Hydrophobic residues 382-386 of antithrombin III, Ala-Ala-Ala-Ser-Thr, serve as the epitope for an antibody which facilitates hydrolysis of the inhibitor by thrombin.

In the presence of a monoclonal antibody raised against the human thrombin-antithrombin III complex, the reaction between thrombin and antithrombin III proceeded to form preferentially a two-chain form of the inhibitor rather than to follow the major pathway of stable acyl complex formation. We thus propose the term "switching antibody" for an antibody that switches the enzyme-inhibitor reaction (Asakura, S., Matsuda, M., Yoshida, N., Terukina, S., and Kihara, H. (1989) J. Biol. Chem. 264, 13736-13739). By analyzing a CNBr fragment of the thrombin-antithrombin III complex that reacts with the antibody we localized the epitope for the antibody to a strongly hydrophobic residue 382-386 peptide segment, Ala-Ala-Ala-Ser-Thr, of the inhibitor, which is also contiguous with a hydrophobic amino acid Ala at its carboxyl terminus. This particular region should be cryptic in nascent antithrombin III, but could have been exposed to provide the reactive site for the antibody at an early stage of the reaction. Thereby a conformational change may have been induced at or near the reactive site of the complex, facilitating hydrolysis of the inhibitor by the enzyme. Interestingly, this hydrophobic region is highly conserved among members of the serpin family.     

Inhibition of bovine factor IXa and factor Xabeta by antithrombin III.

Factor IXa and factor Xabeta are serine proteases which participate in the middle phase of blood coagulation. These two enzymes are inhibited by antithrombin III by the formation of an enzyme-inhibitor complex containing 1 mol of enzyme and 1 mol of antithrombin III. The complex was readily demonstrated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and loss of coagulant or esterase activity at increasing concentrations of inhibitor. The inactivation of factor IXa by antithrombin III was relatively slow, but the reaction was greatly accelerated by the addition of heparin.     

Interaction and thermal stability of fluorescent labeled derivatives of thrombin and antithrombin III

Derivatives of human thrombin and antithrombin III with fluorescent labels covalently attached to their carbohydrate moieties were prepared by reaction of periodate-oxidized proteins with amino derivatives of dansyl, fluorescein and pyrene. The labeled derivatives retained full biological activity, including their ability to form stable enzyme-inhibitor complexes, a reaction whose rate could be monitored by the increase in fluorescence polarization. When the dansyl-labeled derivatives were heated, they exhibited sigmoidal increases in polarization with midpoints near 50 degrees C for thrombin and 60 degrees C for antithrombin III. By contrast, a complex between antithrombin III and dansyl-thrombin showed no change in polarization up to 70 degrees C, suggesting that the individual components are more stable in the complex. These studies show that fluorescent labels attached to carbohydrate moieties of glycoproteins provide convenient probes for monitoring conformational changes and protein-protein interactions with minimum interference by the probe.     

The active site of antithrombin. Release of the same proteolytically cleaved form of the inhibitor from complexes with factor IXa, factor Xa, and thrombin

Reactions between near equimolar amounts of antithrombin and Factors IXa or Xa resulted in the formation of a free proteolytically modified, two-chain form of the inhibitor, in addition to the inactive antithrombin-protease complexes. The modified inhibitor produced by either enzyme was electrophoretically identical with that formed in the reaction with thrombin. As in the latter reaction, the formation of the modified antithrombin by Factor Xa was increased in the presence of heparin, while only small amounts were produced by Factor IXa both in the absence and presence of the polysaccharide. NH2-terminal sequence analyses of the isolated modified inhibitor formed by Factor Xa showed that a single Arg-Ser bond in the COOH-terminal end of the inhibitor had been cleaved. This cleavage site is identical with that identified in free thrombin-modified antithrombin. The purified antithrombin-Factor IXa and antithrombin-Factor Xa complexes were dissociated by ammonia or hydroxylamine into free enzyme and a modified two-chain form of the inhibitor. Electrophoresis studies and NH2-terminal sequence analyses showed that the modified antithrombin obtained from either complex was identical with that produced in free form by the two enzymes and also with the modified inhibitor that is released from the antithrombin-thrombin complex. The fact that identical results were obtained for the reactions between antithrombin and three enzymes with different specificities strongly suggests that the observed Arg-Ser cleavage site is the active site of antithrombin.     

Reaction of porcine pancreatic elastase with 7-substituted 3-alkoxy-4-chloroisocoumarins: design of potent inhibitors using the crystal structure of the complex formed with 4-chloro-3-ethoxy-7-guanidinoisocoumarin

The crystal structure of the acyl enzyme formed upon inhibition of porcine pancreatic elastase (PPE) by 4-chloro-3-ethoxy-7-guanidinoisocoumarin has been determined at a 1.85-A effective resolution. The chlorine atom is still present in this acyl enzyme, in contrast to the previously reported structure of the 7-amino-4-chloro-3-methoxyisocoumarin-PPE complex where the chlorine atom has been replaced by an acetoxy group. The guanidino group forms hydrogen bonds with the carbonyl group and side-chain hydroxyl group of Thr-41, and the acyl carbonyl group has been twisted out of the oxyanion hole. Molecular modeling indicates that the orientation of the initial Michaelis enzyme-inhibitor complex is quite different from that of the acyl enzyme since simple reconstruction of the isocoumarin ring would result in unfavorable interactions with Ser-195 and His-57. Molecular models were used to design a series of new 7-(alkylureido)- and 7-(alkylthioureido)-substituted derivatives of 3-alkoxy-7-amino-4-chloroisocoumarin as PPE inhibitors. All the 3-ethoxyisocoumarins were better inhibitors than those in the 3-methoxy series due to better interactions with the S1 pocket of PPE. The best ureido inhibitor also contained a tert-butylureido group at the 7-position of the isocoumarin. Due to a predicted interaction with a small hydrophobic pocket on the surface of PPE, this isocoumarin and a related phenylthioureido derivative are among the best irreversible inhibitors thus far reported for PPE (kobs/[I] = 8100 M-1 s-1 and 12,000 M-1 s-1). Kinetic studies of the stability of enzyme-inhibitor complexes suggest that many isocoumarins are alkylating the active site histidine at pH 7.5 via a quinone imine methide intermediate, while at pH 5.0, the predominant pathway appears to be simple formation of a stable acyl enzyme derivative.     

Hysteretic enzyme response induced by inhibitory antibodies against human leukocyte elastase

Polyclonal antibodies raised in rabbits against human leukocyte elastase contained two distinct populations of enzyme-inhibiting immunoglobulins. The enzyme-catalyzed reaction in the presence of antibodies (both IgG or monovalent Fab fragments) showed a transient state lasting up to several minutes depending on the inhibitor and substrate concentrations, which was followed by a linear steady-state. The transient was a concave upward or concave downward lag phase depending on whether the enzyme had been preincubated with the antibodies or not, respectively. The kinetic analysis of reaction progress curves showed that both antibody populations were slow inhibitors, which completely and reversibly excluded the substrate from binding to the enzyme. For both antibody populations, the formation of the enzyme-inhibitor complex was characterized by an initial rapid interaction followed by a slow isomerization to a catalytically inactive complex. The apparent pseudo first-order rate constant of the transient slow phase was a hyperbolic function of the inhibitor concentration for both antibodies, from which relevant kinetic constants and the half times for enzyme inactivation could be calculated. For instance, with a total antibody concentration of 1 mg/ml (as IgG), leukocyte elastase was inactivated with t1/2 = 0.31s and 24.8s by the faster and the slower of the two antibodies, respectively. It is suggested that the hysteretic response of the enzyme to the inhibitory action of its antibodies may be due to a kind of memory of the antibody molecule for a special inactive enzyme conformation resulting from inhibition by proteinase inhibitors during the immunization procedure. In turn, the purified antibodies would be able to reversibly induce a slow transition of the enzyme molecule from an active to a substrate-excluding conformation ("induced misfit").     

Preparation and characterization of monolconal antibodies against the human thrombin-antithrombin III complex

Monoclonal antibodies were raised against human thrombin-antithrombin III complex by a hybridoma technique. Among them, five monoclonal antibodies, designated as JITAT-4, -14, -16, -17 and -19, were found to react with thrombin-antithrombin III, but not with its nascent components, α-thrombin or antithrombin III. Their respective immunoglobulin classes are IgG₁ for JITAT-16 and -19, and IgG_2a for JITAT-4, -14 and -17. Besides the thrombin-antithrombin III complex, they all bound to the Factor Xa-antithrombin III complex and the active-site-cleaved two-chain antithrombin III as well. Moreover, the reactivity of these two antibodies to the neoantigens was not affected by heparin, suggesting that their epitopes are independent of heparin-induced conformational changes of antithrombin III. Two of them, JITAT-16 and -17, were categorized as high-affinity antibodies to thrombin-antithrombin III complex, the dissociation constants being 6.7 nM and 4.8 nM, respectively. However, they do not share antigenic determinants. These monoclonal antibodies may allow us to explore more precisely the reaction between antithrombin III and thrombin or its related enzymes.     

S protein modulates the heparin-catalyzed inhibition of thrombin by antithrombin III. Evidence for a direct interaction of S protein with heparin

The interference of S protein with the heparin-catalyzed inhibition of thrombin by antithrombin III was studied in a purified system and in plasma. The effect of S protein to counteract heparin activity was documented by kinetic analysis of the initial phase of the inhibition reaction. Addition of S protein induced a concentration-dependent reduction of the inhibition rate, reflected in a decrease of the apparent pseudo-first-order rate constant by a factor of 5-8 in the presence of a twofold molar excess of S protein over antithrombin III. A non-competitive interaction of S protein with the thrombin--antithrombin-III--heparin inhibition reaction with Ki = 0.6 microM was found. While the association constant of thrombin--antithrombin III in the presence of 0.05 U/ml heparin amounted to 2.5 X 10(8) M-1, an approximately 200-fold decrease of this value was observed in the presence of S protein. The fast formation of the covalent complex between thrombin and antithrombin III in the presence of heparin was impaired as a result of the presence of S protein, as was shown by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. In the absence of heparin the inhibition of thrombin by antithrombin III alone was not influenced by S protein. The heparin-counteracting activity of S protein was found to be mainly expressed in the range of 0.01-0.1 U/ml heparin, thereby shifting the point of 50% inhibition of thrombin from 0.003 U/ml to 0.1 U/ml heparin with a second-order rate constant of k2 = 1.4 X 10(6) M-1. A direct interaction of S protein with heparin was demonstrated by crossed immunoelectrophoresis with purified proteins as well as in plasma and serum. The analysis of plasma and serum by crossed immunoelectrophoresis against rabbit anti-(human S protein) serum revealed an additional cathodal peak in the serum sample, resulting from the interaction of S protein with serum components. These findings not only indicate a direct interaction of S protein with heparin in the onset of the inhibition of thrombin by antithrombin-III--heparin, but also a contribution of S protein during enzyme-inhibitor complex formation.     

Crystal structure of a substrate complex of Mycobacterium tuberculosis beta-ketoacyl-acyl carrier protein synthase III (FabH) with lauroyl-coenzyme A

Beta-ketoacyl-acyl carrier protein synthase III (FabH) catalyzes a two step reaction that initiates the pathway of fatty acid biosynthesis in plants and bacteria. In Mycobacterium tuberculosis, FabH catalyzes extension of lauroyl, myristoyl and palmitoyl groups from which cell wall mycolic acids of the bacterium are formed. The first step of the reaction is an acyl group transfer from acyl-coenzyme A to the active-site cysteine of the enzyme; the second step is acyl chain extension by two carbon atoms through Claisen condensation with malonyl-acyl carrier protein. We have previously determined the crystal structure of a type II, dissociated M.tuberculosis FabH, which catalyzes extension of lauroyl, myristoyl and palmitoyl groups. Here we describe the first long-chain Michaelis substrate complex of a FabH, that of lauroyl-coenzyme A with a catalytically disabled Cys-->Ala mutant of M.tuberculosis FabH. An elongated channel extending from the mutated active-site cysteine defines the acyl group binding locus that confers unique acyl substrate specificity on M.tuberculosis FabH. CoA lies in a second channel, bound primarily through interactions of its nucleotide group at the enzyme surface. The apparent weak association of CoA in this complex may play a role in the binding and dissociation of long chain acyl-CoA substrates and products and poses questions pertinent to the mechanism of this enzyme.     

Sterospecific selection of nucleophilic compounds in the chymotrypsin-catalyzed formation of the peptide bonds]

The course of stereospecific selection of nucleophilic compounds was studied in the reaction of acyl-enzymes interaction with razemic substrate-like nucleophiles, e.g. amino acid esters, by measuring optical rotation or incorporation of labelled D-compounds. It was shown that the acyl-enzymes are not responsible for the stereospecific selection of substrate-like nucleophiles. Since stereospecific selection of nucleophiles occurs in some chymotrypsin-catalyzed reactions, such selection may be produced by chymotrypsin till the formation of an acyl-enzyme compound with the substrate at the enzyme-inhibitor stage (or the Michaelis complex) with nucleophilic compounds. Even under the optimal conditions no absolute stereospecific selection of nucleophiles occurred, as was observed in case of a substrate (a donor of the acyl amino acid residue), undergoing degradation. An essential role of a specific site of nucleophile binding in the reactions of chymotrypsin-catalyzed peptide bond formation, is emphasized.     

Binding of thrombin to thrombomodulin accelerates inhibition of the enzyme by antithrombin III. Evidence for a heparin-independent mechanism

The endothelial cell surface provides a receptor for thrombin-designated thrombomodulin (TM) which regulates thrombin formation and the activity of the enzyme at the vessel wall surface by serving as a potent cofactor for the activation of protein C by thrombin. Heparin-like structures of the vessel wall have been proposed as another regulatory mechanism catalyzing the inhibition of thrombin by antithrombin III. In the present study, the interaction of antithrombin III with the thrombin-TM complex and its interference with heparin and polycations were investigated by using human components and TM isolated from the microvasculature of rabbit lung. Purified TM bound thrombin and acted as a cofactor for protein C activation. The addition of heparin (0.5 unit/mL) to the reaction mixture interfered neither with the binding of thrombin to TM nor with the activation of protein C. However, the polycations protamine (1 unit/mL) as well as polybrene (0.1 mg/mL) affected the thrombin-TM interaction. This was documented by an increase in the Michaelis constant from 8.3 microM for thrombin alone to 19.5 microM for thrombin-TM with the chromogenic substrate compound S-2238 in the presence of 1 unit/mL protamine. When the inhibition of thrombin by antithrombin III was determined, the second-order rate constant k2 = 8.4 X 10(3) M-1 s-1 increased about 8-fold in the presence of TM, implying an accelerative function of TM in this reaction. Although purified TM did not bind to antithrombin III-Sepharose, suggesting the absence of heparin-like structures within the receptor molecule, protamine reversed the accelerative effect of TM in the inhibition reaction.(ABSTRACT TRUNCATED AT 250 WORDS)     

Catalytic pathways of Euphorbia characias peroxidase reacting with hydrogen peroxide

The reaction of Euphorbia characias latex peroxidase (ELP) with hydrogen peroxide as the sole substrate was studied by conventional and stopped-flow spectrophotometry. The reaction mechanism occurs via three distinct pathways. In the first (pathway I), ELP shows catalase-like activity: H2O2 oxidizes the native enzyme to compound I and subsequently acts as a reducing substrate, again converting compound I to the resting ferric enzyme. In the presence of an excess of hydrogen peroxide, compound I is still formed and further reacts in two other pathways. In pathway II, compound I initiates a series of cyclic reactions leading to the formation of compound II and compound III, and then returns to the native resting state. In pathway III, the enzyme is inactivated and compound I is converted into a bleached inactive species; this reaction proceeds faster in samples illuminated with bright white light, demonstrating that at least one of the intermediates is photosensitive. Calcium ions decrease the rate of pathway I and accelerate the rate of pathways II and III. Moreover, in the presence of calcium the inactive stable verdohemochrome P670 species accumulates. Thus, Ca2+ ions seem to be the key for all catalytic pathways of Euphorbia peroxidase.     

Quantitative structure-activity relationships for the pre-steady state acetylcholinesterase inhibition by carbamates

4-Nitrophenyl-N-substituted carbamates (1) are characterized as pseudosubstrate inhibitors of acetylcholinesterase. The first step is formation of the enzyme-inhibitor tetrahedral intermediate with the inhibition constant (Ki), the second step is formation of the carbamyl enzyme with the carbamylation constant (kc), and the third step is hydrolysis of the carbamyl enzyme with decarbamylation constant (kd). According to pre-steady state kinetics the Ki step is divided further into two steps: (1) formation of the enzyme-inhibitor complex with the dissociation constant (KS) and (2) formation of the enzyme-inhibitor tetrahedral intermediate from the complex with the equilibrium constant (k2/k-2). Since the inhibitors are protonated in pH 7.0 buffer solution, the virtual dissociation constant (KS') of the enzyme-protonated inhibitor complex can be calculated from the equation, -log KS'=-log KS-pKa + 14. The -logKS, -log KS', log k2, and log k-2 values are multiply linearly correlated with the Jave equation (log(k/k0)=rho*sigma* + deltaEs + psi pi). For -log KS'-sigma*-Es)pi-correlation, the rho* value of -0.4 indicates that the enzyme-protonated inhibitor complexes have more positive charges than the protonated inhibitors, the delta value of 0.44 suggests that the bulkily substituted inhibitors lessen the reaction due to the difficulty of the inhibitors to enter the narrow enzyme active site gorge, and the psi value of 0.27 implies that the inhibitors with hydrophobic substituents accelerate the inhibitors entering the active site gorge of the enzyme. For log k2/k-2,-sigma*-Es-pi-correlation, the rho* value of 1.1 indicates that the enzyme-protonated inhibitor tetrahedral intermediates have more negative charges than the enzyme-protonated inhibitor complexes, the delta value of 0.15 suggests that the bulkily substituted inhibitors are difficult to bind into a small acyl binding site of the enzyme, and the psi value of -0.3 implies that the inhibitors with hydrophobic substituents resist binding to the hydrophilic acyl binding site of the enzyme.     

Inactivation of bacterial DD-peptidase by beta-sultams

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

Initiation of the extrinsic pathway of blood coagulation: evidence for the tissue factor dependent autoactivation of human coagulation factor VII

Previous studies demonstrated proteolytic activation of human blood coagulation factor VII by an unidentified protease following complex formation with tissue factor expressed on the surface of a human bladder carcinoma cell line (J82). In the present study, an active-site mutant human factor VII cDNA (Ser344----Ala) has been constructed, subcloned, and expressed in baby hamster kidney cells. Mutant factor VII was purified to homogeneity in a single step from serum-free culture supernatants by immunoaffinity column chromatography. Mutant factor VII was fully carboxylated, possessed no apparent clotting activity, and was indistinguishable from plasma factor VII by SDS-PAGE. Cell binding studies indicated that mutant factor VII bound to J82 tissue factor with essentially the same affinity as plasma factor VII and was cleaved by factor Xa at the same rate as plasma factor VII. In contrast to radiolabeled single-chain plasma factor VII that was progressively converted to two-chain factor VIIa on J82 monolayers, mutant factor VII was not cleaved following complex formation with J82 tissue factor. Incubation of radiolabeled mutant factor VII with J82 cells in the presence of recombinant factor VIIa resulted in the time-dependent and tissue factor dependent conversion of single-chain mutant factor VII to two-chain mutant factor VIIa. Plasma levels of antithrombin III had no discernible effect on the factor VIIa catalyzed activation of factor VII on J82 cell-surface tissue factor but completely blocked this reaction catalyzed by factor Xa. These results are consistent with an autocatalytic mechanism of factor VII activation following complex formation with cell-surface tissue factor, which may play an important role in the initiation of extrinsic coagulation in normal hemostasis.     

Mechanism-based inactivation of human leukocyte elastase via an enzyme-induced sulfonamide fragmentation process

We describe herein the design and in vitro biochemical evaluation of a novel class of mechanism-based inhibitors of human leukocyte elastase (HLE) that inactivate the enzyme via an unprecedented enzyme-induced sulfonamide fragmentation cascade. The inhibitors incorporate in their structure an appropriately functionalized saccharin scaffold. Furthermore, the inactivation of the enzyme by these inhibitors was found to be time-dependent and to involve the active site. Biochemical, HPLC, and mass spectrometric studies show that the interaction of these inhibitors with HLE results in the formation of a stable acyl complex and is accompanied by the release of (L) phenylalanine methyl ester. The data are consistent with initial formation of a Michaelis-Menten complex and subsequent formation of a tetrahedral intermediate with the active site serine (Ser(195)). Collapse of the tetrahedral intermediate with tandem fragmentation results in the formation of a highly reactive conjugated sulfonyl imine which can either react with water to form a stable acyl enzyme and/or undergo a Michael addition reaction with an active site nucleophilic residue (His(57)). It is also demonstrated herein that this class of compounds can be used in the design of inhibitors of serine proteases having either a neutral or basic primary substrate specificity. Thus, the results suggest that these inhibitors constitute a potential general class of mechanism-based inhibitors of (chymo)trypsin-like serine proteases.     

Mechanism of the anticoagulant action of heparin

Summary The anticoagulant effect of heparin, a sulfated glycosaminoglycan produced by mast cells, requires the participation of the plasma protease inhibitor antithrombin, also called heparin cofactor. Antithrombin inhibits coagulation proteases by forming equimolar, stable complexes with the enzymes. The formation of these complexes involves the attack by the enzyme of a specific Arg-Ser bond in the carboxy-terminal region of the inhibitor. The complexes so formed are not dissociated by denaturing solvents, which indicates that a covalent bond may contribute to their stability. This bond may be an acyl bond between the active-site serine of the enzyme and the arginine of the cleaved reactive bond of the inhibitor. However, the native complexes dissociate slowly at near-neutral pH into free enzyme and a modified inhibitor, cleaved at the reactive bond. So, antithrombin apparently functions as a pseudo-substrate that traps the enzyme in a kinetically stable complex.The reactions between antithrombin and coagulation proteases are slow in the absence of heparin. However, optimal amounts of heparin accelerate these reactions up to 2 000-fold, thereby efficiently preventing the formation of fibrin in blood. The accelerating effect, and thus the anticoagulant activity, is shown by only about one-third of the molecules in all heparin preparations, while the remaining molecules are almost inactive. The highly active molecules bind tightly to antithrombin, i.e. with a binding constant of slightly below 10⁸ M^–1 at physiological ionic strength, while the relatively inactive molecules bind about a thousand-fold more weakly. The binding of the high-affinity heparin to antithrombin is accompanied by a conformational change in the inhibitor that is detectable by spectroscopic and kinetic methods. This conformational change follows an initial, weak binding of heparin to antithrombin and causes the tight interaction between polysaccharide and inhibitor that is prerequisite to heparin anticoagulant activity. It has also been postulated that the conformational change leads to a more favourable exposure of the reactive site of antithrombin, thereby allowing the rapid interaction with the proteases.Heparin also binds to the coagulation proteases. Recent studies indicate that this binding is weaker and less specific that the binding to antithrombin. Nevertheless, for some enzymes, thrombin, Factor IX_a and Factor XI_a, an interaction between heparin and the protease, in addition to that between the polysaccharide and antithrombin; apparently is involved in the accelerated inhibition of the enzymes. The effect of this interaction may be to approximate enzyme with inhibitor in an appropriate manner. However, the bulk of the evidence available indicates that binding of heparin to the protease alone cannot be responsible for the accelerating effect of the polysaccharide on the antithrombin-protease reaction.Heparin acts as a catalyst in the antithrombin-protease reaction, i.e. it accelerates the reaction in non-stoichiometric amounts and is not consumed during the reaction. This ability can be explained by heparin being released from the antithrombin-protease complex for renewed binding to antithrombin, once the complex has been formed. Such a decresed affinity of heparin for the antithrombin complex, compared to the affinity for antithrombin alone, has been demonstrated.The structure of the antithrombin-binding region in heparin has been investigated following the isolation of oligosaccharides with high affinity for antithrombin. The smallest such oligosaccharide, an octasaccharide, obtained after partial random depolymerization of heparin with nitrous acid, was found to contain a unique glucosamine-3-O-sulfate group, which could not be detected in other portions of the high affinity heparin molecule and which was absent in heparin with low affinity for antithrombin. The actual antithrombin-binding region within this octasaccharide molecule has been identified as a pentasaccharide sequence with he predominant structure: N-acetyl-D-glucosamine(6-O-SO₃)D-glucoronic acidD-glucosamine(N-SO₃;3,6-di-O-SO₃)L-iduronic acid(2-O-SO₃)D-glucosamine(N-SO₃;6-O-SO₃). In addition to the 3-O-sulfate group, both N-sulfate groups as well as the 6-O-sulfate group of the N-acetylated glucosamine unit appear to be essential for the interaction with antithrombin. The remarkably constant structure of this sequence, as compared to other regions of the heparin molecule, suggests a strictly regulated mechanism of biosynthesis.The ability of heparin to potentiate the inhibition of blood coagulation by antithrombin generally decreases with decreasing molecular weight of the polysaccharide. However, individual coagulation enzymes differ markedly with regard to this molecular-weight dependence. Oligosaccharides in the extreme low-molecular weight range, i.e. octa- to dodecasaccharides, with high affinity for antithrombin have high anti-Factor X_a-activity but are virtually unable to potentiate the inhibition of thrombin. Furthermore, such oligosaccharides are ineffective in preventing experimentally induced venous thrombosis in rabbits. Slightly larger oligosaccharides, containing 16 to 18 monosaccharide residues, show significant anti-thrombin as well as antithrombotic activities, yet have little effect on overall blood coagulation. These findings indicate that the affinity of a heparin fragment for antithrombin is not in itself a measure of the ability to prevent venous thrombo-genesis, and that the anti-Factor X_a activity of heparin is only a partial expression of its therapeutic potential as an antithrombotic agent.The biological role of the interaction between heparin and antithrombin is unclear. In addition to a possible function in the regulation of hemostasis, endogenous heparin may serve as a regulator of extravascular serine proteinases. Mouse peritoneal macrophages have been found to synthesize all the enzymes that constitute the extrinsic pathway of coagulation. Moreover, tissue thromboplastin is produced by these cells in response to a functional interaction with activated T-lymphocytes. The inhibition of this extravascular coagulation system by heparin, released from mast cells, may be potentially important in modulating inflammatory reactions.     

Reaction pathway for inhibition of blood coagulation factor Xa by tick anticoagulant peptide.

The reaction pathway for inhibition of human factor Xa (fXa) by recombinant tick anticoagulant peptide (rTAP) was studied by stopped-flow fluorometry. In the presence of the fluorogenic substrate N-tert-butyloxycarbonyl-L-isoleucyl-L-glutamylglycyl-L-arginyl-7-amido-4 - methylcoumarin (B-IEGR-AMC) and under pseudo-first-order conditions, inhibition appears to occur via a two-step process. Initially, a weak enzyme-inhibitor complex forms with a dissociation constant (Ki) of 68 +/- 6 microM. The initial complex then rearranges to a more stable fXa-rTAP complex with a rate constant (k2) of 123 +/- 5 s-1. The apparent second-order rate constant (k2/Ki) describing formation of the stable complex is (1.8 +/- 0.2) x 10(6) M-1 s-1. Studies of the reaction of rTAP with fXa in the presence of the fluorescent active-site probe p-amino-benzamidine (P) revealed a reaction pathway wherein rTAP initially binds to the fXa-P complex in a two-step process prior to displacing P from the active site. These results indicate that rTAP can bind fXa via a site distinct from the active site (an exosite). The subsequent displacement of P from the active site of fXa by rTAP exhibits a dependence on the concentration of P, indicating that rTAP is locked into the active site in a third step.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mechanism of Gly-Pro-pNA cleavage catalyzed by dipeptidyl peptidase-IV and its inhibition by saxagliptin (BMS-477118)

Dipeptidyl peptidase-IV (DPP-IV) is a serine protease with a signature Asp-His-Ser motif at the active site. Our pH data suggest that Gly-Pro-pNA cleavage catalyzed by DPP-IV is facilitated by an ionization of a residue with a pK of 7.2 +/- 0.1. By analogy to other serine proteases this pK is suggestive of His-Asp assisted Ser addition to the P1 carbonyl carbon of the substrate to form a tetrahedral intermediate. Solvent kinetic isotope effect studies yielded a D2Okcat/Km=2.9+/-0.2 and a D2Okcat=1.7+/-0.2 suggesting that kinetically significant proton transfers contribute to rate limitation during acyl intermediate formation (leaving group release) and hydrolysis. A "burst" of product release during pre steady-state Gly-Pro-pNA cleavage indicated rate limitation in the deacylation half-reaction. Nevertheless, the amplitude of the burst exceeded the enzyme concentration significantly (approximately 15-fold), which is consistent with a branching deacylation step. All of these data allowed us to better understand DPP-IV inhibition by saxagliptin (BMS-477118). We propose a two-step inhibition mechanism wherein an initial encounter complex is followed by covalent intermediate formation. Final inhibitory complex assembly (kon) depends upon the ionization of an enzyme residue with a pK of 6.2 +/- 0.1, and we assigned it to the catalytic His-Asp pair which enhances Ser nucleophilicity for covalent addition. An ionization with a pK of 7.9 +/- 0.2 likely reflects the P2 terminal amine of the inhibitor hydrogen bonding to Glu205/Glu206 in the enzyme active site. The formation of the covalent enzyme-inhibitor complex was reversible and dissociated with a koff of (5.5 +/- 0.4) x 10(-5) s(-1), thus yielding a Ki* (as koff/kon) of 0.35 nM, which is in good agreement with the value of 0.6 nM obtained from steady-state inhibition studies. Proton NMR spectra of DPP-IV showed a downfield resonance at 16.1 ppm. Two additional peaks in the 1H NMR spectra at 17.4 and 14.1 ppm were observed upon mixing the enzyme with saxagliptin. Fractionation factors (phi) of 0.6 and 0.5 for the 17.4 and 14.1 ppm peaks, respectively, are suggestive of short strong hydrogen bonds in the enzyme-inhibitor complex.     

Antithrombin III regulates complement activity in vitro

Heparin, a polyion, exerts its main activity to inhibit coagulation through a serine protease inhibitor, antithrombin III. Previous studies have clearly shown that heparin in the absence of antithrombin III also has the capacity to regulate C activity. The present studies examined the ability of purified human antithrombin III to regulate classical and alternative pathways of C, alone and in the presence of heparin. Antithrombin III alone inhibited generation of both pathways in a dose-related manner; antithrombin III at 8 micrograms/10(7) cellular intermediates inhibited generation of the classical and alternative pathway convertases by 60 and 42%, respectively. Antithrombin III and heparin augmented each other's capacity to inhibit generation of both convertases in a dose-related manner. Antithrombin III did not appear to inhibit on the basis of charge because it is only slightly anionic (isoelectric pH value, 5.0); instead, antithrombin III may have acted as a serine protease inhibitor of the proteolytic enzymes of the C cascades. Antithrombin III acted only to inhibit formation of the alternative pathway convertase but had no activity on terminal lysis by this pathway; similarly, antithrombin III inhibited preformed EAC1,4b,2a,3b but had no activity on classical pathway cellular intermediates containing additional components. Finally, antithrombin III inhibited consumption of factor B hemolytic activity in a reaction mixture that also contained factor D and C3b, suggesting that factor D activity was also inhibited. These studies demonstrate the capacity of antithrombin III to regulate C and suggest that, in concert with heparin, antithrombin III may play an important role in the regulation of C in vivo.