Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “Enzyme Reactivators”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 325 records · Page 18Linked to original sources
PubMed2000

Mycobacterium tuberculosis H37Rv comparative gene-expression analysis in synthetic medium and human macrophage.

Mycobacteria are intracellular pathogens that survive and grow in host macrophages. Following phagocytosis, sustained intracellular bacterial growth depends on its ability to avoid destruction by macrophage-mediated host defences such as lysosomal enzymes, reactive oxygen and the reactive nitrogen intermediates. This suggests that the interaction between host cell and microbe is delicately balanced, and can be tipped in favour of either organism. The identification of Mycobacterium tuberculosis H37Rv (MTB) genes expressed within host cells would contribute greatly to the development of new strategies to fight tuberculosis. In the present study, we compared MTB gene expression in the course of intra- (human macrophages) and extracellular growth (Sauton's medium) to ascertain whether differences might occur between gene-expression patterns in the two habitats of replication. Using reverse-transcriptase polymerase chain reaction (RT-PCR) on a group of 14 MTB-Complex-specific genes, we found that MT10Sa (a small stable RNA), 35 kDa (unknown), ahpC (alkyl hydroperoxide reductase, AhpC), sigF (alternative RNA Polymerase sigma factor), and katG (catalase-peroxidase, HPI) genes are expressed in both the environments, while Ag85B, Ag85C (members of the Antigen 85 Complex), rpoV (RNA Polymerase sigma factor) and ESAT6 (early secretory antigen, 6 kDa) are expressed only in the in vitro culture; on the other hand, Ag85A (Antigen 85 Complex), rpoB (RNA Polymerase beta sub-unit), pab (Protein antigen b), invA and invB genes (encoding proteins that show homologies with p60 of Listeria monocytogenes) are expressed only inside the macrophage. Positive RT-PCR products on cDNAs for these genomic regions were not obtained from approximately 1000-fold more bacteria grown in Laboratory Broth. Identification of M. tuberculosis genes expressed in response to phagocytosis by human macrophages increases our basic understanding of the host-pathogen interaction, and helps to identify bacterial factors necessary for in vivo survival and growth.

Colony Count, Microbial↗
PubMed2003

Stabilization and re-activation of trapped enzyme by immobilized heat shock protein and molecular chaperones.

The potential of using immobilized Heat Shock Protein 70 (HSP 70) in combination with other molecular chaperones to ameliorate problems of enzyme denaturation was investigated. Firefly luciferase was used as a model enzyme due to its sensitivity to thermal denaturation, and the availability of a sensitive chemiluminescent assay method for determination of relative activity of this enzyme. Control experiments and development of effective combinations of HSP with other chaperones involved re-activation of enzyme in bulk solution. A combination of HSP 70, alpha-crystallin and reticulocyte lysate (RL) in bulk solution were found to re-activate soluble firefly luciferase to about 60% of the initial activity after the enzyme activity had been reduced to less than 2% by thermal denaturation. HSP 70 that was covalently immobilized onto glass surfaces was also able to re-activate denatured enzyme that was in bulk solution. Over 30% of the initial activity could be regained from heat denatured enzyme when using immobilized HSP in the presence of other chaperones. The activity of soluble enzyme decayed to negligible values in a period of days when stored at room temperature. In the presence of immobilized HSP and chaperones, activity stabilized at about 10% of the initial activity even after many weeks. The results suggest that immobilized molecular chaperones such as HSP 70 may provide some potential for stabilization and re-activation of enzymes that are trapped in thin aqueous films for applications in biosensors and reactors.

Animals↗
PubMed2003

An amperometric bi-enzyme sensor for determination of formate using cofactor regeneration.

A biosensor for detection of formate at submicromolar concentrations has been developed by co-immobilizing formate dehydrogenase (FDH, E.C. 1.2.1.2), salicylate hydroxylase (SHL, E.C. 1.14.13.1) and NAD(+) linked to polyethylene glycol (PEG-NAD(+)) in a poly(vinyl alcohol) (PVA) matrix in front of a Clark-electrode. The principle of the bi-enzyme scheme is as follows: formate dehydrogenase converts formate into carbon dioxide using PEG-NAD(+). Corresponding PEG-NADH produced is then oxidized to PEG-NAD(+) by salicylate hydroxylase using sodium salicylate and oxygen. The oxygen consumption is monitored with the Clark-electrode. The advantages of this biosensor approach are the effective re-oxidation of PEG-NADH, and the entrapment of PEG-NAD(+) resulting in avoiding the addition of expensive cofactor to the working medium for each measurement. This bi-enzyme sensor has achieved a linear range of 1-300 microM and a detection limit of 1.98 x 10(-7) M for formate (S/N=3), with the response time of 4 min. The working stability is limited to 7 days due to the inactivation of the enzymes. Only sodium salicylate was needed in milli-molar amounts.

Biosensing Techniques↗
PubMed1995

Intracellular signaling in neutrophil priming and activation.

In order for neutrophils to function effectively in host defense, they have evolved specific attributes including the ability to migrate to the site of inflammation and release an array of toxic products including proteolytic enzymes, reactive oxygen species, and cationic proteins. While these compounds are intended for killing invading pathogens, if released inappropriately, they may also contribute to tissue damage. Such inflammatory tissue injury may be important in the pathogenesis of a variety of clinical disorders including arthritis, ischemia-reperfusion tissue injury, the systemic inflammatory response syndrome (SIRS), and the acute respiratory distress syndrome (ARDS). Despite the importance of neutrophil function in host defense and dysfunction in disease states, much remains unknown about the intracellular signaling pathways regulating neutrophil activity. This review will focus on the signaling molecules regulating leukocyte 'effector' functions including receptors, GTP-binding proteins, phospholipases, polyphosphoinositide metabolism, and protein kinases and phosphatases.

Animals↗
PubMed2005

Selective molecular recognition, C-H bond activation, and catalysis in nanoscale reaction vessels.

Supramolecular chemistry represents a way to mimic enzyme reactivity by using specially designed container molecules. We have shown that a chiral self-assembled M4L6 supramolecular tetrahedron can encapsulate a variety of cationic guests with varying degrees of stereoselectivity. Reactive iridium guests can be encapsulated, and the C-H bond activation of aldehydes occurs with the host cavity controlling the ability of substrates to interact with the metal center based upon size and shape. In addition, the host container can act as a catalyst by itself. By restricting reaction space and preorganizing the substrates into reactive conformations, it accelerates the sigmatropic rearrangement of enammonium cations.

Catalysis↗
PubMedDate not supplied

Drug delivery systems employing 1,6-elimination: releasable poly(ethylene glycol) conjugates of proteins.

Using lysozyme as a representative protein substrate that loses its activity when PEGylation takes place on the epsilon-amino group of lysine residues, various amounts of a novel releasable PEG linker (rPEG) were conjugated to the protein. rPEG-lysozyme conjugates were relatively stable in pH 7.4 buffer for over 24 h. However, regeneration of native protein from the rPEG conjugates occurred in a predictable manner during incubation in high pH buffer or rat plasma, as demonstrated by enzymatic activity and structural characterization. The rates of regeneration were also correlated with PEG number: native lysozyme was released more rapidly from the monosubstituted conjugate than from the disubstituted conjugate, suggesting possible steric hindrance to the approach of cleaving enzymes. Recovery of normal activity and structure for the regenerated native lysozyme was shown by a variety of assays.

Animals↗
PubMed1995

Influence of alpha-subunits on the high-pressure stability of apo and holo beta 2-subunits in the bienzyme complex tryptophan synthase from Escherichia coli.

At a hydrostatic pressure of up to 2 kbar, the isolated alpha-subunit of tryptophan synthase from Escherichia coli proved to be a stable enzyme by virtue of specific activity as well as UV absorption and fluorescence emission spectra. The protein can therefore be regarded as a suitable effector for the investigation of structure-function relationships in the dimeric beta 2-subunit under the influence of high hydrostatic pressure. Complete deactivation of the beta 2-component in the alpha 2 beta 2 bienzyme complex occurs above 1300 bar (midpoint of transition for alpha apo beta 2, 790 bar; for alpha 2 holo beta 2, 1057 bar). Sucrose (13%) shifts both midpoints of transition to values higher by about 300 bar. As shown by sucrose gradient centrifugation and limited trypsinolysis, deactivation of the beta 2-dimer is paralleled by dissociation into denatured beta-chains. At 10 degrees C, the corresponding dissociation constants K at 1 bar as well as the reaction volumes of dissociation delta V are calculated as 4.2 x 10(-9) M and -196 mL/mol for the apo-beta 2-component and as 9.8 x 10(-19) M and -632 mL/mol for the holo-beta 2-component in the bienzyme complex. Furthermore, large negative activation volumes are determined, reflecting the rate increase with increasing pressure: -89 mL/mol for the apo-beta 2-dimer and -195 mL/mol for the holo-beta 2-dimer.(ABSTRACT TRUNCATED AT 250 WORDS)

Centrifugation, Density Gradient↗
PubMed1984

Adenosinetriphosphatase site stoichiometry in sarcoplasmic reticulum vesicles and purified enzyme.

The stoichiometry of phosphorylation (catalytic) sites in sarcoplasmic reticulum vesicles ( SRV ) and SR ATPase purified by differential solubilization with deoxycholate was found to be 4.77 +/- 0.4 and 6.05 +/- 0.18 nmol/mg of protein, respectively, when phosphorylation was carried out under conditions permitting 32P labeling of nearly all sites. Assuming that each site corresponds to a single 115K ATPase chain, the observed site stoichiometry accounts only for 55% and 70% of the total protein. Failure to obtain higher phosphorylation levels was due to the presence of nonspecific protein contaminants in SRV or to the presence of inactive aggregates in the ATPase purified with deoxycholate. This was demonstrated by dissolving SRV and purified ATPase with lithium dodecyl sulfate, subjecting them to molecular sieve HPLC, and collecting the elution fractions for determination of protein, measurement of 32P-labeled sites, and electrophoretic analysis. In fact, in the specific elution peak containing the 115K ATPase chains, phosphorylation levels were 6.62 +/- 0.33 and 7.03 +/- 0.18 in SRV and purified ATPase, corresponding to 68% and 86% of the protein in the specific elution peak. An alternate purification method was then developed, based on solubilization of SRV with dodecyl octaethylene glycol monoether ( C12E8 ), separation of delipidated ATPase by anion-exchange chromatography, and enzyme reactivation with phosphatidylcholine. This preparation yields 7.3 +/- 0.44 nmol of phosphorylation site/mg of protein of the SRV fraction before HPLC.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Triphosphatases↗
PubMed2003

Spectroscopic characterization of soybean lipoxygenase-1 mutants: the role of second coordination sphere residues in the regulation of enzyme activity.

Lipoxygenases are non-heme iron enzymes, which catalyze the stereo- and regiospecific hydroperoxidation of unsaturated fatty acids. Spectroscopic studies on soybean lipoxygenase have shown that the ferrous form of the enzyme is a mixture of five- and six-coordinate species (40 and 60%, respectively). Addition of substrate leads to a purely six-coordinate form. A series of mutations in the second coordination sphere (Q697E, Q697N, Q495A, and Q495E) were generated, and the structures of the mutants were solved by crystallography [Tomchick et al. (2001) Biochemistry 40, 7509-7517]. While this study clearly showed the contribution of H-bond interactions between the first and the second coordination spheres in catalysis, no correlation with the coordination environment of the Fe(II) was observed. A recent study using density-functional theory [Lehnert and Solomon (2002) J. Biol. Inorg. Chem. 8, 294-305] indicated that coordination flexibility, involving the Asn694 ligand, is regulated via H-bond interactions. In this paper, we investigate the solution structures of the second coordination sphere mutants using CD and MCD spectroscopy since these techniques are more sensitive indicators of the first coordination sphere ligation of Fe(II) systems. Our data demonstrate that the iron coordination environment directly relates to activity, with the mutations that have the ability to form a five-coordinate/six-coordinate mixture being more active. We propose that the H-bond between the weak Asn694 ligand and the Gln697 plays a key role in the modulation of the coordination flexibility of Asn694, and thus, is crucial for the regulation of enzyme reactivity.

Amino Acid Substitution↗
PubMed2005

Structural basis for tumor pyruvate kinase M2 allosteric regulation and catalysis.

Four isozymes of pyruvate kinase are differentially expressed in human tissue. Human pyruvate kinase isozyme M2 (hPKM2) is expressed in early fetal tissues and is progressively replaced by the other three isozymes, M1, R, and L, immediately after birth. In most cancer cells, hPKM2 is once again expressed to promote tumor cell proliferation. Because of its almost ubiquitous presence in cancer cells, hPKM2 has been designated as tumor specific PK-M2, and its presence in human plasma is currently being used as a molecular marker for the diagnosis of various cancers. The X-ray structure of human hPKM2 complexed with Mg(2+), K(+), the inhibitor oxalate, and the allosteric activator fructose 1,6-bisphosphate (FBP) has been determined to a resolution of 2.82 A. The active site of hPKM2 is in a partially closed conformation most likely resulting from a ligand-induced domain closure promoted by the binding of FBP. In all four subunits of the enzyme tetramer, a conserved water molecule is observed on the 2-si face of the prospective enolate and supports the hypothesis that a proton-relay system is acting as the proton donor of the reaction (1). Significant structural differences among the human M2, rabbit muscle M1, and the human R isozymes are observed, especially in the orientation of the FBP-activating loop, which is in a closed conformation when FBP is bound. The structural differences observed between the PK isozymes could potentially be exploited as unique structural templates for the design of allosteric drugs against the disease states associated with the various PK isozymes, especially cancer and nonspherocytic hemolytic anemia.

Allosteric Regulation↗
PubMed2005

Inactivation of bacterial DD-peptidase by beta-sultams.

N-Acyl-beta-sultams are time-dependent, irreversible active site-directed inhibitors of Streptomyces R61 DD-peptidase. The rate of inactivation is first order with respect to beta-sultam concentration, and the second-order rate constants show a dependence on pH similar to that for the hydrolysis of a substrate. Inactivation is due to the formation of a stable 1:1 enzyme-inhibitor complex as a result of the active site serine being sulfonylated by the beta-sultam as shown by ESI-MS analysis and by X-ray crystallography. A striking feature of the sulfonyl enzyme is that the inhibitor is not bound to the oxyanion hole but interacts extensively with the "roof" of the active site where the Arg 285 is located.

Anti-Bacterial Agents↗
PubMed2005

X-ray absorption investigation of a unique protein domain able to bind both copper(I) and copper(II) at adjacent sites of the N-terminus of Haemophilus ducreyi Cu,Zn superoxide dismutase.

The N-terminal metal binding extension of the Cu,Zn superoxide dismutase from Haemophilus ducreyi is constituted by a histidine-rich region followed by a methione-rich sequence which shows high similarity with protein motifs involved in the binding of Cu(I). X-ray absorption spectroscopy experiments selectively carried out with peptides corresponding to the two metal binding regions indicate that both sequences can bind either Cu(II) or Cu(I). However, competition experiments demonstrate that Cu(II) is preferred by histidine residues belonging to the first half of the motif, while the methionine-rich region preferentially binds Cu(I) via the interaction with three methionine sulfur atoms. Moreover, we have observed that the rate of copper transfer from the peptides to the active site of a copper-free form of the Cu,Zn superoxide dismutase mutant lacking the N-terminal extension depends on the copper oxidation state and on the residues involved in metal binding, histidine residues being critically important for the efficient transfer. Differences in the enzyme reactivation rates in the presence of mixtures of the two peptides when compared to those obtained with the single peptides suggest that the two halves of the N-terminal domain functionally interact during the process of copper transfer, possibly through subtle modifications of the copper coordination environment.

Binding Sites↗
PubMed2001

Mechanism of reaction of hydrogen peroxide with horseradish peroxidase: identification of intermediates in the catalytic cycle.

The mechanism of the reaction of horseradish peroxidase isoenzyme C (HRPC) with hydrogen peroxide to form the reactive enzyme intermediate compound I has been studied using electronic absorbance, rapid-scan stopped-flow, and electron paramagnetic resonance (EPR) spectroscopies at both acid and basic pH. The roles of the active site residues His42 and Arg38 in controlling heterolytic cleavage of the H(2)O(2) oxygen-oxygen bond have been probed with site-directed mutant enzymes His42 --> Leu (H42L), Arg38 --> Leu (R38L), and Arg38 --> Gly (R38G). The biphasic reaction kinetics of H42L with H(2)O(2) suggested the presence of an intermediate species and, at acid pH, a reversible second step, probably due to a neutral enzyme-H(2)O(2) complex and the ferric-peroxoanion-containing compound 0. EPR also indicated the formation of a protein radical situated more than approximately 10 A from the heme iron. The stoichiometry of the reaction of the H42L/H(2)O(2) reaction product and 2,2'-azinobis(3-ethylbenzothiazolinesulfonic acid) (ABTS) was concentration dependent and fell from a value of 2 to 1 above 0.7 mM ABTS. These data can be explained if H(2)O(2) undergoes homolytic cleavage in H42L. The apparent rate of compound I formation by H42L, while low, was pH independent in contrast to wild-type HRPC where the rate falls at acid pH, indicating the involvement of an ionizable group with pK(a) approximately 4. In R38L and R38G, the apparent pK(a) was shifted to approximately 8 but there is no evidence that homolytic cleavage of H(2)O(2) occurs. These data suggest that His42 acts initially as a proton acceptor (base catalyst) and then as a donor (acid catalyst) at neutral pH and predict the observed slower rate and lower efficiency of heterolytic cleavage observed at acid pH. Arg38 is influential in lowering the pK(a) of His42 and additionally in aligning H(2)O(2) in the active site, but it does not play a direct role in proton transfer.

Arginine↗
PubMed2005

The hydrogen chemistry of the FeMo-co active site of nitrogenase.

The chemical mechanism by which nitrogenase enzymes catalyze the hydrogenation of N(2) (and other multiply bonded substrates) at the N(c)Fe(7)MoS(9)(homocitrate) active site (FeMo-co) is unknown, despite the accumulation of much data on enzyme reactivity and the influences of key amino acids surrounding FeMo-co. The mutual influences of H(2), substrates, and the inhibitor CO on reactivity are key experimental tests for postulated mechanisms. Fundamental to all aspects of mechanism is the accumulation of H atoms (from e(-) + H(+)) on FeMo-co, and the generation and influences of coordinated H(2). Here, I argue that the first introduction of H is via a water chain terminating at water 679 (PDB structure , Azotobacter vinelandii) to one of the mu(3)-S atoms (S3B) of FeMo-co. Next, using validated density functional calculations of a full chemical representation of FeMo-co and its connected residues (alpha-275(Cys), alpha-442(His)), I have characterized more than 80 possibilities for the coordination of up to three H atoms, and H(2), and H + H(2), on the S2A, Fe2, S2B, Fe6, S3B domain of FeMo-co, which is favored by recent targeted mutagenesis results. Included are calculated reaction profiles for movements of H atoms (between S and Fe, and between Fe and Fe), for the generation of Fe-H(2), for association and dissociation of Fe-H(2) at various reduction levels, and for H/H(2) exchange. This is new hydrogen chemistry on an unprecedented coordination frame, with some similarities to established hydrogen coordination chemistry, and with unexpected and unprecedented structures such as Fe(S)(3)(H(2))(2)(H) octahedral coordination. General principles for the hydrogen chemistry of FeMo-co include (1) the stereochemical mobility of H bound to mu(3)-S, (2) the differentiated endo- and exo- positions at Fe for coordination of H and/or H(2), and (3) coordinative allosteric influences in which structural and dynamic aspects of coordination at one Fe atom are affected by coordination at another Fe atom, and by H on S atoms. Evidence of end-differentiation in FeMo-co is described, providing a rationale for the occurrence of Mo. The reactivity results are discussed in the context of the Thorneley-Lowe scheme for nitrogenase reactions, and especially the scheme for the HD reaction (2H(+) + 2e(-) + D(2) --> 2HD), using a model containing an H-entry site and at least two coordinative sites on FeMo-co. I propose that S3B is the H-entry site, suggest details for the H(+) shuttle to S3B and subsequent movement of H atoms around FeMo-co preparatory to the binding and hydrogenation of N(2) and other substrates, and suggest how H could be transferred to an alkyne substrate. I propose that S2B (normally hydrogen bonded to alpha-195(His)) has a modulatory function and is not an H-entry site. Finally, the recent first experimental trapping of a hydrogenated intermediate with EPR and ENDOR characterization is discussed, leading to a consensual model for the intermediate.

Binding Sites↗
PubMed2006

Creating advanced multifunctional biosensors with surface enzymatic transformations.

This paper summarizes our recent work on the coupling of surface enzyme chemistry and bioaffinity interactions on biopolymer microarrays for the creation of multiplexed biosensors with enhanced selectivity and sensitivity. The surface sensitive techniques of surface plasmon resonance imaging (SPRI) and surface plasmon fluorescence spectroscopy (SPFS) are used to detect the surface enzymatic transformations in real time. Three specific examples of novel coupled surface bioaffinity/surface enzymatic processes are demonstrated: (i) a surface enzymatic amplification method utilizing the enzyme ribonuclease H (RNase H) in conjunction with RNA microarrays that permits the ultrasensitive direct detection of genomic DNA at a concentration of 1 fM without labeling or PCR amplification, (ii) the use of RNA-DNA ligation chemistry to create renewable RNA microarrays from single stranded DNA microarrays, and (iii) the application of T7 RNA polymerase for the on-chip replication of RNA from double stranded DNA microarray elements. In addition, a simple yet powerful theoretical framework that includes the contributions of both enzyme adsorption and surface enzyme kinetics is used to quantitate surface enzyme reactivity. This model is successfully applied to SPRI and SPFS measurements of surface hydrolysis reactions of RNase H and Exonuclease III (Exo III) on oligonucleotide microarrays.

DNA↗
PubMed2003

Refolding of a recombinant full-length non-structural (NS3) protein from hepatitis C virus by chromatographic procedures.

The non-structural protein 3 (NS3) of Hepatitis C virus (HCV) was expressed as inactive aggregates in Escherichia coli. The protein was refolded by chromatographic techniques of which ion exchange chromatography was best for crude samples and gel filtration best for partially purified samples. Immobilized metal ion affinity chromatography showed intermediate performance. Gradient procedures enhanced the recovery of active NS3 protein.

Chromatography↗
PubMed1977

The arrangement of substrate and organophosphorus-inhibitor leaving groups in acetylcholinesterase active site.

The leaving groups of serine esterase substrates and organophosphorus inhibitors bind to different loci at the enzyme active site. Kinetic proof of this general conclusion is presented in the case of acetylcholinesterase reactions. The proposed model for acetylcholinesterase active site provides a new stereochemical mechanism for the phosphorylated enzyme reactivation.

Acetates↗
PubMed1991

Aminoacetone synthase from goat liver. Involvement of arginine residue at the active site and on the stability of the enzyme.

The arginine-specific reagents phenylglyoxal and butane-2,3-dione inactivated goat liver aminoacetone synthase with pseudo-first-order kinetics, with the rate dependent on modifier concentration. Phenylglyoxal and butane-2,3-dione appeared to react with one arginine residue per enzyme molecule. The inactivated enzyme could be re-activated by Tris, suggesting additional evidence of modification of the arginine residue. Acetyl-CoA, one of the substrates, completely protected the enzyme from inactivation. Glycine gave partial protection. Protection by substrates against inactivation by phenylglyoxal and butane-2,3-dione suggested the presence of an essential arginine residue at the substrate-binding region. Experiments with [7-14C]phenylglyoxal in the presence of acetyl-CoA showed that only the arginine residue at the active site could be modified by phenylglyoxal. The stability of the enzyme is dependent on the presence of both EDTA and Mg2+.

Acetyl Coenzyme A↗