Inhibition Rate Constants Research Articles

Expression, purification, and characterization of recombinant rat plasminogen activator inhibitor-1

Summary Plasminogen activator inhibitor-1 (PAI-1) is a physiologically important regulatory protein of the fibrinolytic system. To study in vivo its influence on a variety of biological events (thrombosis, atherosclerosis, tumour progression) a number of different experimental models in various animals, including rats, have been established. A major drawback of these models is the lack of the purified endogenous proteins and/or specific reagents for their detection in each particular species. In this study we describe the expression, in Escherichia coli (E. coli) cells, the purification, and the characterization of rat PAI-1. As expected, sodium dodecyl sulphate polyacrylamide gel electrophoresis revealed a protein with an apparent molecular mass of ∼45 kDa. Recombinant rat PAI-1 had a specific inhibitory activity of 434 000±74 000 U/mg (mean±SD, n=11) towards human tissue-type plasminogen activator (t-PA), corresponding to 58±10% of the theoretical maximum value. Evaluation of the reaction products formed upon interaction between recombinant rat PAI-1 and its target proteinases t-PA or urokinase-type plasminogen activator revealed the presence of large amounts of covalent complex, small amounts of cleaved PAI-1 and residual latent PAI-1. Active recombinant rat PAI-1 converted spontaneously to the latent form with a half-life of 2±0.2h (n=6). The second-order rate constant of inhibition of human t-PA by recombinant rat PAI-1 was two-fold lower compared to that by recombinant human PAI-1 (0.6±0.02×107M−1s−1 vs 1.4±0.14×107M−1s−1, respectively, P The current data demonstrate the biochemical equivalence between rat PAI-1 and human PAI-1. The availability of purified recombinant rat PAI-1 will allow the development of research tools required to evaluate the in vivo role of PAI-1 in various rat models.

Expression and Characterization of Recombinant Porcine Plasminogen Activator Inhibitor-1

Porcine models are, among other animal models, very suitable for in vivo investigations in the vascular field especially with respect to the possible relationship between atherosclerosis and thrombosis. In order to use this model to define the in vivo role of PAI-1, the characterization of porcine PAI-1 and its availability for the generation of immunological tools are a prerequisite. Porcine plasminogen activator inhibitor-1 (poPAI-1) cDNA was isolated from a cDNA library prepared from cultured porcine aortic cells and characterized in comparison with PAI-1 cDNA's from other species including human, bovine, rabbit, rat and murine. Subsequently the DNA sequence coding the mature protein was cloned into an appropriate vector for expression in Escherichia coli and recombinant porcine PAI-1 was purified and characterized. On SDS-PAGE the apparent molecular weight was estimated to be 45 kDa, identical to the molecular weight of human PAI-1. The purified recombinant porcine PAI-1 (rpoPAI-1) had a specific activity of 508,800 +/- 800 U/mg (mean +/- SD, n = 3) towards human tissue-type plasminogen activator (ht-PA) and a functional half-life in vitro of 2.1 +/- 0.8 h (n = 3). Incubation with a two fold molar excess of ht-PA (n = 3) or human urokinase-type plasminogen activator (hu-PA, n = 2) followed by analysis by SDS-PAGE revealed reaction products corresponding to active (71 +/- 7% resp. 96 +/- 3.6%), latent (12 +/- 0.4% resp. 2.6 +/- 2.4%) and substrate (16.6 +/- 6.8% resp. 1.5 +/- 1.3) forms. Inactivated samples of porcine PAI-1 could be reactivated with guanidinium chloride up to 52% of its original specific activity towards t-PA and u-PA. The second order rate constant of inhibition of ht-PA was 1.64 +/- 0.37 10(7)M-1 s-1 (n = 9). In gel filtration rpoPAI-1 in buffer eluted at a volume corresponding to 24 kDa, whereas in the presence of porcine plasma, the molecular form containing PAI-1 activity eluted at a volume corresponding to 330 kDa, presumably as a consequence of binding of active PAI-1 to vitronectin. Taken together, these data demonstrate that no obvious functional differences exist between human and porcine PAI-1.

Biochemical determination of insecticides via cholinesterases. 1. Acetylcholinesterase from rat brain: functional expression using a baculovirus system, and biochemical characterization

Using a baculovirus-insect cell system, acetylcholinesterase from rat brain (rbAChE) was actively expressed and biochemically characterized and compared to the non-recombinant, tissue-derived rbAChE, establishing biochemical identity between both enzymes. Inhibition kinetics were similar for both enzymes using reversible inhibitors specific to the active and peripheral site. Inhibition rate constants of both enzymes treated with paraoxon as a reference insecticide were identical for both enzymes. As neither the catalytic parameters nor inhibition kinetics revealed any significant difference among the tissue derived and the recombinant enzyme, the baculovirus-insect expression system can be used for the preparation of recombinant rbAChE and mutants thereof.

On the Inhibition Mechanism of Sarcoplasmic or Endoplasmic Reticulum Ca2+-ATPases by Cyclopiazonic Acid

Ca2+-ATPase inhibition by stoichiometric and substoichiometric concentrations of cyclopiazonic acid was studied in sarcoplasmic reticulum preparations from rabbit fast-twitch muscle. The apparent affinity of the nonphosphorylated enzyme for ATP showed a Kd of approximately 3 microM in the absence of cyclopiazonic acid and approximately 28 microM in the presence of the drug. Fractional saturation of the enzyme by cyclopiazonic acid was accompanied by the appearance of two ATP-binding populations (enzyme with and without drug) and a progressive increase in the half-maximal concentration for saturating the ATP-binding sites. Enzyme turnover in the presence of stoichiometric concentrations of cyclopiazonic acid displayed lower apparent affinity for ATP and lower maximal hydrolytic activity than in the absence of the drug. When cyclopiazonic acid is in the substoichiometric range, the observed kinetic parameters will correspond to the simultaneous contribution of two different reaction cycles sustained by the enzyme with and without drug. The inhibition could be elicited by adding ATP to allow the enzyme turnover when cyclopiazonic acid was preincubated with the enzyme in the presence of Ca2+. The onset of inhibition during enzyme cycling was observed over a period of seconds, revealing the existence of a low inhibition rate constant. It is concluded that cyclopiazonic acid decreases enzyme affinity for ATP in non-turnover conditions by approximately one order of magnitude. This allows enzyme cycling after drug binding, provided that a high ATP concentration is used. Cyclopiazonic acid and ATP do not compete for the same binding site.

INDIRECT PARASYMPATHOMIMETIC ACTIVITY OF THE CLASS III ANTIARRHYTHMIC SUBSTANCE D/ L-SOTALOL IN VITRO: REVERSIBLE INHIBITION OF CHOLINESTERASE ISOENZYMES FROM BLOOD AND THE HUMAN CENTRAL NERVOUS SYSTEM

Inhibitory effects of the class III antiarrhythmic compound D/ L-sotalol on acetylcholinesterase (AChE; EC 3.1.1.7) isoenzymes of both erythrocytes and the human caudate nucleus and on serum cholinesterase (ChE; EC 3.1.1.8) were studied in vitrousing a spectrophotometric kinetic assay with acetylthiocholine (ASCh) as substrate. Sotalol concentrations in the assays varied from 0.32 to 3.2m M. All isoenzymes studied were inhibited by D/ L-sotalol in a reversible and concentration-dependent manner. Double reciprocal plots of the reaction velocity against varying ASCh concentrations revealed that D/ L-sotalol reduced substrate affinity (apparent Michaelis constant, KM, increased) of serum ChE, but did not change the enzyme's maximal rate of ASCh hydrolysis (V max). Thus, D/ L-sotalol inhibition of serum ChE was of the competitive type (rate constant for reversible competitive inhibition: K i=0.51m M). In contrast, D/ Lsotalol reduced the maximal reaction velocity of the AChE isoenzyme from the central nervous system (caudate nucleus), but had no influence on substrate affinity of the enzyme (K Mwith ASCh unchanged) indicating purely non-competitive inhibition kinetics (rate constant of reversible non-competitive inhibition: K i′=0.44m M). D/ L-sotalol inhibition of erythrocyte AChE was of mixed competitive/non-competitive type (K i=0.31m M, K i′=0.49m M). Non-competitive D/ L-sotalol inhibition of caudate nucleus AChE and the non-competitive component of erythrocyte AChE inhibition cannot be overcome by increased concentrations of the cholinergic transmitter acetylcholine (ACh). Peak D/ L-sotalol plasma levels as described in the literature for both humans (15μ M) and experimental animals (dogs: 18μ M; rats: 260μ M) as well as maximal myocardial concentrations of the substance (dogs: 46μ M; rats: 478μ M) are in the range of about 2% to 100% of the sotalol inhibition rate constants determined in the present paper for cholinesterase isoenzymes in vitro. Thus, D/ L-sotalol inhibition of ACh hydrolysis in vivomay contribute to both the well known antiarrhythmic potential and proarrhythmic side effects of the compound.

Requirement of Lysine Residues Outside of the Proposed Pentasaccharide Binding Region for High Affinity Heparin Binding and Activation of Human Antithrombin III

Variant forms of human antithrombin III with glutamine or threonine substitutions at Lys114, Lys125, Lys133, Lys136, and Lys139 were expressed in insect cells to evaluate their roles in heparin binding and activation. Recombinant native ATIII and all of the variants had very similar second order rate constants for thrombin inhibition in the absence of heparin, ranging from 1.13 x 10(5) M-1min-1 to 1.66 x 10(5) M-1min-1. Direct binding studies using 125I-flouresceinamine-heparin yielded a Kd of 6 nM for the recombinant native ATIII and K136T, whereas K114Q and K139Q bound heparin so poorly that a Kd could not be determined. K125Q had a moderately reduced affinity. Heparin binding affinity correlated directly with heparin cofactor activity. Recombinant native ATIII was nearly identical to plasma-purified ATIII, whereas K114Q and K139Q were severely impaired in heparin cofactor activity. K125Q and K136T were only slightly impaired. Based on these data, Lys114 and Lys139, which are outside of the putative pentasaccharide binding site, play pivotal roles in the high affinity binding of heparin to ATIII and the activation of thrombin inhibitory activity.

Inhibition of neutrophil elastase by the α1-proteinase inhibitor-immunoglobulin A complex

Neutrophil elastase is thought to be involved in cartilage destruction occurring in rheumatoid arthritis despite the local presence of α 1-proteinase inhibitor. Part of synovial fluid α 1-proteinase inhibitor forms a mixed disulfide with immunoglobulin A, which has been postulated to lack inhibitory activity. We show here that the immunoglobulin-inhibitor complex tightly inhibits neutrophil elastase and cathepsin G, bovine pancreatic trypsin and chymotrypsin, and porcine pancreatic elastase. Although the rate constant of inhibition of neutrophil elastase by immunoglobulin A-bound α 1-proteinase inhibitor ( k ass = 9.2 × 10 5 M −1 · s −1) is about 10-fold lower than that measured with the free inhibitor, it is high enough to enable efficient inhibition of elastase in vivo.

Sequence requirements in the reactive-center loop of plasminogen-activator inhibitor-1 for recognition of plasminogen activators.

Plasminogen activator inhibitor-1 (PAI-1) is a member of the serpin superfamily of proteins and is the fast acting inhibitor of both urinary plasminogen activator and tissue-type plasminogen activator. We have assessed the functional significance of reactive center residues on the carboxy-terminal side of the cleavage site of recombinant human PAI-1. Using site-directed mutagenesis, the P1'-P5' residues (P1' is the first residue on the carboxy-terminal side of the protease cleavage site) of the wild-type PAI-1 reactive center sequence were replaced with the corresponding sequences of plasminogen activator inhibitor-2, antithrombin, alpha 2-antiplasmin and protease nexin I. Rate constants of inhibition of the serine proteases urinary plasminogen activator, tissue-type plasminogen activator, plasmin and thrombin by the variants were determined. The results suggest a crucial role for both reactive center length and sequence in the inhibition of plasminogen activators by PAI-1. Analysis of substitutions at positions P4' and P5' both confirms and extends our previous work demonstrating a favorable electrostatic interaction between these residues and tissue-type plasminogen activator. None of the variants show dramatic increases in the rate constants of inhibition of other serine proteases, suggesting that these residues alone are not sufficient to confer protease specificity on PAI-1. Apparently, the determinants of the rapid inhibitory specificity of PAI-1 are localized to the P1'-P5' region of the reactive center and these residues act synergistically to produce the exquisite specificity of PAI-1 for plasminogen activators.

Heparin Stimulation of the Inhibition of Activated Protein C and other Enzymes by Human Protein C Inhibitor - Influence of the Molecular Weight of Heparin and Ionic Strength

The ability of unfractionated (UF) heparin and low-molecular-weight heparin (LMWH) to potentiate the inhibition of fibrinolytic and coagulation factors by protein C inhibitor (PCI) was studied. Inhibition of activated protein C (APC), urokinase plasminogen activator (uPA), tissue plasminogen activator (tPA), thrombin, factor Xa (Xa), factor XIa (XIa) and plasma kallikrein (KK) by PCI was found to be dependent on the size of the polysaccharide. In general, maximal stimulation was reached with UF heparin, except in the case of KK. Differences in heparin stimulation were more pronounced for thrombin, APC, uPA, tPA and XIa, whereas inactivation of Xa by PCI was less dependent on the presence of heparin, and kallikrein showed higher potentiation with LMWH than with UF heparin. The second-order rate constants for enzyme inhibition by PCI were strongly dependent on the ionic strength, and, in general, with an ionic strength higher than 0.15 the heparin stimulation of the inhibition reactions was drastically reduced. These results may explain the large discrepancies in the literature on the effect of heparin on the stimulation of enzyme inhibition by PCI. They also show that LMWH is less efficient in stimulating the PCI inhibition of APC, uPA and tPA, which could contribute to the antithrombotic effect of these enzymes.

Tryptophan 60-D in the B-insertion loop of thrombin modulates the thrombin-antithrombin reaction.

In a recent study it was demonstrated that thrombin des-PPW reacts with antithrombin (AT) very poorly. In this study it is shown that a Trp to Ala (W60A) mutant of thrombin also reacts with AT at a lower rate than thrombin. The inhibition kinetics were studied by the slow-binding kinetic approach. In both the presence and absence of heparin, the pseudo-first-order rate constant of thrombin inhibition (kobs) increased linearly with AT concentration, indicating that inhibition, in the concentration range covered, conforms to a bimolecular reaction E+I-->K assn E-I. Only the second-order association rate constant (kassn) for thrombin can be estimated [6.8 +/- 2.7) x 10(3) M-1 s-1 in the absence of heparin and (4.1 +/- 1.2) x 10(6) M-1 s-1 in the presence of heparin]. With W60A and des-PPW, the kobs of inhibition increased hyperbolically as a function of AT concentration, indicating that the inhibition is a two-step process according to E+I<==>K init E.I-->k2 E-I. The kinetic constants for W60A were estimated to be Kinit = 13.6 +/- 3.3 microM and k2 = 0.007 +/- 0.001 s-1 in the absence of heparin and K init = 13.6 +/- 3.1 nM and k2 = 0.008 +/- 0.002 s-1 in the presence of heparin. AT inhibited des-PPW very slowly [K assn = (2.9 +/- 0.7) x 10(1) M-1 s-1], but heparin accelerated the reaction approximately 20,000-fold and made it possible to demonstrate a two-step reaction mechanism for des-PPW with K init = 10.4 +/- 2.3 nM and k2 = 0.006 +/- 0.001 s-1. In contrast to thrombin, an active AT-binding pentasaccharide enhanced the inhibition of des-PPW approximately 15-fold. These results indicate that (1) in contrast to thrombin, the heparin-induced conformational change in AT is required for optimal inhibition of des-PPW and (1) Trp60 is essential for normal thrombin-AT reaction. On the basis of these results, a modified model for thrombin-AT interaction is proposed.

Antithrombin-mediated Inhibition of Factor Vlla-Tissue Factor Complex by the Synthetic Pentasaccharide Representing the Heparin Binding Site to Antithrombin

We examined the effect of the synthetic pentasaccharide representing the minimal binding site of heparin to antithrombin on the antithrombin-mediated inactivation of factor VIIa bound to tissue factor. This effect was compared to the effect of unfractionated heparin. Using purified recombinant human coagulation factors and either a clotting or an amidolytic assay for the determination of the residual activity of factor VIIa, we showed that the pentasaccharide was an efficient antithrombin-dependent inhibitor of the coagulant activity of tissue factor-factor VIIa complex. In our experimental conditions, assuming a mean MW of 14,000 for heparin, the molar pseudo-first order rate constants for ATIII-mediated FVIIa inhibition by ATIII-binding heparin and by the synthetic pentasaccharide were found to be similar with respective values of 104,000 +/- 10,500 min-1 and 112,000 +/- 12,000 min-1 (mean +/- s.e.m., n = 3).

Characterization and Treatment of the Toxicity of O-isobutyl S-[2-(diethylamino)ethyl]methylphosphonothioate, a Structural Isomer of VX, in Guinea Pigs

O-isobutyl S-[2-(diethylamino)ethyl]methylphosphonothioate (VR), a structural isomer of the chemical warfare agent O-ethyl S-[2(diisopropylamino)ethyl] methylphosphonothioate (VX) was characterized with respect to its toxicity, acetylcholinesterase (AChE) inhibition, oxime reactivation, and antidotal treatment. VR was highly toxic in guinea pigs, with an s.c. LD 50 of 11.3 μLg/kg. The bimolecular rate constant for inhibition of AChE by VR was 1.4 x 10 8 M -1 min -1 , which is similar to the AChE inhibition rate constants of VX and of other chemical warfare agents whose toxicity is attributed to their inhibition of AChE. Complete in vitro reactivation of VR-inhibited AChE was produced by oximes, but reactivation with the bispyridinium oximes 1-(((4-(aminocarbonyl)pyridino)methoxy methyl)-2-((hydroximino)-methyl)pyridinium dichloride (HI-6), 1-(((4-(aminocarbonyl)pyridino)methoxy)-methyl)-2,4-bis((hydroxyimino)methyl)pyridinium dimethanesulfonate (HLo7), and toxogonin dichloride (TOXO) occurred at a much higher rate than with the monopyridinium oxime 2-pyridine aldoxime methylchloride (2-PAM). The reactivation rates with HLo7, HI-6, and TOXO were, respectively, 21-, 5-, and 3-fold greater than with 2-PAM. The aging of VR-inhibited AChE to an oxime-resistant state occurred slowly with a half-life of 43 h. Treatment of guinea pigs with equimolar (145 μLmol/kg) amounts of HI-6 (55 mg/kg) or 2-PAM (25 mg/kg) in combination with atropine (16 mg/kg) i.m. 1 min after VR exposure produced protective ratios of 43.9 for HI-6 and 6.5 for 2-PAM. Carbamate pretreatment prior to these oxime/atropine treatments caused no increase in their protective ratios against VR. These observations suggest that VR is a highly toxic anticholinesterase agent that can be effectively treated with an appropriate oxime/atropine regimen.

Thermokinetic studies of the irreversible inhibition of single-substrate, enzyme-catalyzed reactions

A thermokinetic ratio method for the irreversible inhibition of single-substrate enzymecatalyzed reactions is proposed in this paper. By analyzing a measured curve this method can be used to calculate the apparent rate constant of the inhibited reaction without letting the reaction go to completion. Using the LKB-2107 batch microcalorimeter, the arginase-catalyzed hydrolysis of L-arginine in the presence of p-chloromercuribenzoate (PCMB) has been studied and PCMB established as an irreversible mixed inhibitor. The second-order rate constants for inhibition of arginase by PCMB in the absence and presence of L-arginine have been determined by this ratio method to be k EI = 94.4 M − s −1 and k ESI = 35.2 M −1 s −1, respectively, at 298.15 K. Chemical modification with PCMB indicates that arginase contains three reactive cysteinyl residues at most but these residues are not present at the active site of arginase.

Measurement of individual rate constants of irreversible inhibition of a cysteine proteinase by an epoxysuccinyl inhibitor

The apparent binding constant (K i) and the first order rate constant of inactivation (k 3) have been obtained for EP-475 inhibition of papain at partially saturating concentrations of inhibitor and various substrate concentrations by global analysis of the data. The value of k 3 (k 3 = 0.71 ± 0.25 s −1) is larger whereas K i (2.4 ± 0.91 μM) is comparable to or higher than those values for most nucleofuge methylketone inhibitors.

Selective activation of heparin cofactor II by a sulfated polysaccharide isolated from the leaves of Artemisia princeps

While checking anticoagulant activities in crude fractions from Wakan-Yakus (traditional herbal drugs), we detected antithrombin activity in the polysaccharide fraction of the leaves of Artemisia princeps Pamp. A sulfated polysaccharide purified from the crude fractions by ion-exchange chromatography on DEAE-cellulose and gel filtration on Sepharose 6B potentiated the heparin cofactor II (HC II)-dependent antithrombin activity but not the antithrombin activity of antithrombin III (AT III). The polysaccharide enhanced the HC II-thrombin reaction more than 6000-fold. The apparent second-order rate constant of thrombin inhibition by HC II increased from 3.8 x 10(4) (in the absence of the polysaccharide) to 2.5 x 10(8) M-1 min-1 in the presence of 25-125 micrograms/ml of the polysaccharide. In human plasma, the polysaccharide accelerated the formation of thrombin-HC II complex. The stimulating effect on HC II-dependent antithrombin activity was almost totally abolished by treatment with chondroitinase AC I, heparinase or heparitinase, while chondroitinase ABC or chondroitinase AC II had little or no effect. These results suggest that the polysaccharide is a glycosaminoglycan-like material with properties that are quite distinct from heparin or dermatan sulfate.

Elimination of glycosylation heterogeneity affecting heparin affinity of recombinant human antithrombin III by expression of a β-like variant in baculovirus-infected insect cells

In order to promote homogeneity of recombinant antithrombin III interactions with heparin, an asparagine-135 to alanine substitution mutant was expressed in baculovirus-infected insect cells. The N135A variant does not bear an N-linked oligosaccharide on residue 135 and is therefore similar to the beta isoform of plasma antithrombin. Purified bv.hat3.N135A is homogeneous with respect to molecular mass, charge and elution from immobilized heparin. Second-order rate constants for thrombin and factor Xa inhibition determined in the absence and presence of heparin are in good agreement with values established for plasma antithrombin and these enzymes. Based on far- and near-UV CD, bv.hat3.N135A has a high degree of conformational similarity to plasma antithrombin. Near-UV CD, absorption difference and fluorescence spectroscopy studies indicate that it also undergoes an identical or very similar conformational change upon heparin binding. The Kds of bv.hat3.N135A for high-affinity heparin and pentasaccharide were determined and are in good agreement with those of the plasma beta-antithrombin isoform. The demonstrated similarity of bv.hat3.N135A and plasma antithrombin interactions with target proteinases and heparins suggest that it will be a useful base molecule for investigating the structural basis of antithrombin III heparin cofactor activity.

Trypanothione reductase from Leishmania donovani. Purification, characterisation and inhibition by trivalent antimonials.

Trypanothione reductase was purified to homogeneity from Leishmania donovani promastigotes transfected with the expression plasmid pTEX-LdTR. The physical, spectral and kinetic properties were found to be similar to those obtained from other pathogenic trypanosomatids. The substrates trypanothione disulfide and NADPH exhibit Michaelis-Menten saturation kinetics with Km values of 36 microM and 9 microM, respectively, the former yielding a kcat/Km of 5.0 x 10(6) M-1 s-1. Like other trypanothione reductases, the leishmania enzyme is unable to use glutathione disulfide as substrate. Both trypanothione reductase and the analogous mammalian enzyme, glutathione reductase, are inhibited by trivalent but not pentavalent anti-leishmanial antimonials. Inhibition by trivalent sodium antimonyl gluconate (Triostam) occurs in a time-dependent manner, with the pseudo-first-order rate constants of inhibition being linearly related to drug concentration. Inhibition proceeds until an apparent equilibrium between active enzyme/free drug and inactive enzyme-drug complex is reached. MelT, an adduct of melarsen oxide and dihydrotrypanothione which is a competitive inhibitor of the disulfide binding site of trypanothione reductase, confers protection against Triostam. Prior reduction of the catalytically active disulfide bridge by NADPH is essential for inhibition. Spectral analysis shows that the broad absorbance band centred on 530 nm, characteristic of the charge-transfer complex in the two-electron-reduced EH2 enzyme, is lost upon addition of Triostam. Further spectral changes resemble those associated with reduction of the FAD prosthetic group to FADH2. Inhibition by Triostam is readily reversed by dilution or addition of the dithiols 2,3-dimercaptopropanol, 2,3-dimercaptosuccinate or dithiothreitol, but not dihydrotrypanothione, suggesting that this trypanosomatid-unique metabolite is unlikely to protect the enzyme from inhibition in whole cells. A mechanism consistent with these observations is proposed.

Trypanothione Reductase from Leishmania donovani. Purification, Characterisation and Inhibition by Trivalent Antimonials

Trypanothione reductase was purified to homogeneity from Leishmania donovani promastigotes transfected with the expression plasmid pTEX-LdTR. The physical, spectral and kinetic properties were found to be similar to those obtained from other pathogenic trypanosomatids. The substrates trypanothione disulfide and NADPH exhibit Michaelis-Menten saturation kinetics with Km values of 36 μM and 9 μM, respectively, the former yielding a kcat/Km of 5.0×106 M−1 s−1. Like other trypanothione reductases, the leishmania enzyme is unable to use glutathione disulfide as substrate. Both trypanothione reductase and the analogous mammalian enzyme, glutathione reductase, are inhibited by trivalent but not pentavalent anti-leishmanial antimonials. Inhibition by trivalent sodium antimonyl gluconate (Triostam) occurs in a time-dependent manner, with the pseudo-first-order rate constants of inhibition being linearly related to drug concentration. Inhibition proceeds until an apparent equilibrium between active enzyme/free drug and inactive enzyme-drug complex is reached. MelT, an adduct of melarsen oxide and dihydrotrypanothione which is a competitive inhibitor of the disulfide binding site of trypanothione reductase, confers protection against Triostam. Prior reduction of the catalytically active disulfide bridge by NADPH is essential for inhibition. Spectral analysis shows that the broad absorbance band centred on 530 nm, characteristic of the charge-transfer complex in the two-electron-reduced EH2 enzyme, is lost upon addition of Triostam. Further spectral changes resemble those associated with reduction of the FAD prosthetic group to FADH2. Inhibition by Triostam is readily reversed by dilution or addition of the dithiols 2,3-dimercaptopropanol, 2,3-dimercaptosuccinate or dithiothreitol, but not dihydrotrypanothione, suggesting that this trypanosomatid-unique metabolite is unlikely to protect the enzyme from inhibition in whole cells. A mechanism consistent with these observations is proposed.

Role of the Thrombin Insertion Loop 144-155. Study of Thrombin Mutations W148G, K154E and a Thrombin-Based Synthetic Peptide

Thrombin is a multifunctional serine protease that plays a critical role in hemostasis. Crystallographic studies revealed that the insertion loop, residues 144-155 (human thrombin B chain numbering) located on the surface of thrombin, might be involved in the access of substrates to the active-site of the enzyme. This loop has also been proposed as a potential candidate for a binding site for thrombomodulin and selected thrombin substrates. In order to examine this hypothesis, we have introduced single amino acid substitutions into the loop 144-155 (W148G, K154E). These point mutations did not result in major changes in thrombin specificity. However, the mutant thrombins presented slight modifications in their catalytic activity on the tripeptidic substrate H-D-Lys-(epsilon-benzyloxycarbonyl)-Pro-Arg-NH-nitroanilide ([K154E]thrombin) or tosyl-Gly-Pro-Arg-NH-nitroanilide ([W148G]thrombin), and in the second-order rate constants of inhibition by antithrombin III ([K154E]thrombin) and ([W148G]thrombin) compared to recombinant wild-type thrombin. Kinetics of fibrinogen hydrolysis were minimally affected by the K154E mutation and were not affected by the W148G mutation. Neither of the mutations affected thrombin interaction with hirudin or its C-terminal tail, protein C activation by thrombin or thrombin-thrombomodulin, or platelet activation. We also examined the properties of a synthetic peptide corresponding to the sequence T147-S158. The synthetic peptide T147-S158 did not inhibit thrombin interaction with fibrin, thrombomodulin or protein C. Together, our results indicate that the thrombin loop 144-155 is indirectly involved in the catalytic function of the enzyme, most probably by limiting the access of the substrates to the catalytic site, and argue against the presence of a recognition exosite for fibrin(ogen), thrombomodulin or platelets within the loop.

Role of the Thrombin Insertion Loop 144-155. Study of Thrombin Mutations W148G, K154E and a Thrombin-Based Synthetic Peptide

Thrombin is a multifunctional serine protease that plays a critical role in hemostasis. Crystallographic studies revealed that the insertion loop, residues 144–155 (human thrombin B chain numbering) located on the surface of thrombin, might be involved in the access of substrates to the active-site of the enzyme. This loop has also been proposed as a potential candidate for a binding site for thrombomodulin and selected thrombin substrates. In order to examine this hypothesis, we have introduced single amino acid substitutions into the loop 144–155 (W148G, K154E). These point mutations did not result in major changes in thrombin specificity. However, the mutant thrombins presented slight modifications in their catalytic activity on the tripeptidic substrate H-d-Lys-(e-benzyloxycarbonyl)-Pro-Arg-NH-nitroanilide {[K154E]thrombin} or tosyl-Gly-Pro-Arg-NH-nitroanilide {[W148G]thrombin}, and in the second-order rate constants of inhibition by antithrombin III {[K154E]thrombin} and {[W148G]thrombin} compared to recombinant wild-type thrombin. Kinetics of fibrinogen hydrolysis were minimally affected by the K154E mutation and were not affected by the W148G mutation. Neither of the mutations affected thrombin interaction with hirudin or its C-terminal tail, protein C activation by thrombin or thrombin-thrombomodulin, or platelet activation. We also examined the properties of a synthetic peptide corresponding to the sequence T147-S158. The synthetic peptide T147-S158 did not inhibit thrombin interaction with fibrin, thrombomodulin or protein C. Together, our results indicate that the thrombin loop 144–155 is indirectly involved in the catalytic function of the enzyme, most probably by limiting the access of the substrates to the catalytic site, and argue against the presence of a recognition exosite for fibrin(ogen), thrombomodulin or platelets within the loop.