B segment was replaced with the corresponding region of MAO-A, termed BA 151–366B, lacked catalytic activity. The lack of catalytic activity was not due to aberrant expression but rather an inactive protein as demonstrated by Western blot analysis. These results demonstrate that MAO-B amino acids 152–366 contain a domain(s) that confers substrate and inhibitor selectivity.

Stromelysin expression regulates collagenase activation in human fibroblasts. Dissociable control of two metalloproteinases by interferon-gamma.

The expression of collagenolytic activity by cells represents the rate-limiting step in the turnover of collagen during remodeling. The collagenase gene is transcriptionally activated in normal dermal or rheumatoid synovial fibroblasts by interleukin-1 beta (IL-1 beta), resulting in secretion of trypsin-activatable procollagenase measuring in the range of 2.0-5.0 units/10(6) cells/48 h in the 14C-fibril assay. The addition of interferon-gamma (IFN-gamma; 50-100 units/ml) inhibits the expression of collagenase activity by 45-80% in these cells. The IL-1 beta induction of procollagenase protein was not altered by IFN-gamma, as judged by Western blot analysis using a monoclonal antibody to collagenase and by gelatin zymography, and procollagenase mRNA was also unaltered, as assessed by Northern blot analysis. Because collagenolytic activity is also controlled by the quantity of tissue inhibitor of metalloproteinases present, its expression was examined by Western blot analysis using a polyclonal antibody to tissue inhibitor of metalloproteinases and by reverse gelatin zymography. Tissue inhibitor of metalloproteinase protein was found to be unaltered or slightly less abundant in conditioned media from cultures treated with IL-1 beta and IFN-gamma when compared with that from cultures treated with IL-1 beta alone. However, the expression of the metalloproteinase activator of procollagenase, stromelysin, was found to be significantly inhibited by the addition of IFN-gamma. Addition of purified activated stromelysin to these conditioned media completely reconstituted collagenolytic activity. These observations demonstrate in an intact system that stromelysin is a specific activator necessary for the development of collagenolytic activity. Despite stromelysin's lack of catalytic activity against collagen, its expression can serve as a control point in the regulation of collagenolysis.

873 α-L-IDURONIDASE IN LEUKOCTYES AND PLASMA: ISOZYMES OF INTEREST IN BONE MARROW TRANSPLANTATION FOR HURLER SYNDROME

Three mechanisms are proposed to account for metabolic correction in Hurler syndrome after bone marrow transplantation. Of these, endogenous enzyme replacement is predicated upon the availability and catalytic activity of iduronidase (Idu) in marrow-derived cells and plasma. To characterize enzyme available after transplantation, we studied Idu from normal individuals utilizing the 4-methylumbelliferyl-α-L-iduronide substrate. Idu specific activities were determined for leukocyte subpopulations (mean, n=18): granulocytes 54.7 nmoles/mg-hr, lymphocytes 61.2, and monocytes 47.5. Of further interest mere physicochemical characteristics of Idu. In vitro, leukocyte and plasma Idu activities, with optima at pH 3.0-3.4, had less than 1% of maximal activity at pH 7.3. We also quantitated Idu isozymes after separation by DEAE chromatography since enzyme affinity for DEAE has been related to cellular binding and endocytosis. In leukocytes, high-affinity Idu B was the predominant form comprising approximately 90% of total activity. In plasma, Idu B was likewise the major component contributing 64-76% total activity. We conclude that Idu is available in transplantable leukocytes; however, lack of catalytic activity at neutral pH suggests that glycosaminoglycan (GAG) degradation in plasma is insignificant in vivo. Since only lysosomal GAG catabolism is likely to be physiologically important, cellular uptake of enzyme may be crucial. Leukocytes and plasma contain high-affinity Idu B available for endocytosis by deficient cells.

The alpha-subunit of mouse 7 S nerve growth factor is an inactive serine protease.

Tryptic and cyanogen bromide peptides accounting for approximately 85% of the amino acid sequence of the alpha-subunit of mouse 7 S nerve growth factor have been isolated and extensively sequenced. The partial structure revealed a high degree of identity with the gamma-subunit (greater than 80%), which is an arginine esteropeptidase of the serine protease family. However, the alpha-subunit does not cleave synthetic arginine ester or peptide substrates nor is it labeled by diisopropylfluorophosphate. The lack of catalytic activity may result from a Gly----His substitution near the active site serine or from the blocked NH2 terminus.

Role of Oxalacetate in the Regulation of Mammalian Succinate Dehydrogenase

Abstract In most procedures for the isolation of membrane-bound or soluble succinate dehydrogenase, the enzyme is obtained largely in the deactivated form. As judged by the quantity of oxalacetate enzymatically determined in perchlorate extracts, the deactivated form contains tightly bound oxalacetate in 1:1 proportion to the enzyme (equated with histidyl flavin content) in particles. A somewhat lower ratio has been obtained for soluble preparations of the enzyme. The oxalacetate is bound very tightly: it is not displaced by succinate or malonate in the cold, nor released by precipitation or gel exclusion of the enzyme, and it does not react with malate dehydrogenase. It is released, however, on denaturation of the enzyme with perchloric acid or under conditions which favor conversion of the enzyme to the activated form. If the enzyme is freed from oxalacetate by activation with bromide and gel exclusion, treatment of the oxalacetate-free fully active enzyme with very low concentrations of l- or d-malate or oxalacetate leads to deactivation and concomitant formation of tightly bound oxalacetate in the same molar ratio found in the enzyme as isolated from heart mitochondria. Dissociation of the complex formed in this manner requires the same experimental conditions as those needed for dissociation of the complex as found in the isolated enzyme. The activation energies for activation and for release of oxalacetate are the same but vary with the type of activator used. The first order rate constant for activation by a variety of agents is about twice that measured for the dissociation of tightly bound oxalacetate. Moreover, with some activators oxalacetate release shows a considerable lag behind recovery of catalytic activity. It is suggested that the presence of oxalacetate in the deactivated enzyme is not the cause of its lack of catalytic activity but a consequence of the deactivated conformation. Further observations are described which suggest that a deactivation-activation cycle may also occur in the absence of oxalacetate.

Lipid peroxidation in rat tissue homogenates: Interaction of iron and ascorbic acid as the normal catalytic mechanism

Iron and ascorbic acid appear to be the normal catalytic components responsible for the lipid peroxidation reaction in aerobically incubated rat tissue homogenates. The amounts of each present in the catalytically-active fractions of rat liver, brain, testis, and kidney are appropriate to explain the lipid peroxidation reaction measured. Utilization of ascorbic acid as part of the normal catalytic mechanism is indicated by the following: The catalytic activity of the tissue soluble phase occurs only in the small molecule fraction eluted from Sephadex, and ascorbic acid occurs only in this fraction; the extent of catalysis by the small molecule fractions of the soluble phases from several tissues is proportional to their ascorbic acid content; and pH effect on lipid peroxidation is the same for both soluble-phase and ascorbic acid catalysis. Utilization of iron as part of the normal catalytic mechanism is indicated by EDTA inhibition studies and by measurements of pH effects. Previous studies have demonstrated the lack of catalytic activity by cations other than iron for the lipid peroxidation reaction in homogenates. Lipid peroxidation is inhibited at high tissue concentration and the inhibition is due to components occurring in the large molecule fraction of the soluble phase.

A Chemical Model for Electrolytic Oxidation of Iodate

DURING an investigation of the electrolytic production of periodate from iodate, the oxidation of iodate was expressed mathematically as a chemical reaction in which gaseous oxygen formation competed with the desired reaction for the available supply of a common precursor material1. In writing the equations, liberties, which could be mathematically rationalized, were taken with the classical forms of the rate equation, but these liberties resulted in equations that could only approximate real counterparts. In particular, it was stated that “sometimes, in the early stages of the oxidation, the reaction proceeds at a very slow rate. This is attributed to an initial lack of catalytic activity at the surface …”. The question has always remained, whether or not the classical equations, which were modified mainly to facilitate solution, would not better express the total behaviour of an operating cell. Another intriguing question has been the identity and quantitative behaviour of the precursor material. Now the set of classical equations has been solved on an analogue computer and insight has been gained into the electrode processes which could occur.