PubMed Health⌕ Search

Biomedical subjects

I Björk

Publications and source records attributed to I Björk.

At least 19 recordsLinked to original sources
PubMed2000

The high stability of cruzipain against pH-induced inactivation is not dependent on its C-terminal domain.

Unlike mammalian lysosomal cysteine proteases, the trypanosomal cysteine protease cruzipain contains a 130-amino acid residue C-terminal domain, in addition to the catalytic domain, and it is stable at neutral pH. The endogenous (with C-terminal domain) and recombinant (without C-terminal domain) cruzipains exhibit similar stabilities at both acid (k(inac)=3.1x10(-3) s(-1) and 4.4x10(-3) s(-1) at pH 2.75 for endogenous and recombinant cruzipain, respectively) and alkaline pH (k(inac)=3.0x10(-3) s(-1) and 3. 7x10(-3) s(-1) at pH 9.15 for endogenous and recombinant cruzipain, respectively). The pH-induced inactivation, which is a highly pH dependent first order process, is irreversible and accompanied by significant changes of secondary and tertiary structure as revealed by circular dichroism measurements. The different stability of cruzipain as compared to related proteases, is therefore due mainly to the different number, nature and distribution of charged residues within the catalytic domain and not due to addition of the C-terminal domain.

Animals↗
PubMed1999

Importance of the second binding loop and the C-terminal end of cystatin B (stefin B) for inhibition of cysteine proteinases.

The importance of residues in the second hairpin loop and the C-terminal end of mammalian cystatin B for binding of proteinases was elucidated by mutagenesis of the bovine inhibitor. Bovine cystatin B was modeled onto the crystal structure of the human inhibitor in complex with papain with minimal structural changes. Substitution of the two deduced contact residues in the second hairpin loop, Leu-73 and His-75, with Gly resulted in appreciably reduced affinities for papain and cathepsins H and B. These losses indicated that the two residues together contribute 20-30% of the free energy of binding of cystatin B to these enzymes and that Leu-73 is responsible for most of this contribution. In contrast, the small decrease in the affinity for cathepsin L suggested that the second hairpin loop is less important for inhibition of this proteinase. Replacement of the contact residue in the C-terminal end, Tyr-97, with Ala resulted in losses in affinity for papain and cathepsins L and H that were consistent with Tyr-97 contributing 6-12% of the energy of binding of cystatin B to these enzymes. However, this substitution minimally affected the affinity for cathepsin B, indicating that the C-terminal end is of limited importance for binding of this proteinase. All affinity decreases were due predominantly to increased dissociation rate constants. These results show that both the second hairpin loop and the C-terminal end of cystatin B contribute to anchoring the inhibitor to target proteinases, each of the two regions interacting with a different domain of the enzyme. However, the relative contributions of these two interactions vary with the proteinase.

Amino Acid Sequence↗
PubMed1999

The role of Arg46 and Arg47 of antithrombin in heparin binding.

Heparin greatly accelerates the reaction between antithrombin and its target proteinases, thrombin and factor Xa, by virtue of a specific pentasaccharide sequence of heparin binding to antithrombin. The binding occurs in two steps, an initial weak interaction inducing a conformational change of antithrombin that increases the affinity for heparin and activates the inhibitor. Arg46 and Arg47 of antithrombin have been implicated in heparin binding by studies of natural and recombinant variants and by the crystal structure of a pentasaccharide-antithrombin complex. We have mutated these two residues to Ala or His to determine their role in the heparin-binding mechanism. The dissociation constants for the binding of both full-length heparin and pentasaccharide to the R46A and R47H variants were increased 3-4-fold and 20-30-fold, respectively, at pH 7.4. Arg46 thus contributes only little to the binding, whereas Arg47 is of appreciable importance. The ionic strength dependence of the dissociation constant for pentasaccharide binding to the R47H variant showed that the decrease in affinity was due to the loss of both one charge interaction and nonionic interactions. Rapid-kinetics studies further revealed that the affinity loss was caused by both a somewhat lower forward rate constant and a greater reverse rate constant of the conformational change step, while the affinity of the initial binding step was unaffected. Arg47 is thus not involved in the initial weak binding of heparin to antithrombin but is important for the heparin-induced conformational change. These results are in agreement with a previously proposed model, in which an initial low-affinity binding of the nonreducing-end trisaccharide of the heparin pentasaccharide induces the antithrombin conformational change. This change positions Arg47 and other residues for optimal interaction with the reducing-end disaccharide, thereby locking the inhibitor in the activated state.

Amino Acid Substitution↗
PubMed1999

A pre-steady-state kinetic analysis of substrate binding to human recombinant deoxycytidine kinase: a model for nucleoside kinase action.

Deoxycytidine kinase (dCK) is an enzyme with broad substrate specificity which can phosphorylate pyrimidine and purine deoxynucleosides, including important antiviral and cytostatic agents. In this study, stopped-flow experiments were used to monitor intrinsic fluorescence changes induced upon binding of various phosphate donors (ATP, UTP, and the nonhydrolyzable analogue AMP-PNP) and the acceptor dCyd to recombinant dCK. Monophasic kinetics were observed throughout. The nucleotides as well as dCyd bound to the enzyme by a two-step mechanism, involving a rapid initial equilibrium step, followed by a protein conformational change that is responsible for the fluorescence change. The bimolecular association rate constants for nucleotide binding [(4-10) x 10(3) M-1 s-1] were 2-3 orders of magnitude lower than those for dCyd binding [(1.3-1.5 x 10(6) M-1 s-1]. This difference most likely is due predominantly to the large difference in the forward rate constants of the conformational changes (0.04-0.26 s-1 vs 560-710 s-1). Whereas the kinetics of the binding of ATP, UTP, and AMP-PNP to dCK showed some differences, UTP exhibiting the tightest binding, no significant differences were observed for the binding of dCyd to dCK in the presence or absence of phosphate donors. However, the binding of dCyd to dCK in the presence of ATP or UTP was accompanied by a 1.5- or 3-fold higher quenching amplitude as compared with dCyd alone or in the presence of AMP-PNP. We conclude that ATP and UTP induce a conformational change in the enzyme, thereby enabling efficient phosphoryl transfer.

Adenosine Triphosphate↗
PubMed1999

The affinity and kinetics of inhibition of cysteine proteinases by intact recombinant bovine cystatin C.

Recent studies have shown that the bovine cysteine proteinase inhibitor, cystatin C, is synthesized as a preprotein containing a 118-residue mature protein. However, the forms of the inhibitor isolated previously from bovine tissues had shorter N-terminal regions than expected from these results, and also lower affinity for proteinases than human cystatin C. In this work, we report the properties of recombinant, full-length bovine cystatin C having a complete N-terminal region. The general characteristics of this form of the inhibitor, as reflected by the isoelectric point, the far-ultraviolet circular dichroism spectrum, the thermal stability and the changes of tryptophan fluorescence on interaction with papain, resembled those of human cystatin C. The affinity and kinetics of inhibition of papain and cathepsins B, H and L by the bovine inhibitor were also comparable with those of the human inhibitor, although certain differences were apparent. Notably, the affinity of bovine cystatin C for cathepsin H was somewhat weaker than that of human cystatin C, and bovine cystatin C bound to cathepsin L with about a four-fold higher association rate constant than the human inhibitor. This rate constant is comparable with the highest values reported previously for cystatin-cysteine proteinase reactions. The full-length, recombinant bovine cystatin C bound appreciably more tightly to proteinases than the shorter form characterized previously. Digestion of the recombinant inhibitor with neutrophil elastase resulted in forms with truncated N-terminal regions and appreciably decreased affinity for papain, consistent with the forms of bovine cystatin C isolated previously having arisen by proteolytic cleavage of a mature, full-length inhibitor.

Animals↗
PubMed1999

The contribution of N-terminal region residues of cystatin A (stefin A) to the affinity and kinetics of inhibition of papain, cathepsin B, and cathepsin L.

The affinity and kinetics of binding of three N-terminally truncated variants of the cysteine proteinase inhibitor cystatin A to cysteine proteinases were characterized. Deletion of Met-1 only minimally altered the inhibitory properties of the protein. However, deletion also of Ile-2 resulted in reduced affinities of 900-, >/=3-, and 200-fold for papain and cathepsins L and B, respectively. Further truncation of Pro-3 substantially increased the inhibition constants to approximately 0.5 microM for papain and cathepsin L and to 60 microM for cathepsin B, reflecting additionally 2 x 10(3)-, 2 x 10(4)-, and 400-fold decreased affinities, respectively. The reductions in affinity shown by the latter mutant indicate that the N-terminal region contributes about 40% of the total free energy of binding of cystatin A to cysteine proteinases. Moreover, Pro-3 and to a lesser extent Ile-2 are the residues responsible for this binding energy. The reduced affinities for papain and cathepsin L were due only to higher dissociation rate constants, whereas both lower association and higher dissociation rate constants contributed to the decreased affinity for cathepsin B. These differential effects indicate that the N-terminal portion of cystatin A primarily functions by stabilizing the complexes with enzymes having easily accessible active-site clefts, e.g., papain and cathepsin L. In contrast, the N-terminal region is required also for an initial binding of cystatin A to cathepsin B, presumably by promoting the displacement of the occluding loop and allowing facile interaction of the rest of the inhibiting wedge with the active-site cleft of the enzyme.

Animals↗
PubMed1999

Acidic pH as a physiological regulator of human cathepsin L activity.

Human cysteine protease cathepsin L was inactivated at acid pH by a first-order process. The inactivation rate decreased with increasing concentrations of a small synthetic substrate, suggesting that substrates stabilize the active conformation. The substrate-independent inactivation rate constant increased with organic solvent content of the buffer, consistent with internal hydrophobic interactions, disrupted by the organic solvent, also stabilizing the enzyme. Circular dichroism showed that the inactivation is accompanied by large structural changes, a decrease in alpha-helix content being especially pronounced. The high activation energy of the reaction at pH 3.0 (200 kJ.mol-1) supported such a major conformational change occurring. The acid inactivation of cathepsin L was irreversible, consistent with the propeptide being needed for proper folding of the enzyme. Aspartic protease cathepsin D was shown to cleave denatured, but not active cathepsin L, suggesting a potential mechanism for in-vivo regulation and turnover of cathepsin L inside lysosomes.

Amino Acid Sequence↗
PubMed1999

The epidermal growth factor precursor. A calcium-binding, beta-hydroxyasparagine containing modular protein present on the surface of platelets.

Various human body fluids and secretions contain a soluble form of the epidermal growth factor (EGF) precursor. The EGF precursor molecule contains eight EGF modules in addition to EGF itself. Using monoclonal antibodies specific for the EGF modules 7 and 8, we have purified the soluble form of the EGF precursor from human urine to homogeneity. The protein was shown to have a molecular mass of about 160 kDa and the N-terminal sequence SAPNHWSXPE. EGF modules 2, 7 and 8 of the precursor have the consensus sequence for post-translational beta-hydroxylation of Asp/Asn residues. We identified the presence of erythro-beta-hydroxy-aspartic acid (Hya) in acid hydrolysates of the EGF precursor (2.4 M.M protein-1). As the DNA sequence encodes Asn in the corresponding position, the Hya represents erythro-beta-hydroxyasparagine (Hyn). The Hyn-containing modules have a consensus calcium-binding motif immediately N-terminal of the first Cys residue. The synthetic EGF module 2 (residues 356-395) of the EGF precursor was found to bind calcium with low affinity, Kd approximately 3.5 mM, i.e. similar to the affinity of other isolated calcium-binding EGF modules. EGF module 7, when part of the intact protein, was found to bind Ca2+ with a Kd approximately 0.2 microM, i.e. approximately 10(4)-fold higher than that of isolated EGF modules presumably due to the influence of neighboring modules. We have detected EGF precursor in platelet-rich plasma and demonstrated it to be associated to platelets. The platelets were found to have 30-160 EGF molecules each.

Amino Acid Sequence↗
PubMed1999

Hydrophobic sequences can substitute for the wild-type N-terminal sequence of cystatin A (stefin A) in tight binding to cysteine proteinases selection of high-affinity N-terminal region variants by phage display.

A phage-display library of the cysteine-proteinase inhibitor, cystatin A, was constructed in which variants with the four N-terminal amino acids randomly mutated were expressed on the surface of filamenteous phage. Screening of this library for binding to papain gave predominantly variants with a glycine residue in position 4. This finding is in agreement with previous conclusions that glycine in this position is essential for tight binding of cystatin A to cysteine proteinases by allowing optimal interaction of the N-terminal region of the inhibitor with the enzyme. In contrast, the first three residues of the variants obtained by the screening were more variable. Two variants were identified with similar affinities for papain as the wild-type inhibitor, but with these residues, Val-Phe-Thr- or Ile-Leu-Leu, differing appreciably from those of the wild-type, Met-Ile-Pro. Other sequences of the N-terminal region, presumably mainly hydrophobic, can thus substitute for the wild-type sequence and contribute similar energy to the inhibitor-proteinase interaction. The two variants binding tightly to papain differed in their affinity for cathepsin B, demonstrating that cystatin variants with increased selectivity for a particular target cysteine proteinase can be obtained by phage-display technology.

Amino Acid Sequence↗
PubMed1999

A novel strategy to generate biologically active neo-glycosaminoglycan conjugates.

Heparin and heparan sulfate are structurally related polysaccharides with a variety of biological effects/functions. Most of these effects are due to interactions, of varying specificity, between the negatively charged polysaccharide chains and proteins. While such interactions generally involve a single saccharide domain of decasaccharide size or less, ternary complexes of two protein molecules binding to separate domains on a single polysaccharide chain are known to occur. To facilitate studies on domain organization and its importance for biological function a strategy was developed to chemically conjugate defined heparin oligomers in linear and chemoselective fashion. The procedure requires that the oligosaccharide to provide the reducing-terminal domain of the conjugate is generated by lyase degradation of a parent polysaccharide, whereas the nonreducing-terminal domain is obtained through deaminative cleavage with nitrous acid. The applicability of the method was demonstrated by constructing a conjugate composed of two heparin 12-mers, of which the reducing-terminal component contained the antithrombin-binding region, whereas the nonreducing-terminal domain did not. Contrary to any of the unconjugated oligomers, the product was found to efficiently promote the inactivation of thrombin by antithrombin.

Anticoagulants↗
PubMed1998

Inactivation of papain by antithrombin due to autolytic digestion: a model of serpin inactivation of cysteine proteinases.

Cross-class inhibition of cysteine proteinases by serpins differs from serpin inhibition of serine proteinases primarily in that no stable serpin-cysteine proteinase complex can be demonstrated. This difference in reaction mechanism was elucidated by studies of the inactivation of the cysteine proteinases, papain and cathepsin L, by the serpin antithrombin. The two proteinases were inactivated with second-order rate constants of (1.6+/-0.1)x10(3) and (8.6+/-0. 4)x10(2) M-1.s-1 respectively. An antithrombin to papain inactivation stoichiometry of approximately 3 indicated extensive cleavage of the inhibitor concurrent with enzyme inactivation, a behaviour verified by SDS/PAGE. N-terminal sequence analyses showed cleavage predominantly at the P2-P1 bond, but also at the P2'-P3' bond of antithrombin. The papain band in SDS/PAGE progressively disappeared on reaction of the enzyme with increasing amounts of antithrombin, but no band representing a stable antithrombin-papain complex appeared. SDS/PAGE with 125I-labelled papain showed that the disappearance of papain was caused by cleavage of the enzyme into small fragments. These results suggest a mechanism in which papain attacks a peptide bond in the reactive-bond loop of antithrombin adjacent to that involved in serine proteinase inhibition. The reaction proceeds, similarly to that between serpins and serine proteinases, to form an inactive acyl-intermediate complex, although with the substrate pathway dominating in the papain reaction. In this complex, papain is highly susceptible to proteolysis and is degraded by still active papain, which greatly decreases the lifetime of the complex and results in liberation of fragmented, inactive enzyme. This model may have relevance also for the inactivation of physiologically or pathologically important cysteine proteinases by serpins.

Antithrombins↗
PubMed1998

Mechanism of heparin activation of antithrombin: evidence for an induced-fit model of allosteric activation involving two interaction subsites.

The anticoagulant activation of the serpin antithrombin by heparin pentasaccharide DEFGH was previously shown to involve trisaccharide DEF first binding and inducing activation of the serpin, followed by disaccharide GH binding and stabilizing the activated state [Petitou et al. (1997) Glycobiology 7, 323-327; Desai et al. (1998) J. Biol. Chem. 273, 7478-7487]. In the present study, the role of conformational changes and charged residues of the GH disaccharide in the allosteric activation mechanism was investigated with variant pentasaccharides modified in the GH disaccharide. Perturbation of the conformational equilibrium of iduronate residue G through replacement of the nonessential 3-OH of this residue with -H resulted in parallel decreases in the fraction of residue G in the skew boat conformer (from 64 to 24%) and in the association constant for pentasaccharide binding to antithrombin [(2.6 +/- 0.3)-fold], consistent with selective binding of the skew boat conformer to the serpin. Introduction of an additional sulfate group to the 3-OH of residue H flanking a putative charge cluster in the GH disaccharide greatly enhanced the affinity for the serpin by approximately 35-fold with only a small increase in the fraction of residue G in the skew boat conformation (from 64 to 85%). The salt dependence of binding, together with a recent X-ray structure of the antithrombin-pentasaccharide complex, suggested that the majority of the enhanced affinity of the latter pentasaccharide was due to direct electrostatic and hydrogen-bonding interactions of the H residue 3-O-sulfate with antithrombin. All variant pentasaccharides produced a normal enhancement of antithrombin fluoresence and normal acceleration of factor Xa inhibition by the serpin at saturating levels, indicating that conformational activation of antithrombin was not affected by the pentasaccharide modifications. Rapid kinetic studies were consistent with the altered affinities of the variant pentasaccharides resulting mostly from perturbed interactions of the reducing-end GH disaccharide with the activated antithrombin conformation and minimally to an altered binding of the nonreducing-end DEF trisaccharide to the native serpin conformation. Together, these results support a model in which the conformational flexibility of the G residue facilitates conversion to the skew boat conformer and thereby allows charged groups of the GH disaccharide to bind and stabilize the activated antithrombin conformation that is induced by the DEF trisaccharide.

Allosteric Regulation↗
PubMed1998

The role of Gly-4 of human cystatin A (stefin A) in the binding of target proteinases. Characterization by kinetic and equilibrium methods of the interactions of cystatin A Gly-4 mutants with papain, cathepsin B, and cathepsin L.

The importance of the evolutionarily conserved Gly-4 residue for the affinity and kinetics of interaction of cystatin A with several cysteine proteinases was assessed by site-directed mutagenesis. Even the smallest replacement, by Ala, resulted in approximately 1000-, approximately 10- and approximately 6000-fold decreased affinities for papain, cathepsin L, and cathepsin B, respectively. Substitution by Ser gave further 3-8-fold reductions in affinity, whereas the largest decreases, >10(5)-fold, were observed for mutations to Arg and Glu. The kinetics of inhibition of papain by the mutants with small side chains, Ala and Ser, were compatible with a one-step bimolecular reaction similar to that with wild-type cystatin A. The decreased affinities of these mutants for papain and cathepsin L were due exclusively to increased dissociation rate constants, but the reduced affinities for cathepsin B were due also to decreased association rate constants. The latter finding indicates that the intact N-terminal region serves as a guide directing cystatin A to the active site of cathepsin B, as has been proposed for cystatin C. The kinetics of binding of the mutants with charged side chains, Arg and Glu, to papain were consistent with a two-step binding mechanism, in which the mutant side chains are accommodated in the complex by a conformational change. The NMR solution structure of the Ala and Trp mutants showed only minor changes compared with wild-type cystatin A, indicating that the large reductions in affinity for proteinases are not due to altered structures of the mutants. Instead, a side chain larger than a hydrogen atom at position 4 affects the interaction with the proteinase most likely by interfering with the binding of the N-terminal region.

Binding, Competitive↗
PubMed1998

Mechanism of heparin activation of antithrombin. Role of individual residues of the pentasaccharide activating sequence in the recognition of native and activated states of antithrombin.

To determine the role of individual saccharide residues of a specific heparin pentasaccharide, denoted DEFGH, in the allosteric activation of the serpin, antithrombin, we studied the effect of deleting pentasaccharide residues on this activation. Binding, spectroscopic, and kinetic analyses demonstrated that deletion of reducing-end residues G and H or nonreducing-end residue D produced variable losses in pentasaccharide binding energy of approximately 15-75% but did not affect the oligosaccharide's ability to conformationally activate the serpin or to enhance the rate at which the serpin inhibited factor Xa. Rapid kinetic studies revealed that elimination of the reducing-end disaccharide marginally affected binding to the native low-heparin-affinity conformational state of antithrombin but greatly affected the conversion of the serpin to the activated high-heparin- affinity state, although the activated conformation was still favored. In contrast, removal of the nonreducing- end residue D drastically affected the initial low-heparin-affinity interaction so as to favor an alternative activation pathway wherein the oligosaccharide shifted a preexisiting equilibrium between native and activated serpin conformations in favor of the activated state. These results demonstrate that the nonreducing-end residues of the pentasaccharide function both to recognize the native low-heparin-affinity conformation of antithrombin and to induce and stabilize the activated high-heparin-affinity conformation. Residues at the reducing-end, however, poorly recognize the native conformation and instead function primarily to bind and stabilize the activated antithrombin conformation. Together, these findings establish an important role of the heparin pentasaccharide sequence in preferential binding and stabilization of the activated conformational state of the serpin.

Antithrombin III↗
PubMed1998

Two-step mechanism of inhibition of cathepsin B by cystatin C due to displacement of the proteinase occluding loop.

Stopped-flow kinetics showed that the inhibition of the lysosomal cysteine proteinase, cathepsin B, by its endogenous inhibitor, cystatin C, occurs by a two-step mechanism, in which an initial, weak interaction is followed by a conformational change. The initial interaction most likely involves binding of the N-terminal region of the inhibitor to the proteinase. Considerable evidence indicates that the subsequent conformational change is due to the inhibitor displacing the occluding loop of the proteinase that partially obscures the active site. The presence of this loop, which allows the enzyme to function as an exopeptidase, thus complicates the inhibition mechanism, rendering cathepsin B much less susceptible than other cysteine proteinases to inhibition by cystatins.

Binding Sites↗
PubMed1998

Calcium binding to the first EGF-like module of human factor IX in a recombinant fragment containing residues 1-85. Mutations V46E and Q50E each manifest a negligible increase in calcium affinity.

The first EGF-like module of human coagulation factor IX contains a single functionally important calcium ion binding site. We have now shown the dissociation constant for this site to be approximately 160 microM in a recombinant protein fragment consisting of residues 1-85 in human fIX. This represents a approximately 10-fold increase in affinity as compared with the isolated EGF module (residues 46-85). The Gla module (here with Glu instead of Gla) thus increases the affinity of the EGF module calcium ion binding site. Each of two mutations, V46E and Q50E, made to investigate whether the extra negative charge would increase the affinity of the calcium binding site manifested a negligible increase in affinity.

Amino Acid Sequence↗
PubMed1997

Molecular cloning and N-terminal analysis of bovine cystatin C. Identification of a full-length N-terminal region.

The N-terminal region of human cystatin C has been shown to be of crucial importance for the interaction of the inhibitor with cysteine proteinases. However, several studies have been unable to identify the corresponding region in bovine cystatin C, indicating that the binding of proteinases to the bovine inhibitor may not be dependent on this region. With the aim to resolve this apparent discrepancy and to elucidate the relation of bovine cystatin C to other cystatins, we have isolated a cDNA clone encoding bovine precystatin C. The sequence of this cDNA was similar to that of the human inhibitor and showed a putative signal peptidase cleavage site consistent with the N-terminal regions of the bovine and human inhibitors being of comparable size. This suggestion was verified by determination of the relative molecular mass of the mature bovine inhibitor isolated from cerebrospinal fluid under conditions minimising proteolysis. The N-terminal of the purified inhibitor was blocked, but the sequence of the N-terminal peptide produced by digestion with endopeptidase LysC could be unequivocally determined by tandem mass spectroscopy. Together, these results show that bovine cystatin C has 118 residues, in contrast with 110-112 residues reported previously, and has an N-terminal region analogous to that of human cystatin C. This region presumably is of similar importance for tight binding of target proteinases as in the human inhibitor.

Amino Acid Sequence↗
PubMed1997

Inactivation of thrombin by antithrombin is accompanied by inactivation of regulatory exosite I.

Exosite I of the blood clotting proteinase, thrombin, mediates interactions of the enzyme with certain inhibitors, physiological substrates and regulatory proteins. Specific binding of a fluorescein-labeled derivative of the COOH-terminal dodecapeptide of hirudin ([5F] Hir54-65) to exosite I was used to probe changes in the function of the regulatory site accompanying inactivation of thrombin by its physiological serpin inhibitor, antithrombin. Fluorescence-monitored equilibrium binding studies showed that [5F]Hir54-65 and Hir54-65 bound to human alpha-thrombin with dissociation constants of 26 +/- 2 nM and 38 +/- 5 nM, respectively, while the affinity of the peptides for the stable thrombin-antithrombin complex was undetectable (>/=200-fold weaker). Kinetic studies showed that the loss of binding sites for [5F]Hir54-65 occurred with the same time-course as the loss of thrombin catalytic activity. Binding of [5F] Hir54-65 and Hir54-65 to thrombin was correlated quantitatively with partial inhibition of the rate of the thrombin-antithrombin reaction, maximally decreasing the bimolecular rate constants 1.7- and 2.1-fold, respectively. These results support a mechanism in which thrombin and the thrombin-Hir54-65 complex can associate with antithrombin and undergo formation of the covalent thrombin-antithrombin complex at modestly different rates, with inactivation of exosite I leading to dissociation of the peptide occurring subsequent to the rate-limiting inactivation of thrombin. This mechanism may function physiologically in localizing the activity of thrombin by allowing inactivation of thrombin that is bound in exosite I-mediated complexes with regulatory proteins, such as thrombomodulin and fibrin, without prior dissociation of these complexes. Concomitant with inactivation of thrombin, the thrombin-antithrombin complex may be irreversibly released due to exosite I inactivation.

Antithrombin III↗