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E Di Cera

Publications and source records attributed to E Di Cera.

At least 55 records · Page 3Linked to original sources
PubMed1996

Release of fibrinopeptides by the slow and fast forms of thrombin.

The release of fibrinopeptides A and B by the slow and fast forms of thrombin was studied over the temperature range from 5 to 45 degrees C and the salt concentration range from 100 to 800 mM. The sequential mechanism for the release of fibrinopeptides originally proposed by Shafer was found to be obeyed under all conditions examined. The origin of preferential binding of fibrinogen and fibrin I to the fast form of thrombin in the transition state is in the second-order rate constant for association, k(l). In the case of fibrinogen, the values of k(l) for interaction with the fast and slow forms at 25 degrees C are 19 +/- 4 and 2.5 +/- 0.3 microM(-1) s(-1), with an activation energy of about 10 kcal/mol in both forms. In the case of fibrin I, the analogous values of k(l) are 9.1 +/- 0.7 and 2.5 +/- 0.2 microM(-1) s(-1), and the activation energy is about 4.5 kcal/mol in both forms. The mechanism of recognition of fibrinogen and fibrin I by thrombin entails a diffusion-controlled step with a small energy barrier. Analysis of the temperature dependence of the coupling free energy for allosteric switching indicates that the preferential interaction of fibrinogen and fibrin I with the fast form of thrombin in the transition state is entropy-driven, signaling a contribution of the hydrophobic effect to the slow-->fast transition. The salt dependence of the release of fibrinopeptides shows a constant coefficient Gamma(salt) = d ln(k(cat)/K(m))/d ln [salt] in the concentration range examined. Interestingly, the value of Gamma(salt) is independent of the salt used (NaCl, ChCl, or NaF) and is -1.5 +/- 0.1 for fibrinopeptide A and -2.5 +/- 0.1 for fibrinopeptide B. Hence, Gamma(salt) reflects predominantly the electrostatic contribution to the formation of the transition state, with a larger contribution seen in the interaction of thrombin with fibrin I. It is concluded that the interaction of thrombin with fibrinogen and fibrin I, leading to the release of fibrinopeptides A and B, is driven by electrostatic forces that presumably favor the correct preorientation of the enzyme and the substrate to form a productive complex in the transition state. This electrostatic-steering effect, also reported for thrombin-hirudin interaction, leads to a diffusion-controlled encounter with a very small energy barrier. Once the complex is formed, the enzyme switches to the fast form as a result of entropic factors presumably linked to water release from a more extended surface of recognition. While the release of fibrinopeptides as a function of salt concentration was being studied, an important observation was made on the role of Cl- in the formation of the fibrin clot. This anion drastically and specifically reduces the thickness of fibrin fibers, as judged by the 10-fold decrease in the equilibrium turbidity of clots developed in NaCl as compared to the turbidity of clots developed in NaF. Hence, the transition from a "coarse" to a "fine" clot induced by an increase in ionic strength as first described by Ferry is, instead, due to the specific binding of Cl- to intermediates in the ensuing polymerization. In fact, no change in the clotting curve is observed when the ionic strength is changed with NaF.

Electrochemistry↗
PubMed1996

Valence screening of water in protein crystals reveals potential Na+ binding sites.

Identification of Na+ binding sites in protein crystals is complicated by comparable electron density of this monovalent cation and water. Valence calculations can predict the location of metal ion binding sites in proteins with high precision. These calculations were used to screen 332,242 water molecules in 2742 protein structures reported in the Protein Data Bank (PDB), searching for molecules with Na+/- specific valence values V(Na+) > or = 1.0 v.u., as expected for a bound Na ion. Thirty-three water molecules (<0.01% of the total) were found be have V(Na+) > or = 1.0 v.u. and to be located within 3.5 A from at least two protein oxygen atoms. These water molecules, with a high Na+ -specific valence, do not have valences specific for other cations, like Li+, K+, Mg2+ or Ca2+. They belong to nine different proteins (deoxyribonuclease I, enolase, hen egg-white lysozyme, human lysozyme, phospholipase A2, proteinase A, rubredoxin, thrombin and phage T4 lysozyme) and appear with similar coordination geometry, typically octahedral, in the same place in multiple crystal structure determinations of the same protein. In the case of thrombin, the water molecule singled out by valence calculations is, in fact, a bound Na ion as demonstrated by molecular replacement with Rb+. Valence calculations provide an accurate screening of water in protein crystals and may help identify Na+ binding sites of functional importance.

Algorithms↗
PubMed1996

Theory of allosteric effects in serine proteases.

The classical Botts-Morales theory for the action of a modifier on the catalytic properties of an enzyme has been extended to deal with allosteric effects in serine proteases. The exact analytical solution derived for the linkage scheme at steady state provides a rigorous framework for the study of many biologically relevant systems, including enzymes activated by monovalent cations and cofactor-controlled protease-zymogen interactions in blood coagulation. When the enzyme obeys Michaelis-Menten kinetics, the exact solution of the kinetic linkage scheme simplifies considerably. Of particular importance for practical applications is a simple equation expressing the dependence of the specificity constant of the enzyme, kcat/Km, on the concentration of the modifier, from which the equilibrium binding constant for the formation of the enzyme-modifier complex can be estimated. Analysis of the allosteric changes in thrombin activity induced by thrombomodulin and Na+ in terms of this equation yields accurate determinations of the equilibrium binding constants for both effectors.

Acylation↗
PubMed1995

Identification of residues linked to the slow-->fast transition of thrombin.

Residues energetically linked to the allosteric transition of thrombin from its anticoagulant slow form to the procoagulant fast form have been identified by site-directed mutagenesis. The energetics of recognition by the two forms of the enzyme were probed by using a synthetic chromogenic substrate, fibrinogen, and hirudin. The thrombin residues E39, W60d, E192, D221, and D222 are linked to the slow-->fast transition and are part of an "allosteric core" through which events originating at the Na+ binding loop propagate to other regions of the enzyme. The thrombin residues Y76, W96, W148, and R173 lie at the periphery of the allosteric core, affect recognition of fibrinogen and hirudin to the same extent in both forms, and are not linked to the slow-->fast transition.

Allosteric Regulation↗
PubMed1995

Thermodynamic investigation of hirudin binding to the slow and fast forms of thrombin: evidence for folding transitions in the inhibitor and protease coupled to binding.

Temperature dependent studies of the interaction of the clotting enzyme thrombin with the potent natural inhibitor hirudin reveal a large negative heat capacity change of -1.7(+/- 0.2) kcal/mol per K associated with the formation of the thrombin-hirudin complex, independent of the allosteric state of the enzyme. Binding of N-terminal fragments of hirudin (hir1-49 and hir1-43) is characterized by heat capacity changes of -1.2(+/- 0.1) and -0.9(+/- 0.1) kcal/mol per K, respectively. The magnitude of these heat capacity changes is unprecedented for protease-inhibitor interactions. A thermodynamic analysis based on observed heat capacity and entropy changes predicts that binding is accompanied by substantial coupled folding transitions in both hirudin and thrombin. In the absence of a structure of free thrombin, analysis of differences in the predicted number of residues which fold upon binding hirudin and its fragments leads to the following structural model: three surface loops in thrombin (W60d, W148 and fibrinogen binding loops) are disordered in the free state and fold upon formation of the thrombin-hirudin complex. Molecular dynamics simulations, run over a time scale of 5 ps, are consistent with the hypothesis of large scale coupled folding transitions in both hirudin and thrombin upon formation of the complex. Comparison of the thermodynamics for the interaction of hirudin with the slow and fast forms of thrombin allows dissection of the coupling free energy for allosteric switching. The coupling free energy for the slow-->fast transition increases linearly, in absolute value, with temperature. The coupling enthalpy and entropy terms for hirudin were found to be delta Hoc = 12(+/- 1) kcal/mol and delta Soc = 47(+/- 4) cal/mol per K. Preferential interaction with the fast form is therefore due to the balance of two opposite forces, both quite large in magnitude. The contribution of enthalpic effects opposes the slow-->fast transition and stabilizes binding to the slow form. The contribution of entropic effects favors the slow-->fast transition and stabilizes binding to the fast form. In the physiological temperature range the entropic effects prevail and result in preferential binding of hirudin to the fast form. The region of thrombin recognizing the N-terminal domain of hirudin contains most of the residues that are energetically linked to the slow-->fast transition. This region is part of the "allosteric core" of thrombin and includes the W60d loop, shaping the specificity site S2, and the Na+ binding loop connecting the last two beta-strands of the B chain.

Allosteric Regulation↗
PubMed1995

Binding of fibrinogen A alpha 1-50-beta-galactosidase fusion protein to thrombin stabilizes the slow form.

The interaction of fibrinogen A alpha1-50-beta-galactosidase fusion protein with the slow and fast forms of thrombin was studied and compared to thrombin-fibrinogen interaction under identical solution conditions. At equilibrium, the affinity of the fusion protein for the slow form of thrombin is 3 times higher than its affinity for the fast form. The fusion protein and fibrinogen have the same affinity for the fast form. On the other hand, the affinity of the fusion protein for the slow form of thrombin is 40 times tighter than that of fibrinogen. In the transition state, binding of the fusion protein has the same properties as fibrinogen, with the fast form showing higher specificity. The N-terminal fragment of the fibrinogen A alpha chain thus contains residues that are responsible for the preferential binding of the fusion protein to the slow form at equilibrium and to the fast form in the transition state. If this fragment binds to thrombin in a similar way for fibrinogen and the fusion protein, then the N-terminal domains of the B beta and gamma chains of fibrinogen, that are not present in the fusion protein, must play a key role in the binding of fibrinogen to thrombin at equilibrium. These chains may destabilize binding to the slow form by nearly 2.4 kcal/mol, thereby favoring binding of fibrinogen to the fast form. We propose that the three chains of fibrinogen play different roles in the thrombin-fibrinogen interaction, with the A alpha chain containing residues for preferential binding to the fast form in the transition state and the B beta and gamma chains containing residues that destabilize binding to the slow form at equilibrium.

Enzyme Stability↗
PubMed1995

Crystal structure of the large fragment of Thermus aquaticus DNA polymerase I at 2.5-A resolution: structural basis for thermostability.

The crystal structure of the large fragment of the Thermus aquaticus DNA polymerase (Klentaq1), determined at 2.5-A resolution, demonstrates a compact two-domain architecture. The C-terminal domain is identical in fold to the equivalent region of the Klenow fragment of Escherichia coli DNA polymerase I (Klenow pol I). Although the N-terminal domain of Klentaq1 differs greatly in sequence from its counterpart in Klenow pol I, it has clearly evolved from a common ancestor. The structure of Klentaq1 reveals the strategy utilized by this protein to maintain activity at high temperatures and provides the structural basis for future improvements of the enzyme.

Amino Acid Sequence↗
PubMed1995

The Na+ binding site of thrombin.

Thrombin is an allosteric serine protease existing in two forms, slow and fast, targeted toward anticoagulant and procoagulant activities. The slow --> fast transition is induced by Na+ binding to a site contained within a cylindrical cavity formed by three antiparallel beta-strands of the B-chain (Met180-Tyr184a, Lys224-Tyr228, and Val213-Gly219) diagonally crossed by the Glu188-Glu192 strand. The site is shaped further by the loop connecting the last two beta-strands and is located more than 15 A away from the catalytic triad. The cavity traverses through thrombin from the active site to the opposite surface and contains Asp189 of the primary specificity site near its midpoint. The bound Na+ is coordinated octahedrally by the carbonyl oxygen atoms of Tyr184a, Arg221a, and Lys224, and by three highly conserved water molecules in the D-Phe-Pro-Arg chloromethylketone thrombin. The sequence in the Na+ binding loop is highly conserved in thrombin from 11 different species and is homologous to that found in other serine proteases involved in blood coagulation. Mutation of two Asp residues flanking Arg221a (D221A/D222K) almost abolishes the allosteric properties of thrombin and shows that the Na+ binding loop is also involved in direct recognition of protein C and antithrombin.

Allosteric Regulation↗
PubMed1995

An allosteric switch controls the procoagulant and anticoagulant activities of thrombin.

Thrombin is an allosteric enzyme existing in two forms, slow and fast, that differ widely in their specificities toward synthetic and natural amide substrates. The two forms are significantly populated in vivo, and the allosteric equilibrium can be affected by the binding of effectors and natural substrates. The fast form is procoagulant because it cleaves fibrinogen with higher specificity; the slow form is anticoagulant because it cleaves protein C with higher specificity. Binding of thrombomodulin inhibits cleavage of fibrinogen by the fast form and promotes cleavage of protein C by the slow form. The allosteric properties of thrombin, which has targeted two distinct conformational states toward its two fundamental and competing roles in hemostasis, are paradigmatic of a molecular strategy that is likely to be exploited by other proteases in the blood coagulation cascade.

Allosteric Regulation↗
PubMed1994

Predicting Ca(2+)-binding sites in proteins.

The coordination shell of Ca2+ ions in proteins contains almost exclusively oxygen atoms supported by an outer shell of carbon atoms. The bond-strength contribution of each ligating oxygen in the inner shell can be evaluated by using an empirical expression successfully applied in the analysis of crystals of metal oxides. The sum of such contributions closely approximates the valence of the bound cation. When a protein is embedded in a very fine grid of points and an algorithm is used to calculate the valence of each point representing a potential Ca(2+)-binding site, a typical distribution of valence values peaked around 0.4 is obtained. In 32 documented Ca(2+)-binding proteins, containing a total of 62 Ca(2+)-binding sites, a very small fraction of points in the distribution has a valence close to that of Ca2+. Only 0.06% of the points have a valence > or = 1.4. These points share the remarkable tendency to cluster around documented Ca2+ ions. A high enough value of the valence is both necessary (58 out of 62 Ca(2+)-binding sites have a valence > or = 1.4) and sufficient (87% of the grid points with a valence > or = 1.4 are within 1.0 A from a documented Ca2+ ion) to predict the location of bound Ca2+ ions. The algorithm can also be used for the analysis of other cations and predicts the location of Mg(2+)- and Na(+)-binding sites in a number of proteins. The valence is, therefore, a tool of pinpoint accuracy for locating cation-binding sites, which can also be exploited in engineering high-affinity binding sites and characterizing the linkage between structural components and functional energetics for molecular recognition of metal ions by proteins.

Algorithms↗
PubMed1994

Molecular recognition by thrombin. Role of the slow-->fast transition, site-specific ion binding energetics and thermodynamic mapping of structural components.

The interaction of thrombin with the potent natural inhibitor hirudin is controlled in a complex fashion by the binding of Na+ and Cl- to the enzyme and allosteric transitions. Binding of hirudin is positively linked to Na+ binding, but is opposed in a competitive fashion by the binding of Cl-. Since Na+ binding induces the slow-->fast transition of thrombin, it follows from linkage principles that hirudin binds to the fast form with higher affinity. Hence, the slow-->fast transition is a key component of molecular recognition of hirudin by thrombin. We propose a three-step mechanism for molecular recognition of hirudin by thrombin, which is also relevant for recognition of fibrinogen and possibly the platelet receptor and thrombomodulin. First, the C-terminal acidic tail of hirudin binds to the fibrinogen recognition site of thrombin displacing one Cl ion from the thrombin surface. Then, the enzyme undergoes a conformational transition that gives rise to increased accessibility of the catalytic pocket to small synthetic substrates through movement of the Trp148 loop. The changes in the catalytic moiety triggered allosterically by binding to the fibrinogen recognition site are linked to the uptake of Na+ and are similar to, if not identical with, those observed in the Na(+)-induced slow-->fast transition. Finally, the compact N-terminal domain is accommodated in the region surrounding the catalytic pocket. Hirudin binding is also used as a probe of site-specific ion-binding interactions of Na+ and Cl- with the enzyme, characterized by cooperativity between the Na+ and Cl- binding domains. The structural components directly involved or linked to Na+ and Cl- binding have been explored in terms of free energy perturbations of the binding of hirudin and a number of ligands. The fibrinogen recognition site stores most of the free energy of coupling with Cl- binding, while regions surrounding the access to the catalytic pocket provide most of the free energy of coupling with Na+ binding and the slow-->fast transition of thrombin. It is concluded that thrombin is an allosteric enzyme that exists in two forms, slow and fast, and that the allosteric transition slow-->fast represents the key element of molecular recognition of important physiological substrates. The allosteric nature of the enzyme also enables different structural domains to communicate and give rise to biological function.

Amino Acid Sequence↗
PubMed1994

Thermodynamic basis of site-specific cooperativity.

Cooperative phenomena in biological macromolecules arise from the interaction of many distinct subsystems, such as structural domains or binding sites. Cooperative properties of the system as a whole, like protein folding or allosteric transitions, are subject to the restrictions imposed by thermodynamic stability. These restrictions, however, do not apply in the case of individual subsystems open to interactions with the rest of the macromolecule. The site-specific properties of such subsystems can be understood in general thermodynamic terms from those of a multicomponent system under particular conditions. The analogy provides a thermodynamic basis for site-specific cooperativity.

Binding Sites↗
PubMed1994

A simple activity assay for thrombin and hirudin.

Cloning of the thrombin cDNA has made it possible to study thrombin function by site-directed mutagenesis. Quantitative results from studies of thrombin mutants are often hindered by difficulties in assaying the enzyme activity. The high enzyme concentrations required for activity determination by standard methods limit their usefulness to thrombin mutants that cannot be readily produced in large quantities. We have developed a novel method using the synthetic substrate S-2238 and hirudin, a tight-binding inhibitor of thrombin, that allows for the active-site titration of thrombin at concentrations as low as 20 pM, with an error of < or = 5%. In addition, hirudin activity can be determined by this method to concentrations as low as 40 pM, with an error of < or = 5%.

Binding Sites↗
PubMed1993

Thrombin-fibrinogen interaction: pH dependence and effects of the slow-->fast transition.

A recently developed strategy capable of measuring the equilibrium dissociation constant for thrombin-fibrinogen interaction has been used to explore the pH dependence of the interaction and the effects of thrombin conformational transitions. The dependence of fibrinogen binding to thrombin in the pH range 6-10 is bell-shaped and remarkably similar to that obtained in the case of the small synthetic amide substrate tosyl-Gly-Pro-Arg-p-nitroanilide-AcOH. Since the synthetic substrate contains no groups that can ionize in the pH range 6-10, the bell-shaped curve must reflect ionization reactions of two groups of the enzyme with pK1 = 7.53 +/- 0.09 and pK2 = 8.80 +/- 0.09. These groups can be identified as the catalytic histidine, His57, and the amino terminus of the B chain, Ile16, respectively. Deprotonation of His57 in the acidic region is important for optimal binding, while protonation of Ile16 in the alkaline region is critical for the formation of a salt bridge with Asp194, which guarantees the conformational stability of the enzyme. The loss of binding free energy at low (< 7.0) and high (> 9.0) pH values is linked to protonation of His57 and deprotonation of Ile16, respectively. The first 51 residues of the A alpha chain of fibrinogen are known to be necessary and sufficient for optimal recognition by thrombin, but none of them contributes to the pH dependence of fibrinogen binding in the pH range examined. Hence, the two possible ionizable groups of the A alpha chain, i.e., the amino terminus Alal and His24, make no contacts with the thrombin surface.(ABSTRACT TRUNCATED AT 250 WORDS)

Acylation↗
PubMed1993

Chemical compensation in macromolecular bridge-binding to thrombin.

The binding energetics of eight synthetic peptides capable of interfering with thrombin function have been studied by steady-state measurements and clotting assays. The synthetic peptides are bifunctional inhibitors consisting of three domains: (i) a fragment of the C-terminus of recombinant hirudin, hir55-65, which binds to the fibrinogen-recognition site of thrombin; (ii) a small active site inhibitor, Ac-(DF)PRP, binding to the catalytic pocket of the enzyme, and (iii) a linker spanning these two portions with variable length and chemical composition. All these synthetic peptides are competitive inhibitors of fibrinogen. On the other hand, a linker of at least 13 carbon atoms is required for full competitive inhibition of the hydrolysis by thrombin of small synthetic substrates, which only bind to the catalytic pocket of the enzyme. The best inhibitory effect is observed with a linker of 13 carbon atoms, with a value of KI in the nanomolar range. Studies conducted as a function of temperature, in the range 15-40 degrees C, have revealed the enthalpic and entropic components of inhibitor binding to thrombin. Chemical compensation is observed for all synthetic peptides that bridge-bind to the fibrinogen-recognition site and the catalytic pocket of the enzyme thereby inhibiting in a competitive fashion either fibrinogen binding or the hydrolysis of small synthetic substrates. The extrathermodynamic relationship between delta H and delta G also includes the enthalpy and free energy of binding for the natural substrate fibrinogen and the potent natural inhibitor hirudin, measured under identical solution conditions. Preferential binding of hirudin over fibrinogen is an entropy-driven process.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗