Irreversible Complex Formation Research Articles

Investigating the effect of fusion partners on the enzymatic activity and thermodynamic stability of poly(ethylene terephthalate) degrading enzymes.

Plastics are a cornerstone of the modern world, yet the durable material properties that we have come to depend upon have made them recalcitrant environmental pollutants. Biological solutions in the form of engineered enzymes offer low energy and sustainable approaches to recycle and upcycle plastic waste, uncoupling their production and end of life from fossil fuels and greenhouse gases. These enzymes however, encounter immense challenges acting on plastics: facing hydrophobic surfaces, molecular crowding, and high levels of substrate heterogeneity. There have been mixed reports about the benefits of fusing partner domains to polyethylene terephthalate (PET) degrading enzymes, with moderate improvements identified under specific conditions, but no clarity into the factors that underlie the mechanisms. Here, we use the SpyCatcher003:SpyTag003 technology, which demonstrates a profound 47 °C shift in Tm upon irreversible complex formation, to investigate the influence of the thermal stability of the fusion partner on a range of PETases selected for their optimal reaction temperatures. We find that the thermal stability of the fusion partner does not have a positive correlation on the activity of the enzymes or their evident kinetic and thermal stabilities. Instead, it appears that the fusion to less stable SpyCatcher003 tends to increase the measured activation energy of unfolding compared to the more stable complex and wildtype enzymes. Despite this, the fusions to SpyCatcher003 do not show significantly better catalytic activity on PET films, with or without SpyTag003, and were found to be sometimes disruptive. The approach we highlight here, in using a fusion partner with controllable melting temperature, allowed us to dissect the impact of the stability of a fusion partner on enzyme properties. Although fusion stability did not appear to be coupled with identifiable trends in enzymatic activities, careful analysis of the unfolding pathways, and solid and solution activities of a wider range of enzymes may yield a more detailed understanding.

Cyclic boronates as versatile scaffolds for KPC-2 β-lactamase inhibition.

Klebsiella pneumoniae carbapenemase-2 (KPC-2) is a serine-β-lactamase (SBL) capable of hydrolysing almost all β-lactam antibiotics. We compare KPC-2 inhibition by vaborbactam, a clinically-approved monocyclic boronate, and VNRX-5133 (taniborbactam), a bicyclic boronate in late-stage clinical development. Vaborbactam inhibition is slowly reversible, whereas taniborbactam has an off-rate indicating essentially irreversible complex formation and a 15-fold higher on-rate, although both potentiate β-lactam activity against KPC-2-expressing K. pneumoniae. High resolution X-ray crystal structures reveal closely related binding modes for both inhibitors to KPC-2, with differences apparent only in positioning of the endocyclic boronate ester oxygen. The results indicate the bicyclic boronate scaffold as both an efficient, long-lasting, KPC-2 inhibitor and capable of supporting further iterations that may improve potency against specific enzyme targets and pre-empt the emergence of inhibitor resistant KPC-2 variants.

Steroidal antibiotics are antimetabolites of Acanthamoeba steroidogenesis with phylogenetic implications

Pathogenic organisms may be sensitive to inhibitors of sterol biosynthesis, which carry antimetabolite properties, through manipulation of the key enzyme, sterol methyltransferase (SMT). Here, we isolated natural suicide substrates of the ergosterol biosynthesis pathway, cholesta-5,7,22,24-tetraenol (CHT) and ergosta-5,7,22,24(28)-tetraenol (ERGT), and demonstrated their interference in Acanthamoeba castellanii steroidogenesis: CHT and ERGT inhibit trophozoite growth (EC50 of 51 nM) without affecting cultured human cell growth. Washout experiments confirmed that the target for vulnerability was SMT. Chemical, kinetic, and protein-binding studies of inhibitors assayed with 24-AcSMT [catalyzing C28-sterol via Δ24(28)-olefin production] and 28-AcSMT [catalyzing C29-sterol via Δ25(27)-olefin production] revealed interrupted partitioning and irreversible complex formation from the conjugated double bond system in the side chain of either analog, particularly with 28-AcSMT. Replacement of active site Tyr62 with Phe or Leu residues involved in cation-π interactions that model product specificity prevented protein inactivation. The alkylating properties and high selective index of 103 for CHT and ERGT against 28-AcSMT are indicative of a new class of steroidal antibiotic that, as an antimetabolite, can limit sterol expansion across phylogeny and provide a novel scaffold in the design of amoebicidal drugs. Animal studies of these suicide substrates can further explore the potential of their antibiotic properties.

Theoretical study of the structure and acidity of condensed tannin monomers

Low level of polyphenols absorption is due among other things to the formation of irreversible complexes between non-heminic iron (Fe2+, Fe3+) and these polyphenols through their hydroxyl groups. Understanding of these complexes formation mechanisms led us to explore hydroxyl groups acidity of these polyphenols monomers (catechin, epicatechin, gallocatechin and epigallocatechin) mainly found in food. Quantum chemistry modelling, more precisely the functional density theory (DFT) method, associated to 6-311G (d, p) base was used in gas phase. Spectroscopic, thermodynamic descriptors and geometric parameters resulting from calculations showed a slight stability of catechin compared to epicatechin and gallocatechin compared to epigallocatechin. As for the acidity of these monomers hydroxyl groups, the results show that it decreases similarly as follows: O4’ > O5> O7 > O5’> O3 for Catechin and epicatechin; O3’ > O5> O4’ > O7 > O5’ > O3 and O3’ > O4’ > O5 > O7 > O5’> O3 respectively for gallocatechin and epigallocatechin.Keywords: Catechin, Epicatechin, Gallocatechin, Epigallocatechin, Acidity, DFT.

Molecular cloning, genomic structure, polymorphism analysis and recombinant expression of a α1-antitrypsin like gene from swamp eel, Monopterus albus

Alpha-1-antitrypsin (AAT) is a highly polymorphic glycoprotein antiprotease, involved in the regulation of human immune response. Beyond some genomic characterization and a few protein characterizations, the function of teleost AAT remains uncertain. In this study we cloned an AAT-like gene from a swamp eel liver identifying four exons and three introns, and the full-length cDNA. The elucidated swamp eel AAT amino acid sequence showed high homology with known AATs from other teleosts. The swamp eel AAT was examined both in ten healthy tissues and in four bacterially-stimulated tissues resulting in up-regulation of swamp eel AAT at different times. Swamp eel AAT transcripts were ubiquitously but unevenly expressed in ten tissues. Further, the mature peptide sequence of swamp eel AAT was subcloned and transformed into E. coli with the recombinant proteins successfully inhibiting bovine trypsin activity. Analysis of recombinant AAT showed equimolar formation of irreversible complexes with proteinases, high stability at pH 7.0–10.0 and temperatures below 55 °C. Serum AAT protein level significantly increased in response to inflammation with AAT anti-sera, and, NF-κB, apolipoprotein A1 and transferrin gene expression were dramatically decreased over 72 h post recombinant AAT injection. Lastly, examination of swamp eel AAT allelic polymorphism identified all alleles in both healthy and diseased stock except allele*g, found only in diseased stock, but without statistical difference between the distribution frequency of allele*g in the two stocks. These results are crucial to our ongoing study of the role of teleost AAT in the innate immune system.

The putative role of Rhipicephalus microplus salivary serpins in the tick-host relationship

Inflammation and hemostasis are part of the host's first line of defense to tick feeding. These systems are in part serine protease mediated and are tightly controlled by their endogenous inhibitors, in the serpin superfamily (serine protease inhibitors). From this perspective ticks are thought to use serpins to evade host defenses during feeding. The cattle tick Rhipicephalus microplus encodes at least 24 serpins, of which RmS-3, RmS-6, and RmS-17 were previously identified in saliva of this tick. In this study, we screened inhibitor functions of these three saliva serpins against a panel of 16 proteases across the mammalian defense pathway. Our data confirm that Pichia pastoris-expressed rRmS-3, rRmS-6, and rRmS-17 are likely inhibitors of pro-inflammatory and pro-coagulant proteases. We show that rRmS-3 inhibited chymotrypsin and cathepsin G with stoichiometry of inhibition (SI) indices of 1.8 and 2.0, and pancreatic elastase with SI higher than 10. Likewise, rRmS-6 inhibited trypsin with SI of 2.6, chymotrypsin, factor Xa, factor XIa, and plasmin with SI higher than 10, while rRmS-17 inhibited trypsin, cathepsin G, chymotrypsin, plasmin, and factor XIa with SI of 1.6, 2.6, 2.7, 3.4, and 9.0, respectively. Additionally, we observed the formation of irreversible complexes between rRmS-3 and chymotrypsin, rRmS-6/rRmS-17 and trypsin, and rRmS-3/rRmS-17 and cathepsin G, which is consistent with typical mechanism of inhibitory serpins. In blood clotting assays, rRmS-17 delayed plasma clotting by 60 s in recalcification time assay, while rRmS-3 and rRmS-6 did not have any effect. Consistent with inhibitor function profiling data, 2.0 μM rRmS-3 and rRmS-17 inhibited cathepsin G-activated platelet aggregation in a dose-responsive manner by up to 96% and 95% respectively. Of significant interest, polyclonal antibodies blocked inhibitory functions of the three serpins. Also notable, antibodies to Amblyomma americanum, Ixodes scapularis, and Rhipicephalus sanguineus tick saliva proteins cross-reacted with the three R. microplus saliva serpins, suggesting the potential of these proteins as candidates for universal anti-tick vaccines.

In vitro evaluation of potential complexation between bovine insulin and bovine serum albumin

The objective of this study was to examine the possible binding of bovine insulin (BI) with bovine serum albumin (BSA) to form a new potential diabetogenic irreversible complex protein. Several preparations of BSA and BI were prepared. Both capillary electrophoresis and spectrophotometric analysis were undertaken to test the possibility of complexation between BI and BSA. HPLC was used to test whether the potential complex of BI and BSA is reversible or irreversible. The optimum deviation between the real and calculated absorbances was observed at a BI/BSA ratio of 2. Moreover, the migration time of BI decreased substantially with increasing ratio of BI to BSA until it became almost constant at equal molar ratio of BI/BSA. While the majority of the 2:1 BI-BSA sample detached during the HPLC analysis, which confirms the reversible character of BI-BSA binding, the HPLC chromatogram also emphasizes the formation of an irreversible complexation between the two proteins. This study provides evidence of the formation of reversible and irreversible new BI-BSA complexes under physiological conditions. This highlights the importance of examining the possible diabetogenicity of BI-BSA complex in genetically susceptible people.

Prostate-specific antigen kallikrein complexes and acute myocardial infarction

Recently, attention has focused on prostate-specific antigen kallikrein and the cardiovascular system. The finding of diminished PSA during acute myocardial infarction and the correlation of variation of PSA with coronary lesions and occurrence of major adverse cardiac events have opened a possible new intriguing scenario. As with many stories in biology, the tale of PSA can be more complex than was first appreciated. In the bloodstream, the proteolytic activity of PSA is inhibited by the formation of irreversible complexes with serum protease inhibitors and other acute-phase proteins. We noticed that the formation of irreversible PSA complexes (reflected by the increased bound PSA) has significant correlation with high-sensitivity C-reactive protein (hsCRP) and that seems to play a crucial role in the adverse event prevention.

A steady-state competition model describes the modulating effects of thrombomodulin on thrombin inhibition by plasminogen activator inhibitor-1 in the absence and presence of vitronectin.

Thrombomodulin (TM) slows down the interaction rate between thrombin and plasminogen activator inhibitor 1 (PAI-1). We now show that the 12-fold reduced inhibition rate in the presence of TM does not result from an altered distribution between PAI-1 cleavage and irreversible complex formation. Surface plasmon resonance (SPR) revealed an over 200-fold reduced affinity of TM for thrombin-VR1tPA as compared to thrombin, demonstrating the importance of the VR1 loop in the interaction of thrombin with both TM and PAI-1. Furthermore, in contrast to ATIII, PAI-1 was not able to bind the thrombin/TM complex demonstrating complete competitive binding between PAI-1 and TM. Kinetic modeling on the inhibitory effect of TM confirms a mechanism that involves complete steric blocking of the thrombin/PAI-1 interaction. Also, it accurately decribes the biphasic inhibition profile resulting from the substantial reduction of the extremely fast rate of reversible Michaelis complex formation, which is essential for efficient inhibition of thrombin by PAI-1. Vitronectin (VN) is shown to partially relieve TM inhibitory action only by vastly increasing the initial rate of interaction between free thrombin and PAI-1. In addition, SPR established that solution-phase PAI-1/VN complexes and non-native VN (extracellular matrix form) bind TM directly via the chondroitin sulphate moiety of TM. Collectively, these results show that VR1 is a subsite of exosite 1 on thrombin's surface, which regulates exclusive binding of either PAI-1 or TM. This competition will be physiologically significant in controlling the mitogenic activity of thrombin during vascular disease.

Serpins of oat (Avena sativa) grain with distinct reactive centres and inhibitory specificity.

Most proteinase inhibitors from plant seeds are assumed to contribute to broad-spectrum protection against pests and pathogens. In oat (Avena sativa L.) grain the main serine proteinase inhibitors were found to be serpins, which utilize a unique mechanism of irreversible inhibition. Four distinct inhibitors of the serpin superfamily were detected by native PAGE as major seed albumins and purified by thiophilic adsorption and anion exchange chromatography. The four serpins OSZa-d are the first proteinase inhibitors characterized from this cereal. An amino acid sequence close to the blocked N-terminus, a reactive centre loop sequence, and the second order association rate constant (ka') for irreversible complex formation with pancreas serine proteinases at 24 degrees C were determined for each inhibitor. OSZa and OSZb, both with the reactive centre scissile bond P1-P1' Thr downward arrow Ser, were efficient inhibitors of pancreas elastase (ka' > 105M-1 s-1). Only OSZb was also an inhibitor of chymotrypsin at the same site (ka' = 0.9 x 105M-1 s-1). OSZc was a fast inhibitor of trypsin at P1-P1' Arg downward arrow Ser (ka' = 4 x 106M-1 s-1); however, the OSZc-trypsin complex was short-lived with a first order dissociation rate constant kd = 1.4 x 10-4 s-1. OSZc was also an inhibitor of chymotrypsin (ka' > 106M-1 s-1), presumably at the overlapping site P2-P1 Ala downward arrow Arg, but > 90% of the serpin was cleaved as substrate. OSZd was cleaved by chymotrypsin at the putative reactive centre bond P1-P1' Tyr downward arrow Ser, and no inhibition was detected. Together the oat grain serpins have a broader inhibitory specificity against digestive serine proteinases than represented by the major serpins of wheat, rye or barley grain. Presumably the serpins compensate for the low content of reversible inhibitors of serine proteinases in oats in protection of the grain against pests or pathogens.

Inhibitory serpins from rye grain with glutamine as P 1 and P 2 residues in the reactive center

Six of seven serpins detected in grains of rye ( Secale cereale) were purified and characterized. The amino acid sequence close to the blocked N-terminus, the reactive center loop sequence and the second order association rate constant ( k a′) for irreversible complex formation with chymotrypsin were determined for each serpin. Three of four serpins containing the unusual reactive center P 2–P 1′ QQ↓S and one with P 2–P 1′ PQ↓M were equally efficient inhibitors of chymotrypsin ( k a′∼10 5 M −1 s −1). One serpin with P 2–P 1′ PY↓M was a faster inhibitor ( k a′∼10 6 M −1 s −1). Similar but differently organized glutamine-rich reactive centers were recently found in grain serpins cloned from wheat [Østergaard et al. (2000) J. Biol. Chem. 275, 33272] but not from barley. The prolamin storage proteins of cereal grains contain similar sequences in their glutamine-rich repeats. A possible adaption of hypervariable serpin reactive centers late in Triticeae cereal evolution as defence against insects feeding on cereal grains is discussed.

Site-directed Mutagenesis and Characterization of Uracil-DNA Glycosylase Inhibitor Protein: ROLE OF SPECIFIC CARBOXYLIC AMINO ACIDS IN COMPLEX FORMATION WITH ESCHERICHIA COLI URACIL-DNA GLYCOSYLASE

Bacteriophage PBS2 uracil-DNA glycosylase inhibitor (Ugi) protein inactivates uracil-DNA glycosylase (Ung) by acting as a DNA mimic to bind Ung in an irreversible complex. Seven mutant Ugi proteins (E20I, E27A, E28L, E30L, E31L, D61G, and E78V) were created to assess the role of various negatively charged residues in the binding mechanism. Each mutant Ugi protein was purified and characterized with respect to inhibitor activity and Ung binding properties relative to the wild type Ugi. Analysis of the Ugi protein solution structures by nuclear magnetic resonance indicated that the mutant Ugi proteins were folded into the same general conformation as wild type Ugi. All seven of the Ugi proteins were capable of forming a Ung.Ugi complex but varied considerably in their individual ability to inhibit Ung activity. Like the wild type Ugi, five of the mutants formed an irreversible complex with Ung; however, the binding of Ugi E20I and E28L to Ung was shown to be reversible. The tertiary structure of [13C,15N]Ugi in complex with Ung was determined by solution state multi-dimensional nuclear magnetic resonance and compared with the unbound Ugi structure. Structural and functional analysis of these proteins have elucidated the two-step mechanism involved in Ung.Ugi association and irreversible complex formation.

Interaction of slightly crosslinked gels of poly(diallyldimethylammonium bromide) with sodium dodecyl sulfate and cetylpyridinium bromide

AbstractThe interaction of slightly crosslinked gels of poly(diallyldimethylammonium bromide) (PDADMAB) with both the anionic surfactant sodium dodecyl sulfate (SDS) and the cationic surfactant cetylpyridinium bromide (CPB; system. name: N‐hexadecylpyridinium bromide) in aqueous medium has been investigated. The absorption of the anionic surfactant by the oppositely charged gel leads to contraction or collapse of the network. The absorption of the cationic surfactant by the gel‐SDS complexes results in a reswelling of the network. By means of IR and UV spectroscopy it has been shown that in the presence of the anionic detergent the cationic network absorbs the cationic surfactant. It has been shown that both reversible and irreversible complex formation in the gel phase is possible depending on the initial ratio between the components. Small‐angle‐X‐ray scattering experiments demonstrated that the complexes may have a regular microstructure.

Experimental study of intramolecular crosslinking of poly N ϵ-methacryloyl- L-lysine by copper ions

The intramolecular crosslinking of a polyelectrolyte by divalent metal ions has been investigated for poly-N ϵ-methacryloyl- L-lysine and copper (II) ions. It is shown that in the 4·5–11·0 pH range irreversible complex formation takes place, and is accompanied by a change in hydrodynamic properties of the macromolecules. The structure of the polymer-ligand (metal ion) complexes has been determined, and their behaviour in aqueous solutions investigated. Agreement between the results of theoretical calculations and experimental determination of change in the macromolecular coil dimensions during complexation reactions shows that the model used for theoretical calculations fits particular reactions of intramolecular crosslinking.

Evidence against the occurrence of artifacts due to carrier ampholyte-protein binding during isoelectric focusing

The formation of irreversible complexes between carrier ampholyte components and proteins was investigated by gel filtration of mixtures of proteins and radioactively labelled ampholytes. Experiments were performed both with purified proteins (albumin, ferritin, β-glucuronidase) and with a complex mixture of proteins (serum); in no case was binding of ampholytes to proteins detected. Thus the results argue against the occurrence in isoelectric focusing of proteins of artifacts due to such complex formation.

Acetyl Coenzyme A Carboxylase: IV. BIOTINYL PROSTHETIC GROUP-INDEPENDENT MALONYL COENZYME A DECARBOXYLATION AND CARBOXYL TRANSFER: GENERALIZATION TO OTHER BIOTIN ENZYMES

Abstract Liver acetyl-CoA carboxylase, a biotin-enzyme which catalyzes the ATP-dependent carboxylation of acetyl-CoA (acceptor) to form malonyl-CoA (carboxylated acceptor), decarboxylates malonyl-CoA by a biotin-dependent, as well as a biotin-independent mechanism. Neither ADP, Pi, nor divalent metal ion are required for either of these abortive decarboxylations. The biotin-dependent reaction is blocked by avidin and is activated synergistically by acetyl-CoA and citrate, an allosteric effector of the carboxylase. However, the biotin-independent process is unaffected by avidin and citrate, and is inhibited slightly by acetyl-CoA. The occurrence of this abortive reaction indicates that the enzyme promotes the labilization of the free carboxyl group of malonyl-CoA and that insertion of a proton in its place is possible without the participation of the biotinyl prosthetic group. This phenomenon appears to be a property of biotin-dependent carboxylases since pig heart propionyl-CoA carboxylase, the biotin-free carboxyltransferase component of Escherichia coli acetyl-CoA carboxylase, and avian liver pyruvate carboxylase catalyze similar biotin-independent decarboxylations of their respective carboxylated acceptor substrates. Liver acetyl-CoA carboxylase also catalyzes carboxyl transfer from [3-14C]malonyl-CoA to free d-biotin, a model for the second half-reaction in the over-all carboxylation process. The acid-labile radioactive carboxylation product was stabilized by methylation with diazomethane, and the ester formed identified as 1'-N-carboxy-d-biotin dimethyl ester by carrier recrystallization with the authentic compound. The model reaction does not involve the covalently bound biotinyl prosthetic group, since the essentially irreversible complex formation of this group with avidin does not block carboxyl transfer. While the reaction is specific in that l-biotin and imidazolidone-2 are inactive, certain closely related biotin derivatives including d-homobiotin and biocytin (e-N-d-biotinyl-l-lysine) are somewhat better acceptor substrates than d-biotin. The fact that citrate blocks carboxyl transfer to free d-biotin and biocytin with both avidin-complexed and native carboxylase without affecting the rate of malonyl-CoA decarboxylation is compatible with earlier evidence implicating the biotinyl prosthetic group as the focal point of citrate-induced conformational changes.