In silico studies on the role of mutant Y337A to reactivate tabun inhibited mAChE with K048.

Organophosphorus compound (OP) tabun is resistant to reactivate by many oxime drugs after the formation of OP-conjugate with AChE. The reactivation of tabun-inhibited mAChE and site-directed mutants by bispyridinium oxime, K048 (N-[4-(4-hydroxyiminomethylpyridinio)butyl]-4-carbamoylpyridinium dibromide) showed that the mutations significantly poor the overall reactivation efficacy of K048. We have unravelled the lowered efficacy of K048 with the tabun-mutant mAChE(Y337A) using docking and steered molecular dynamics (SMD) simulations. The computed results showed some interesting features for the interaction of drug molecule K048 with tabun-mAChE(wild-type) and tabun-mutant mAChE(Y337A). The SMD simulations showed that the active pyridinium ring of K048 is directed towards the phosphorus atom conjugated to the active serine (SUN203) of tabun-mAChE(wild-type). The cradle shaped residues Tyr337-Phe338 present in the choline binding site stabilize the active pyridinium ring of K048 with π-π interaction and the residue Trp86 involved in T-shaped cation-π interaction. However, in the case of tabun-mutant mAChE(Y337A).K048 conjugate, the replacement of aromatic Tyr337 with the aliphatic alanine unit in the choline binding site, however, loses one of the π-π interaction between the active pyridinium ring of K048 and the Tyr337. The placement of aliphatic alanine unit resulted in the displacement of the side chain of Phe338 towards the His447. Such displacement is causing the inaccessibility of the drug towards the phosphorus atom conjugated to the active serine (SUN203) of tabun-mutant mAChE(Y337A). Furthermore, the unbinding of the K048 with SMD studies showed that the active pyridinium ring of the drug undergoes a complete turn along the gorge axis and is directed away from the phosphorus atom conjugated to the active serine of the tabun-mutant mAChE(Y337A). Such effects inside the gorge of tabun-mutant mAChE(Y337A) would lower the efficacy of the drug molecule (K048) for the reactivation process. The binding free energy computed for the tabun-mAChE(wild-type) and tabun-mutant mAChE(Y337A) with K048 showed that the drug molecule prefers to bind strongly with the former enzyme (∼30 kJ/mol) than the later one.

Molecular architectures and functions of radical enzymes and their (re)activating proteins.

Certain proteins utilize the high reactivity of radicals for catalysing chemically challenging reactions. These proteins contain or form a radical and therefore named 'radical enzymes'. Radicals are introduced by enzymes themselves or by (re)activating proteins called (re)activases. The X-ray structures of radical enzymes and their (re)activases revealed some structural features of these molecular apparatuses which solved common enigmas of radical enzymes­i.e. how the enzymes form or introduce radicals at the active sites, how they use the high reactivity of radicals for catalysis, how they suppress undesired side reactions of highly reactive radicals and how they are (re)activated when inactivated by extinction of radicals. This review highlights molecular architectures of radical B12 enzymes, radical SAM enzymes, tyrosyl radical enzymes, glycyl radical enzymes and their (re)activating proteins that support their functions. For generalization, comparisons of the recently reported structures of radical enzymes with those of canonical radical enzymes are summarized here.

Transcription factors TFIIF and TFIIS promote transcript elongation by RNA polymerase II by synergistic and independent mechanisms.

Recent evidence suggests that transcript elongation by RNA polymerase II (RNAPII) is regulated by mechanical cues affecting the entry into, and exit from, transcriptionally inactive states, including pausing and arrest. We present a single-molecule optical-trapping study of the interactions of RNAPII with transcription elongation factors TFIIS and TFIIF, which affect these processes. By monitoring the response of elongation complexes containing RNAPII and combinations of TFIIF and TFIIS to controlled mechanical loads, we find that both transcription factors are independently capable of restoring arrested RNAPII to productive elongation. TFIIS, in addition to its established role in promoting transcript cleavage, is found to relieve arrest by a second, cleavage-independent mechanism. TFIIF synergistically enhances some, but not all, of the activities of TFIIS. These studies also uncovered unexpected insights into the mechanisms underlying transient pauses. The direct visualization of pauses at near-base-pair resolution, together with the load dependence of the pause-entry phase, suggests that two distinct mechanisms may be at play: backtracking under forces that hinder transcription and a backtrack-independent activity under assisting loads. The measured pause lifetime distributions are inconsistent with prevailing views of backtracking as a purely diffusive process, suggesting instead that the extent of backtracking may be modulated by mechanisms intrinsic to RNAPII. Pauses triggered by inosine triphosphate misincorporation led to backtracking, even under assisting loads, and their lifetimes were reduced by TFIIS, particularly when aided by TFIIF. Overall, these experiments provide additional insights into how obstacles to transcription may be overcome by the concerted actions of multiple accessory factors.

Progress in the development of enzyme-based nerve agent bioscavengers.

Acetylcholinesterase is the physiological target for acute toxicity of nerve agents. Attempts to protect acetylcholinesterase from phosphylation by nerve agents, is currently achieved by reversible inhibitors that transiently mask the enzyme active site. This approach either protects only peripheral acetylcholinesterase or may cause side effects. Thus, an alternative strategy consists in scavenging nerve agents in the bloodstream before they can reach acetylcholinesterase. Pre- or post-exposure administration of bioscavengers, enzymes that neutralize and detoxify organophosphorus molecules, is one of the major developments of new medical counter-measures. These enzymes act either as stoichiometric or catalytic bioscavengers. Human butyrylcholinesterase is the leading stoichiometric bioscavenger. Current efforts are devoted to its mass production with care to pharmacokinetic properties of the final product for extended lifetime. Development of specific reactivators of phosphylated butyrylcholinesterase, or variants with spontaneous reactivation activity is also envisioned for rapid in situ regeneration of the scavenger. Human paraoxonase 1 is the leading catalytic bioscavenger under development. Research efforts focus on improving its catalytic efficiency toward the most toxic isomers of nerve agents, by means of directed evolution-based strategies. Human prolidase appears to be another promising human enzyme. Other non-human efficient enzymes like bacterial phosphotriesterases or squid diisopropylfluorophosphatase are also considered though their intrinsic immunogenic properties remain challenging for use in humans. Encapsulation, PEGylation and other modifications are possible solutions to address this problem as well as that of their limited lifetime. Finally, gene therapy for in situ generation and delivery of bioscavengers is for the far future, but its proof of concept has been established.

Enzyme reactivation by hydrogen peroxide in heme-based tryptophan dioxygenase.

An intriguing mystery about tryptophan 2,3-dioxygenase is its hydrogen peroxide-triggered enzyme reactivation from the resting ferric oxidation state to the catalytically active ferrous form. In this study, we found that such an odd Fe(III) reduction by an oxidant depends on the presence of L-Trp, which ultimately serves as the reductant for the enzyme. In the peroxide reaction with tryptophan 2,3-dioxygenase, a previously unknown catalase-like activity was detected. A ferryl species (δ = 0.055 mm/s and ΔE(Q) = 1.755 mm/s) and a protein-based free radical (g = 2.0028 and 1.72 millitesla linewidth) were characterized by Mössbauer and EPR spectroscopy, respectively. This is the first compound ES-type of ferryl intermediate from a heme-based dioxygenase characterized by EPR and Mössbauer spectroscopy. Density functional theory calculations revealed the contribution of secondary ligand sphere to the spectroscopic properties of the ferryl species. In the presence of L-Trp, the reactivation was demonstrated by enzyme assays and by various spectroscopic techniques. A Trp-Trp dimer and a monooxygenated L-Trp were both observed as the enzyme reactivation by-products by mass spectrometry. Together, these results lead to the unraveling of an over 60-year old mystery of peroxide reactivation mechanism. These results may shed light on how a metalloenzyme maintains its catalytic activity in an oxidizing environment.

Diol dehydratase-reactivating factor is a reactivase--evidence for multiple turnovers and subunit swapping with diol dehydratase.

Adenosylcobalamin-dependent diol dehydratase (DD) undergoes suicide inactivation by glycerol, one of its physiological substrates, resulting in the irreversible cleavage of the coenzyme Co-C bond. The damaged cofactor remains tightly bound to the active site. The DD-reactivating factor reactivates the inactivated holoenzyme in the presence of ATP and Mg(2+) by mediating the exchange of the tightly bound damaged cofactor for free intact coenzyme. In this study, we demonstrated that this reactivating factor mediates the cobalamin exchange not stoichiometrically but catalytically in the presence of ATP and Mg(2+). Therefore, we concluded that the reactivating factor is a sort of enzyme. It can be designated DD reactivase. The reactivase showed broad specificity for nucleoside triphosphates in the activation of the enzyme·cyanocobalamin complex. This result is consistent with the lack of specific interaction with the adenine ring of ADP in the crystal structure of the reactivase. The specificities of the reactivase for divalent metal ions were also not strict. DD formed 1:1 and 1:2 complexes with the reactivase in the presence of ADP and Mg(2+). Upon complex formation, one ß subunit was released from the (αß)2 tetramer of the reactivase. This result, together with the similarity in amino acid sequences and folds between the DD ß subunit and the reactivase ß subunit, suggests that subunit displacement or swapping takes place upon formation of the enzyme·reactivase complex. This would result in the dissociation of the damaged cofactor from the inactivated holoenzyme, as suggested by the crystal structures of the reactivase and DD.

Exercise training stimulates ischemia-induced neovascularization via phosphatidylinositol 3-kinase/Akt-dependent hypoxia-induced factor-1 alpha reactivation in mice of advanced age.

BACKGROUND: Exercise stimulates the vascular response in pathological conditions, including ischemia; however, the molecular mechanisms by which exercise improves the impaired hypoxia-induced factor (HIF)-1 alpha-mediated response to hypoxia associated with aging are poorly understood. Here, we report that swimming training (ST) modulates the vascular response to ischemia in aged (24-month-old) mice. METHODS AND RESULTS: Aged wild-type mice (MMP-2(+/+)) that maintained ST (swimming 1 h/d) from day 1 after surgery were randomly assigned to 4 groups that were treated with either vehicle, LY294002, or deferoxamine for 14 days. Mice that were maintained in a sedentary condition served as controls. ST increased blood flow, capillary density, and levels of p-Akt, HIF-1 alpha, vascular endothelial growth factor, Fit-1, and matrix metalloproteinase-2 (MMP-2) in MMP-2(+/+) mice. ST also increased the numbers of circulating endothelial progenitor cells and their function associated with activation of HIF-1 alpha. All of these effects were diminished by LY294002, an inhibitor of phosphatidylinositol 3-kinase; enhanced by deferoxamine, an HIF-1 alpha stabilizer; and impaired by knockout of MMP-2. Finally, bone marrow transplantation confirmed that ST enhanced endothelial progenitor cell homing to ischemic sites in aged mice. CONCLUSIONS: ST can improve neovascularization in response to hypoxia via a phosphatidylinositol 3-kinase-dependent mechanism that is mediated by the HIF-1 alpha/vascular endothelial growth factor/MMP-2 pathway in advanced age.

ALA-D and ALA-D reactivated as biomarkers of lead contamination in the fish Prochilodus lineatus.

ALA-D activity and lead concentrations were measured in blood and liver tissues of the fish Prochilodus lineatus, collected from three locations along the coast of the La Plata River, Argentina. Two of them, Berazategui and Berisso, were located nearby the main ducts that discharge the urban and domestic waste disposal from Buenos Aires and La Plata cities, respectively, while the third station (Atalaya) was free of sewage discharges. For both tissues, the levels of lead in fish from Berazategui and Berisso were higher than those found in the samples from Atalaya. For blood, but not for liver, a significant negative correlation was found between ALA-activity and tissue levels of lead considering all the data. However, no good correlations were observed at each location. Therefore, an enzyme reactivation technique was optimized. The blood enzyme, but not the liver one, could be effectively reactivated with zinc (Zn(II)). The values of the reactivated ALA-D in samples from Berazategui and Berisso, but not from Atalaya, were significantly higher than the original values, indicating that the enzyme was actually inhibited. In addition, the reactivation index showed significant correlations with the blood lead levels. It is proposed that the reactivation index, rather than the ALA-D activity, may reflect better the extent of lead contamination, especially for field monitoring programs where many confounding factors may affect the biomarker response.

Molecular basis for specificities of reactivating factors for adenosylcobalamin-dependent diol and glycerol dehydratases.

Adenosylcobalamin-dependent diol and glycerol dehydratases are isofunctional enzymes and undergo mechanism-based inactivation by a physiological substrate glycerol during catalysis. Inactivated holoenzymes are reactivated by their own reactivating factors that mediate the ATP-dependent exchange of an enzyme-bound, damaged cofactor for free adenosylcobalamin through intermediary formation of apoenzyme. The reactivation takes place in two steps: (a) ADP-dependent cobalamin release and (b) ATP-dependent dissociation of the resulting apoenzyme-reactivating factor complexes. The in vitro experiments with purified proteins indicated that diol dehydratase-reactivating factor (DDR) cross-reactivates the inactivated glycerol dehydratase, whereas glycerol dehydratase-reactivating factor (GDR) did not cross-reactivate the inactivated diol dehydratase. We investigated the molecular basis of their specificities in vitro by using purified preparations of cognate and noncognate enzymes and reactivating factors. DDR mediated the exchange of glycerol dehydratase-bound cyanocobalamin for free adeninylpentylcobalamin, whereas GDR cannot mediate the exchange of diol dehydratase-bound cyanocobalamin for free adeninylpentylcobalamin. As judged by denaturing PAGE, the glycerol dehydratase-DDR complex was cross-formed, although the diol dehydratase-GDR complex was not formed. There were no specificities of reactivating factors in the ATP-dependent dissociation of enzyme-reactivating factor complexes. Thus, it is very likely that the specificities of reactivating factors are determined by the capability of reactivating factors to form complexes with apoenzymes. A modeling study based on the crystal structures of enzymes and reactivating factors also suggested why DDR cross-forms a complex with glycerol dehydratase, and why GDR does not cross-form a complex with diol dehydratase.

Assembly of the phagocyte NADPH oxidase complex: chimeric constructs derived from the cytosolic components as tools for exploring structure-function relationships.

Phagocytes generate superoxide (O2*-) by an enzyme complex known as reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. Its catalytic component, responsible for the NADPH-driven reduction of oxygen to O2*-, is flavocytochrome b559, located in the membrane and consisting of gp91phox and p22phox subunits. NADPH oxidase activation is initiated by the translocation to the membrane of the cytosolic components p47phox, p67phox, and the GTPase Rac. Cytochrome b559 is converted to an active form by the interaction of gp91phox with p67phox, leading to a conformational change in gp91phox and the induction of electron flow. We designed a new family of NADPH oxidase activators, represented by chimeras comprising various segments of p67phox and Rac1. The prototype chimera p67phox (1-212)-Rac1 (1-192) is a potent activator in a cell-free system, also containing membrane p47phox and an anionic amphiphile. Chimeras behave like bona fide GTPases and can be prenylated, and prenylated (p67phox -Rac1) chimeras activate the oxidase in the absence of p47phox and amphiphile. Experiments involving truncations, mutagenesis, and supplementation with Rac1 demonstrated that the presence of intrachimeric bonds between the p67phox and Rac1 moieties is an absolute requirement for the ability to activate the oxidase. The presence or absence of intrachimeric bonds has a major impact on the conformation of the chimeras, as demonstrated by fluorescence resonance energy transfer, small angle X-ray scattering, and gel filtration. Based on this, a "propagated wave" model of NADPH oxidase activation is proposed in which a conformational change initiated in Rac is propagated to p67phox and from p67phox to gp91phox.

Direct evidence for the formation of a complex between 1-cysteine peroxiredoxin and glutathione S-transferase pi with activity changes in both enzymes.

Glutathione S-transferase pi (GST pi) has been shown to reactivate oxidized 1-cysteine peroxiredoxin (1-Cys Prx, Prx VI, Prdx6, and AOP2). We now demonstrate that a heterodimer complex is formed between 1-Cys Prx with a C-terminal His6 tag and GST pi upon incubation of the two proteins at pH 8.0 in buffer containing 20% 1,6-hexanediol to dissociate the homodimers, followed by dialysis against buffer containing 2.5 mM glutathione (GSH) but lacking 1,6-hexanediol. The heterodimer can be purified by chromatography on nickel-nitriloacetic acid agarose in the presence of GSH. N-Terminal sequencing showed that equimolar amounts of the two proteins are present in the isolated complex. In the heterodimer, 1-Cys Prx is fully active toward either H2O2 or phospholipid hydroperoxide, while the GST pi activity is approximately 25% of that of the GST pi homodimer. In contrast, the 1-Cys Prx homodimer lacks peroxidase activity even in the presence of free GSH. The heterodimer is also formed in the presence of S-methylglutathione, but no 1-Cys Prx activity is found under these conditions. The yield of heterodimer is decreased in the absence of 1,6-hexanediol or GSH. Rapid glutathionylation of 1-Cys Prx in the heterodimer is detected by immunoblotting. Subsequently, a disulfide-linked dimer is observed on SDS-PAGE, and the free cysteine content is decreased by 2 per heterodimer. The involvement of particular binding sites in heterodimer formation was tested by site-directed mutagenesis of the two proteins. For 1-Cys Prx, neither Cys47 nor Ser32 is required for heterodimer formation but Cys47 is essential for 1-Cys Prx activation. For GST pi, Cys47 and Tyr7 (at or near the GSH-binding site) are needed for heterodimer formation but three other cysteines are not. We conclude that reactivation of oxidized 1-Cys Prx by GST pi occurs by heterodimerization of 1-Cys Prx and GST pi harboring bound GSH, followed by glutathionylation of 1-Cys Prx and then formation of an intersubunit disulfide. Finally, the GSH-mediated reduction of the disulfide regenerates the reduced active-site sulfhydryl of 1-Cys Prx.

Catalytic cycling in beta-phosphoglucomutase: a kinetic and structural analysis.

Lactococcus lactis beta-phosphoglucomutase (beta-PGM) catalyzes the interconversion of beta-d-glucose 1-phosphate (beta-G1P) and beta-d-glucose 6-phosphate (G6P), forming beta-d-glucose 1,6-(bis)phosphate (beta-G16P) as an intermediate. Beta-PGM conserves the core domain catalytic scaffold of the phosphatase branch of the HAD (haloalkanoic acid dehalogenase) enzyme superfamily, yet it has evolved to function as a mutase rather than as a phosphatase. This work was carried out to identify the structural basis underlying this diversification of function. In this paper, we examine beta-PGM activation by the Mg(2+) cofactor, beta-PGM activation by Asp8 phosphorylation, and the role of cap domain closure in substrate discrimination. First, the 1.90 A resolution X-ray crystal structure of the Mg(2+)-beta-PGM complex is examined in the context of previously reported structures of the Mg(2+)-alpha-d-galactose-1-phosphate-beta-PGM, Mg(2+)-phospho-beta-PGM, and Mg(2+)-beta-glucose-6-phosphate-1-phosphorane-beta-PGM complexes to identify conformational changes that occur during catalytic turnover. The essential role of Asp8 in nucleophilic catalysis was confirmed by demonstrating that the D8A and D8E mutants are devoid of catalytic activity. Comparison of the ligands to Mg(2+) in the different complexes shows that a single Mg(2+) coordination site must alternatively accommodate water, phosphate, and the phosphorane intermediate during catalytic turnover. Limited involvement of the HAD family metal-binding loop in Mg(2+) anchoring in beta-PGM is consistent with the relatively loose binding indicated by the large K(m) for Mg(2+) activation (270 +/- 20 microM) and with the retention of activity found in the E169A/D170A double loop mutant. Comparison of the relative positions of cap and core domains in the different complexes indicated that interaction of cap domain Arg49 with the "nontransferring" phosphoryl group of the substrate ligand might stabilize the cap-closed conformation, as required for active site desolvation and alignment of Asp10 for acid-base catalysis. Kinetic analyses of the specificity of beta-PGM toward phosphoryl group donors and the specificity of phospho-beta-PGM toward phosphoryl group acceptors were carried out. The results support a substrate induced-fit mechanism of beta-PGM catalysis, which allows phosphomutase activity to dominate over the intrinsic phosphatase activity. Last, we present evidence that the autophosphorylation of beta-PGM by the substrate beta-G1P accounts for the origin of phospho-beta-PGM in the cell.

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.

The interaction of human tryptase-beta with small molecule inhibitors provides new insights into the unusual functional instability and quaternary structure of the protease.

Human tryptase-beta (HTbeta) is a serine protease with an atypical tetrameric structure and an unusual dependence on heparin binding or high salt for functional and structural stability. In the absence of heparin and at physiological salt, pH, and temperature, HTbeta rapidly loses activity by a reversible process that we have called spontaneous inactivation. The role of tetramer dissociation in this process is controversial. Using small irreversible or competitive inhibitors of HTbeta as stabilizing ligands, we were able to examine tetramer stability under inactivating (decay) conditions in the absence of heparin and to define further the process of spontaneous inactivation. Size exclusion chromatography showed that interaction with inhibitors stabilized the tetramer. Using sedimentation equilibrium, spontaneously inactivated HTbeta (si-HTbeta) was shown to be a destabilized tetramer that dissociates upon dilution and which in the presence of a competitive inhibitor re-formed a stable tetramer. Addition of inhibitors to si-HTbeta rescued catalytic activity as was shown after inhibitor displacement. At high concentrations of si-HTbeta (4-5 microM), the binding of inhibitor alone provided sufficient free energy for complete reactivation and tetramer stabilization, whereas at low si-HTbeta concentration (0.1 microM) where the destabilized tetramer would be mostly dissociated, reactivation required more free energy which was provided by the binding of both an inhibitor and heparin. The results demonstrate that HTbeta is a tetramer in the absence of heparin and that tetramer dissociation is a consequence of and not a prerequisite for inactivation. Heparin binding likely stabilizes the tetramer by favoring a functionally active conformation with stable intersubunit contacts, rather than by simply cross-linking active monomers.

Identification of a reactivating factor for adenosylcobalamin-dependent ethanolamine ammonia lyase.

The holoenzyme of adenosylcobalamin-dependent ethanolamine ammonia lyase undergoes suicidal inactivation during catalysis as well as inactivation in the absence of substrate. The inactivation involves the irreversible cleavage of the Co-C bond of the coenzyme. We found that the inactivated holoenzyme undergoes rapid and continuous reactivation in the presence of ATP, Mg2+, and free adenosylcobalamin in permeabilized cells (in situ), homogenate, and cell extracts of Escherichia coli. The reactivation was observed in the permeabilized E. coli cells carrying a plasmid containing the E. coli eut operon as well. From coexpression experiments, it was demonstrated that the eutA gene, adjacent to the 5' end of ethanolamine ammonia lyase genes (eutBC), is essential for reactivation. It encodes a polypeptide consisting of 467 amino acid residues with predicted molecular weight of 49,599. No evidence was obtained that shows the presence of the auxiliary protein(s) potentiating the reactivation or associating with EutA. It was demonstrated with purified recombinant EutA that both the suicidally inactivated and O2-inactivated holoethanolamine ammonia lyase underwent rapid reactivation in vitro by EutA in the presence of adenosylcobalamin, ATP, and Mg2+. The inactive enzyme-cyanocobalamin complex was also activated in situ and in vitro by EutA under the same conditions. Thus, it was concluded that EutA is the only component of the reactivating factor for ethanolamine ammonia lyase and that reactivation and activation occur through the exchange of modified coenzyme for free intact adenosylcobalamin.

Caspase activation, inhibition, and reactivation: a mechanistic view.

Caspases, a unique family of cysteine proteases, execute programmed cell death (apoptosis). Caspases exist as inactive zymogens in cells and undergo a cascade of catalytic activation at the onset of apoptosis. The activated caspases are subject to inhibition by the inhibitor-of-apoptosis (IAP) family of proteins. This inhibition can be effectively removed by diverse proteins that share an IAP-binding tetrapeptide motif. Recent structural and biochemical studies have revealed the underlying molecular mechanisms for these processes in mammals and in Drosophila. This paper reviews these latest advances.

Calcineurin hydrolysis of para-nitrophenyl phosphorothioate.

para-Nitrophenyl phosphorothioate (pNPT) was hydrolyzed by calcineurin at initial rates slightly, but comparable to rates for para-nitrophenyl phosphate (pNPP). Kinetic characterization yielded higher estimates for both Km and Vmax compared to pNPP. Metal ion activation of phosphorothioate hydrolysis was more promiscuous. Unlike the hydrolysis of with pNPP, Ca2+, Mg2+, and Ba2+ activated calcineurin as well as Mn2+.

Insight into the mechanism of the B12-independent glycerol dehydratase from Clostridium butyricum: preliminary biochemical and structural characterization.

The molecular characterization of a B12-independent glycerol dehydratase from Clostridium butyricum has recently been reported [Raynaud, C., et al. (2003) Proc. Natl. Acad. Sci. U.S.A. 100, 5010-5015]. In this work, we have further characterized this system by biochemical and crystallographic methods. Both the glycerol dehydratase (GD) and the GD-activating enzyme (GD-AE) could be purified to homogeneity under aerobic conditions. In this form, both the GD and GD-AE were inactive. A reconstitution procedure, similar to what has been reported for pyruvate formate lyase activating enzyme (PFL-AE), was employed to reconstitute the activity of the GD-AE. Subsequently, the reconstituted GD-AE could be used to reactivate the GD under strictly anaerobic conditions. We also report here the crystal structure of the inactive GD in the native (2.5 A resolution, Rcryst = 17%, Rfree = 20%), glycerol-bound (1.8 A resolution, Rcryst = 21%, Rfree = 24%), and 1,2-propanediol-bound (2.4 A resolution, Rcryst = 20%, Rfree = 24%) forms. The overall fold of the GD monomer was similar to what has been observed for pyruvate formate lyase (PFL) and anaerobic ribonucleotide reductase (ARNR), consisting of a 10-stranded beta/alpha barrel motif. Clear density was observed for both substrates, and a mechanism for the dehydration reaction is presented. This mechanism clearly supports a concerted pathway for migration of the OH group through a cyclic transition state that is stabilized by partial protonation of the migrating OH group. Finally, despite poor alignment (rmsd approximately 6.8 A) of the 10 core strands that comprise the barrel structure of the GD and PFL, the C-terminal domains of both proteins align well (rmsd approximately 0.7 A) and have structural properties consistent with this being the docking site for the activating enzyme. A single point mutation within this domain, at a strictly conserved arginine residue (R782K) in the GD, resulted in formation of a tight protein-protein complex between the GD and the GD-AE in vivo, thereby supporting this hypothesis.

Apoptosis-associated speck-like protein containing a caspase recruitment domain is a regulator of procaspase-1 activation.

Apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC)/target of methylation-induced silencing/PYCARD represents one of only two proteins encoded in the human genome that contains a caspase recruitment domain (CARD) together with a pyrin, AIM, ASC, and death domain-like (PAAD)/PYRIN/DAPIN domain. CARDs regulate caspase family proteases. We show here that ASC binds by its CARD to procaspase-1 and to adapter proteins involved in caspase-1 activation, thereby regulating cytokine pro-IL-1beta activation by this protease in THP-1 monocytes. ASC enhances IL-1beta secretion into the cell culture supernatants, at low concentrations, while suppressing at high concentrations. When expressed in HEK293 cells, ASC interferes with Cardiak/Rip2/Rick-mediated oligomerization of procaspase-1 and suppresses activation this protease, as measured by protease activity assays. Moreover, ASC also recruits procaspase-1 into ASC-formed cytosolic specks, separating it from Cardiak. We also show that expression of the PAAD/PYRIN family proteins pyrin or cryopyrin/PYPAF1/NALP3 individually inhibits IL-1beta secretion but that coexpression of ASC with these proteins results in enhanced IL-1beta secretion. However, expression of ASC uniformly interferes with caspase-1 activation and IL-1beta secretion induced by proinflammatory stimuli such as LPS and TNF, suggesting pathway competition. Moreover, LPS and TNF induce increases in ASC mRNA and protein expression in cells of myeloid/monocytic origin, revealing another level of cross-talk of cytokine-signaling pathways with the ASC-controlled pathway. Thus, our results suggest a complex interplay of the bipartite adapter protein ASC with PAAD/PYRIN family proteins, LPS (Toll family receptors), and TNF in the regulation of procaspase-1 activation, cytokine production, and control of inflammatory responses.

TIMP-2 (tissue inhibitor of metalloproteinase-2) regulates MMP-2 (matrix metalloproteinase-2) activity in the extracellular environment after pro-MMP-2 activation by MT1 (membrane type 1)-MMP.

The matrix metalloproteinase (MMP)-2 has a crucial role in extracellular matrix degradation associated with cancer metastasis and angiogenesis. The latent form, pro-MMP-2, is activated on the cell surface by the membrane-tethered membrane type 1 (MT1)-MMP, in a process regulated by the tissue inhibitor of metalloproteinase (TIMP)-2. A complex of active MT1-MMP and TIMP-2 binds pro-MMP-2 forming a ternary complex, which permits pro-MMP-2 activation by a TIMP-2-free neighbouring MT1-MMP. It remains unclear how MMP-2 activity in the pericellular space is regulated in the presence of TIMP-2. To address this question, the effect of TIMP-2 on MMP-2 activity in the extracellular space was investigated in live cells, and their isolated plasma membrane fractions, engineered to control the relative levels of MT1-MMP and TIMP-2 expression. We show that both free and inhibited MMP-2 is detected in the medium, and that the net MMP-2 activity correlates with the level of TIMP-2 expression. Studies to displace MT1-MMP-bound TIMP-2 in a purified system with active MMP-2 show minimal displacement of inhibitor, under the experimental conditions, due to the high affinity interaction between TIMP-2 and MT1-MMP. Thus inhibition of MMP-2 activity in the extracellular space is unlikely to result solely as a result of TIMP-2 dissociation from its complex with MT1-MMP. Consistently, immunoblot analyses of plasma membranes, and surface biotinylation experiments show that the level of surface association of TIMP-2 is independent of MT1-MMP expression. Thus low-affinity binding of TIMP-2 to sites distinct to MT1-MMP may have a role in regulating MMP-2 activity in the extracellular space generated by the ternary complex.