Lysine 114 of Antithrombin Is of Crucial Importance for the Affinity and Kinetics of Heparin Pentasaccharide Binding

Artigo Acesso aberto Revisado por pares

Lysine 114 of Antithrombin Is of Crucial Importance for the Affinity and Kinetics of Heparin Pentasaccharide Binding

2001; Elsevier BV; Volume: 276; Issue: 47 Linguagem: Inglês

10.1074/jbc.m105294200

ISSN

1083-351X

Autores

Véronique Arocas, Susan Bock, Srikumar M. Raja, Steven T. Olson, Ingemar Björk,

Tópico(s)

Coagulation, Bradykinin, Polyphosphates, and Angioedema

Resumo

Lys114 of the plasma coagulation proteinase inhibitor, antithrombin, has been implicated in binding of the glycosaminoglycan activator, heparin, by previous mutagenesis studies and by the crystal structure of antithrombin in complex with the active pentasaccharide unit of heparin. In the present work, substitution of Lys114 by Ala or Met was shown to decrease the affinity of antithrombin for heparin and the pentasaccharide by ∼105-fold at I 0.15, corresponding to a reduction in binding energy of ∼50%. The decrease in affinity was due to the loss of two to three ionic interactions, consistent with Lys114 and at least one other basic residue of the inhibitor binding cooperatively to heparin, as well as to substantial nonionic interactions. The mutation minimally affected the initial, weak binding of the two-step mechanism of pentasaccharide binding to antithrombin but appreciably (>40-fold) decreased the forward rate constant of the conformational change in the second step and greatly (>1000-fold) increased the reverse rate constant of this step. Lys114 is thus of greater importance for the affinity of heparin binding than any of the other antithrombin residues investigated so far, viz. Arg47, Lys125, and Arg129. It contributes more than Arg47 and Arg129 to increasing the rate of induction of the activating conformational change, a role presumably exerted by interactions with the nonreducing end trisaccharide unit of the heparin pentasaccharide. However, its major effect, also larger than that of these two residues, is in maintaining antithrombin in the activated state by interactions that most likely involve the reducing end disaccharide unit. Lys114 of the plasma coagulation proteinase inhibitor, antithrombin, has been implicated in binding of the glycosaminoglycan activator, heparin, by previous mutagenesis studies and by the crystal structure of antithrombin in complex with the active pentasaccharide unit of heparin. In the present work, substitution of Lys114 by Ala or Met was shown to decrease the affinity of antithrombin for heparin and the pentasaccharide by ∼105-fold at I 0.15, corresponding to a reduction in binding energy of ∼50%. The decrease in affinity was due to the loss of two to three ionic interactions, consistent with Lys114 and at least one other basic residue of the inhibitor binding cooperatively to heparin, as well as to substantial nonionic interactions. The mutation minimally affected the initial, weak binding of the two-step mechanism of pentasaccharide binding to antithrombin but appreciably (>40-fold) decreased the forward rate constant of the conformational change in the second step and greatly (>1000-fold) increased the reverse rate constant of this step. Lys114 is thus of greater importance for the affinity of heparin binding than any of the other antithrombin residues investigated so far, viz. Arg47, Lys125, and Arg129. It contributes more than Arg47 and Arg129 to increasing the rate of induction of the activating conformational change, a role presumably exerted by interactions with the nonreducing end trisaccharide unit of the heparin pentasaccharide. However, its major effect, also larger than that of these two residues, is in maintaining antithrombin in the activated state by interactions that most likely involve the reducing end disaccharide unit. N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine The plasma proteinase inhibitor of the serpin family, antithrombin, is a major regulator of blood clotting. Its crucial role is evident from many observations that individuals with heterozygous antithrombin deficiency, resulting in lower amounts of active inhibitor, have an increased tendency to develop thrombosis (1Van Boven H.H. Lane D.A. Semin. Hematol. 1997; 34: 188-204PubMed Google Scholar). Moreover, deletion of the gene in mice leads to embryonic lethality, due to fibrin deposition in myocardium and liver and consumptive coagulopathy (2Ishiguro K. Kojima T. Kadomatsu K. Nakayama Y. Takagi A. Suzuki M. Takeda N. Ito M. Yamamoto K. Matsushita T. Kusugami K. Muramatsu T. Saito H. J. Clin. Invest. 2000; 106: 873-878Crossref PubMed Scopus (177) Google Scholar). Antithrombin inhibits most coagulation proteinases, although its main physiological targets are thrombin and factor Xa (1Van Boven H.H. Lane D.A. Semin. Hematol. 1997; 34: 188-204PubMed Google Scholar,3Gettins P.G.W. Patston P.A. Olson S.T. Serpins: Structure, Function, and Biology. R. G. Landes, Austin, TX1996: 15-109Google Scholar, 4Björk I. Olson S.T. Church F.C. Cunningham D.D. Ginsburg D. Hoffman M. Stone S.R. Tollefsen D.M. Chemistry and Biology of Serpins. Plenum Press, New York1997: 17-33Google Scholar). Like all inhibitorily active serpins, antithrombin inactivates target proteinases by a unique mechanism. The proteinase initially recognizes a reactive bond, located in a surface-exposed loop of the serpin, and proceeds to cleave this bond. At the acyl-intermediate stage of this cleavage, the reactive bond loop is opened, which releases the strain on the loop. As a consequence, the liberated N-terminal part of the loop is rapidly inserted into a major β-sheet of the inhibitor, the A sheet. The proteinase is still attached to this segment by an acyl bond and is therefore transported to the opposite pole of the protein. In this location, the proteinase is squeezed against the main body of the inhibitor and is inactivated by the resulting distortion of a large part of its structure, including the active site (3Gettins P.G.W. Patston P.A. Olson S.T. Serpins: Structure, Function, and Biology. R. G. Landes, Austin, TX1996: 15-109Google Scholar, 4Björk I. Olson S.T. Church F.C. Cunningham D.D. Ginsburg D. Hoffman M. Stone S.R. Tollefsen D.M. Chemistry and Biology of Serpins. Plenum Press, New York1997: 17-33Google Scholar, 5Stratikos E. Gettins P.G.W. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 4808-4813Crossref PubMed Scopus (217) Google Scholar, 6Lawrence D.A. Olson S.T. Muhammad S. Day D.E. Kvassman J.O. Ginsburg D. Shore J.D. J. Biol. Chem. 2000; 275: 5839-5844Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar, 7Huntington J.A. Read R.J. Carrell R.W. Nature. 2000; 407: 923-926Crossref PubMed Scopus (948) Google Scholar). In contrast to most serpins, which rapidly inhibit their target proteinases, antithrombin inactivates thrombin and factor Xa at only moderate rates. However, these reactions are greatly accelerated by the sulfated glycosaminoglycan, heparin, which thereby acts as an efficient anticoagulant (3Gettins P.G.W. Patston P.A. Olson S.T. Serpins: Structure, Function, and Biology. R. G. Landes, Austin, TX1996: 15-109Google Scholar, 4Björk I. Olson S.T. Church F.C. Cunningham D.D. Ginsburg D. Hoffman M. Stone S.R. Tollefsen D.M. Chemistry and Biology of Serpins. Plenum Press, New York1997: 17-33Google Scholar). The rate acceleration is due to a specific pentasaccharide region of heparin binding to antithrombin (8Thunberg L. Bäckström G. Lindahl U. Carbohydr. Res. 1982; 100: 393-410Crossref PubMed Scopus (373) Google Scholar, 9Choay J. Petitou M. Lormeau J.C. Sinay P. Casu B. Gatti G. Biochem. Biophys. Res. Commun. 1983; 116: 492-499Crossref PubMed Scopus (596) Google Scholar). This binding occurs by a two-step mechanism, in which an initial, weak complex is formed in a rapid equilibrium in the first step. A conformational change that tightens the binding of the pentasaccharide region and activates the inhibitor is then induced in the second step (10Olson S.T. Srinivasan K.R. Björk I. Shore J.D. J. Biol. Chem. 1981; 256: 11073-11079Abstract Full Text PDF PubMed Google Scholar, 11Olson S.T. Björk I. Sheffer R. Craig P.A. Shore J.D. Choay J. J. Biol. Chem. 1992; 267: 12528-12538Abstract Full Text PDF PubMed Google Scholar). This conformational change is sufficient to accelerate the inhibition of factor Xa, whereas rapid thrombin inhibition is mainly dependent on approximation of enzyme and inhibitor by both binding to the same pentasaccharide-containing heparin chain of at least 18 saccharide units (3Gettins P.G.W. Patston P.A. Olson S.T. Serpins: Structure, Function, and Biology. R. G. Landes, Austin, TX1996: 15-109Google Scholar, 4Björk I. Olson S.T. Church F.C. Cunningham D.D. Ginsburg D. Hoffman M. Stone S.R. Tollefsen D.M. Chemistry and Biology of Serpins. Plenum Press, New York1997: 17-33Google Scholar). The x-ray structure of a complex between antithrombin and a synthetic heparin pentasaccharide (12Jin L. Abrahams J.P. Skinner R. Petitou M. Pike R.N. Carrell R.W. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 14683-14688Crossref PubMed Scopus (638) Google Scholar) indicates that the increased heparin affinity induced in the second binding step involves the D helix of the inhibitor being elongated by 1.5 turns and a new, short α-helix, the P helix, being formed (Fig. 1). Moreover, the enhanced reactivity of antithrombin with factor Xa caused by heparin is most likely due to an increased exposure of the reactive bond loop. The x-ray structure has also identified several basic residues of antithrombin, predominantly Arg47, Lys114, Lys125, and Arg129, that participate in the binding by interacting with negative groups of the pentasaccharide (Fig. 1). The importance of Arg47 and Arg129 for the binding is consistent with the reduced heparin affinities of natural antithrombin variants in which these residues are altered (13Koide T. Odani S. Takahashi K. Ono T. Sakuragawa N. Proc. Natl. Acad. Sci. U. S. A. 1984; 81: 289-293Crossref PubMed Scopus (155) Google Scholar, 14Gandrille S. Aiach M. Lane D.A. Vidaud D. Molho-Sabatier P. Caso R. de Moerloose P. Fiessinger J.N. Clauser E. J. Biol. Chem. 1990; 265: 18997-19001Abstract Full Text PDF PubMed Google Scholar). The roles of the four residues have been further investigated by mutations in recombinant variants of the inhibitor (15Fan B. Turko I.V. Gettins P.G.W. Biochemistry. 1994; 33: 14156-14161Crossref PubMed Scopus (26) Google Scholar, 16Kridel S.J. Chan W.W. Knauer D.J. J. Biol. Chem. 1996; 271: 20935-20941Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar, 17Kridel S.J. Knauer D.J. J. Biol. Chem. 1997; 272: 7656-7660Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar, 18Ersdal-Badju E. Lu A.Q. Zuo Y.C. Picard V. Bock S.C. J. Biol. Chem. 1997; 272: 19393-19400Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar, 19Arocas V. Bock S.C. Olson S.T. Björk I. Biochemistry. 1999; 38: 10196-10204Crossref PubMed Scopus (46) Google Scholar, 20Desai U. Swanson R. Bock S.C. Björk I. Olson S.T. J. Biol. Chem. 2000; 275: 18976-18984Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). Substitution of Arg47, Lys125, and Arg129 led to 20–30-, 30–150-, and 400–2500-fold, respectively, losses of affinity for pentasaccharide and full-length heparin (15Fan B. Turko I.V. Gettins P.G.W. Biochemistry. 1994; 33: 14156-14161Crossref PubMed Scopus (26) Google Scholar, 19Arocas V. Bock S.C. Olson S.T. Björk I. Biochemistry. 1999; 38: 10196-10204Crossref PubMed Scopus (46) Google Scholar, 20Desai U. Swanson R. Bock S.C. Björk I. Olson S.T. J. Biol. Chem. 2000; 275: 18976-18984Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar), reflecting a larger contribution of Arg129 than of the other two residues to the binding. Kinetic studies showed that both Arg47 and Arg129 are involved in the second step of heparin binding (19Arocas V. Bock S.C. Olson S.T. Björk I. Biochemistry. 1999; 38: 10196-10204Crossref PubMed Scopus (46) Google Scholar, 20Desai U. Swanson R. Bock S.C. Björk I. Olson S.T. J. Biol. Chem. 2000; 275: 18976-18984Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar), predominantly by decreasing the reverse rate constant of this step and thus aiding in keeping antithrombin in its activated form. In contrast, they only moderately contribute to increasing the rate of induction of the conformational change. Analogous studies of the role of Lys125 in the kinetics of heparin binding are lacking. Similarly, although mutation of Lys114 has demonstrated the importance of this residue for heparin binding (16Kridel S.J. Chan W.W. Knauer D.J. J. Biol. Chem. 1996; 271: 20935-20941Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar, 17Kridel S.J. Knauer D.J. J. Biol. Chem. 1997; 272: 7656-7660Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar), its quantitative contribution to the binding and role in the binding kinetics are unknown. In this work, we have investigated the role of Lys114 in binding of heparin by mutation of this residue to Ala or Met. We find that these mutations decrease the affinity for pentasaccharide and full-length heparin by ∼105-fold, reflecting a considerably larger contribution to heparin binding of Lys114 than of the other residues investigated so far. Like Arg47 and Arg129, Lys114 does not participate to any appreciable extent in the first step of heparin binding but acts predominantly in the second step. However, it functions by both substantially increasing (>40-fold) the forward rate constant and greatly decreasing (>1000-fold) the reverse rate constant of this step. It is therefore of crucial importance both for induction of the heparin-induced conformational change that activates antithrombin and for locking the inhibitor in the conformationally activated state. Antithrombin variants with substitutions of Lys114 by Ala or Met were produced by site-directed mutagenesis with the previously characterized N135A variant as base molecule and were expressed in a baculovirus system (18Ersdal-Badju E. Lu A.Q. Zuo Y.C. Picard V. Bock S.C. J. Biol. Chem. 1997; 272: 19393-19400Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar, 19Arocas V. Bock S.C. Olson S.T. Björk I. Biochemistry. 1999; 38: 10196-10204Crossref PubMed Scopus (46) Google Scholar, 20Desai U. Swanson R. Bock S.C. Björk I. Olson S.T. J. Biol. Chem. 2000; 275: 18976-18984Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar, 21Ersdal-Badju E. Lu A. Peng X. Picard V. Zendehrouh P. Turk B. Björk I. Olson S.T. Bock S.C. Biochem. J. 1995; 310: 323-330Crossref PubMed Scopus (44) Google Scholar, 22Turk B. Brieditis I. Bock S.C. Olson S.T. Björk I. Biochemistry. 1997; 36: 6682-6691Crossref PubMed Scopus (104) Google Scholar). (It should be noted that a previously reported “K114A/N135A” mutant (18Ersdal-Badju E. Lu A.Q. Zuo Y.C. Picard V. Bock S.C. J. Biol. Chem. 1997; 272: 19393-19400Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar) that had the same heparin affinity as the control was actually K107A/N135A, i.e. had the wild type Lys at position 114. The K114A/N135A mutant used in the present work has the correct sequence and is not the “K114A/N135A” of Ref. 18Ersdal-Badju E. Lu A.Q. Zuo Y.C. Picard V. Bock S.C. J. Biol. Chem. 1997; 272: 19393-19400Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). The N135A, K114A/N135A, and K114M/N135A variants were purified by affinity chromatography on a 5-ml HiTrap Heparin (Amersham Pharmacia Biotech) column at pH 7.4, as detailed in earlier work (18Ersdal-Badju E. Lu A.Q. Zuo Y.C. Picard V. Bock S.C. J. Biol. Chem. 1997; 272: 19393-19400Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar, 19Arocas V. Bock S.C. Olson S.T. Björk I. Biochemistry. 1999; 38: 10196-10204Crossref PubMed Scopus (46) Google Scholar, 20Desai U. Swanson R. Bock S.C. Björk I. Olson S.T. J. Biol. Chem. 2000; 275: 18976-18984Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar, 22Turk B. Brieditis I. Bock S.C. Olson S.T. Björk I. Biochemistry. 1997; 36: 6682-6691Crossref PubMed Scopus (104) Google Scholar). However, to bind the K114A/N135A and K114M/N135A variants to the immobilized heparin, it was necessary to reduce the ionic strength of the filtered lysates by dilution with one-half volume of 20 mm sodium phosphate, 100 μm EDTA, pH 7.4, prior to loading of the columns. These variants were further purified by anion exchange chromatography on a Mono Q HR 5/5 column (Amersham Pharmacia Biotech), eluted with a 30-ml gradient from 0.02 to 0.6m NaCl in 20 mm sodium phosphate, 0.1% (w/v) polyethylene glycol 8000, pH 7.4. A Lys114 to Met antithrombin variant was also produced by site-directed mutagenesis with an N135Q variant as base molecule and was expressed in a baby hamster kidney cell system (23Gettins P.G.W. Fan B. Crews B.C. Turko I.V. Olson S.T. Streusand V.J. Biochemistry. 1993; 32: 8385-8389Crossref PubMed Scopus (75) Google Scholar, 24Turko I.V. Fan B. Gettins P.G.W. FEBS Lett. 1993; 335: 9-12Crossref PubMed Scopus (41) Google Scholar). The N135Q and K114M/N135Q variants were purified by affinity chromatography on heparin-agarose, followed by successive chromatographies on DEAE-Sepharose and Sephacryl S-200 (Amersham Pharmacia Biotech), as described previously (25Olson S.T. Björk I. Shore J.D. Methods Enzymol. 1993; 222: 525-560Crossref PubMed Scopus (267) Google Scholar). The purity of the antithrombin preparations was analyzed by SDS-polyacrylamide gel electrophoresis with the Tricine1 or Laemmli buffer systems (26Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207218) Google Scholar, 27Schägger H. von Jagow G. Anal. Biochem. 1987; 166: 368-379Crossref PubMed Scopus (10480) Google Scholar) and by nondenaturing polyacrylamide gel electrophoresis with the Laemmli buffer system. Concentrations of the three variants were determined from the absorbance at 280 nm with the use of the molar absorption coefficient of plasma antithrombin, 37,700 m−1 cm−1(28Nordenman B. Nyström C. Björk I. Eur. J. Biochem. 1977; 78: 195-203Crossref PubMed Scopus (193) Google Scholar). Human α-thrombin was a gift from Dr. J. Fenton (New York State Department of Health, Albany, NY). Human factor Xa was purified as described elsewhere (29Bock P.E. Craig P.A. Olson S.T. Singh P. Arch. Biochem. Biophys. 1989; 273: 375-388Crossref PubMed Scopus (83) Google Scholar). The synthetic antithrombin-binding normal (9Choay J. Petitou M. Lormeau J.C. Sinay P. Casu B. Gatti G. Biochem. Biophys. Res. Commun. 1983; 116: 492-499Crossref PubMed Scopus (596) Google Scholar) and high affinity (compound 83 in Ref.30van Boeckel C.A.A. Petitou M. Angew. Chem. 1993; 32: 1671-1690Crossref Scopus (378) Google Scholar) pentasaccharides and the nonreducing end trisaccharide unit of the pentasaccharide (DEF in Ref. 31Desai U.R. Petitou M. Björk I. Olson S.T. J. Biol. Chem. 1998; 273: 7478-7487Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar) were generous gifts from Dr. M. Petitou (Sanofi Recherche, Toulouse, France). Full-length heparin with high affinity for antithrombin was isolated as described previously (11Olson S.T. Björk I. Sheffer R. Craig P.A. Shore J.D. Choay J. J. Biol. Chem. 1992; 267: 12528-12538Abstract Full Text PDF PubMed Google Scholar, 22Turk B. Brieditis I. Bock S.C. Olson S.T. Björk I. Biochemistry. 1997; 36: 6682-6691Crossref PubMed Scopus (104) Google Scholar, 32Olson S.T. Björk I. J. Biol. Chem. 1991; 266: 6353-6364Abstract Full Text PDF PubMed Google Scholar) and had a molecular mass of ∼8000 Da (∼26 saccharides) and a reduced polydispersity. All experiments were carried out at 25.0 ± 0.2 °C. The buffer in most experiments was 20 mm sodium phosphate, 100 μm EDTA, 0.1% (w/v) polyethylene glycol 8000, adjusted to pH 6.0 or 7.4. The ionic strength of this buffer is 0.025 and 0.05 at the two pH values, respectively, and NaCl was added if higher ionic strengths were desired. However, 10 mm sodium phosphate, 100 μm EDTA, 0.1% (w/v) polyethylene glycol 8000 was used for measurements at I0.025, pH 7.4. Stoichiometries and dissociation equilibrium constants for the binding of the different heparin forms to the antithrombin variants were measured by titrations monitored by the enhancement of intrinsic protein fluorescence accompanying the interaction, as described previously (19Arocas V. Bock S.C. Olson S.T. Björk I. Biochemistry. 1999; 38: 10196-10204Crossref PubMed Scopus (46) Google Scholar, 20Desai U. Swanson R. Bock S.C. Björk I. Olson S.T. J. Biol. Chem. 2000; 275: 18976-18984Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar, 22Turk B. Brieditis I. Bock S.C. Olson S.T. Björk I. Biochemistry. 1997; 36: 6682-6691Crossref PubMed Scopus (104) Google Scholar, 25Olson S.T. Björk I. Shore J.D. Methods Enzymol. 1993; 222: 525-560Crossref PubMed Scopus (267) Google Scholar). Stoichiometries of full-length heparin binding to the N135A and N135Q variants were measured at I 0.15, pH 7.4, with antithrombin concentrations of 0.1–0.3 μm. Stoichiometries of pentasaccharide or full-length heparin binding to the K114A/N135A, K114M/N135A, and K114M/N135Q variants were determined at I 0.025, pH 6.0, and 1–2 μm antithrombin. Affinities of trisaccharide, pentasaccharide, or full-length heparin binding to the variants were determined at pH 6.0 or 7.4 and different ionic strengths with antithrombin concentrations of 50–500 nm. The data were fitted to the equilibrium binding equation by nonlinear least-squares analysis (25Olson S.T. Björk I. Shore J.D. Methods Enzymol. 1993; 222: 525-560Crossref PubMed Scopus (267) Google Scholar). The kinetics of binding of pentasaccharide or full-length heparin to the N135A and K114A/N135A antithrombin variants were analyzed under pseudo-first order conditions by monitoring the increase in protein fluorescence in an SX-17MV stopped-flow instrument (Applied Biophysics, Leatherhead, United Kingdom) as in earlier work (11Olson S.T. Björk I. Sheffer R. Craig P.A. Shore J.D. Choay J. J. Biol. Chem. 1992; 267: 12528-12538Abstract Full Text PDF PubMed Google Scholar, 19Arocas V. Bock S.C. Olson S.T. Björk I. Biochemistry. 1999; 38: 10196-10204Crossref PubMed Scopus (46) Google Scholar, 20Desai U. Swanson R. Bock S.C. Björk I. Olson S.T. J. Biol. Chem. 2000; 275: 18976-18984Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar, 22Turk B. Brieditis I. Bock S.C. Olson S.T. Björk I. Biochemistry. 1997; 36: 6682-6691Crossref PubMed Scopus (104) Google Scholar). Experiments with both saccharides were done at I 0.075, although at pH 6.0 for the pentasaccharide and at pH 7.4 for full-length heparin. Saccharide concentrations varied between 0.2 and 13 μm and were at least 10-fold higher than antithrombin concentrations. Progress curves were fitted to a single exponential function to give the observed pseudo-first order rate constant, kobs. Four traces were averaged for each rate constant determination, and reported kobs values are averages of at least four such determinations. Stoichiometries of inhibition of active site-titrated human α-thrombin by the antithrombin variants in the absence of heparin were measured essentially as detailed previously (19Arocas V. Bock S.C. Olson S.T. Björk I. Biochemistry. 1999; 38: 10196-10204Crossref PubMed Scopus (46) Google Scholar, 20Desai U. Swanson R. Bock S.C. Björk I. Olson S.T. J. Biol. Chem. 2000; 275: 18976-18984Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar, 25Olson S.T. Björk I. Shore J.D. Methods Enzymol. 1993; 222: 525-560Crossref PubMed Scopus (267) Google Scholar). Briefly, a series of samples of thrombin at a constant concentration of 0.1 or 0.5 μm was incubated with increasing amounts of antithrombin variant in I 0.15, pH 7.4 buffer. The residual activity of the enzyme was then determined after 16 h (for 0.1 μm thrombin) or 1–2 h (for 0.5 μm thrombin) from the initial rate of hydrolysis of the substrate, S-2238 (d-phenylalanyl-l-pipecolyl-l-arginyl-p-nitroanilide; Chromogenix, Mölndal, Sweden). Stoichiometries of thrombin inhibition by the N135A, K114A/N135A, N135Q, and K114M/N135Q variants in the presence of heparin at I 0.05, pH 7.4, were measured by incubating 20 nm thrombin with increasing concentrations of antithrombin variant and 25 nm full-length heparin for 1 h and assaying the residual enzyme activity in the same manner. The inhibition stoichiometries were obtained from linear least-squares fits of plots of residual enzyme activity versus the molar ratio of inhibitor to enzyme (25Olson S.T. Björk I. Shore J.D. Methods Enzymol. 1993; 222: 525-560Crossref PubMed Scopus (267) Google Scholar). Second order rate constants for inhibition of human α-thrombin or factor Xa by the N135A, K114A/N135A, N135Q, and K114M/N135Q variants in the absence and presence of pentasaccharide or full-length heparin were measured under pseudo-first order conditions, essentially as in earlier work (11Olson S.T. Björk I. Sheffer R. Craig P.A. Shore J.D. Choay J. J. Biol. Chem. 1992; 267: 12528-12538Abstract Full Text PDF PubMed Google Scholar, 19Arocas V. Bock S.C. Olson S.T. Björk I. Biochemistry. 1999; 38: 10196-10204Crossref PubMed Scopus (46) Google Scholar, 20Desai U. Swanson R. Bock S.C. Björk I. Olson S.T. J. Biol. Chem. 2000; 275: 18976-18984Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar, 22Turk B. Brieditis I. Bock S.C. Olson S.T. Björk I. Biochemistry. 1997; 36: 6682-6691Crossref PubMed Scopus (104) Google Scholar, 25Olson S.T. Björk I. Shore J.D. Methods Enzymol. 1993; 222: 525-560Crossref PubMed Scopus (267) Google Scholar, 31Desai U.R. Petitou M. Björk I. Olson S.T. J. Biol. Chem. 1998; 273: 7478-7487Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar, 33Björk I. Ylinenjärvi K. Olson S.T. Hermentin P. Conradt H.S. Zettlmeissl G. Biochem. J. 1992; 286: 793-800Crossref PubMed Scopus (53) Google Scholar). Uncatalyzed reactions with thrombin and factor Xa were analyzed at I 0.15 and 0.05, pH 7.4, and such reactions with factor Xa were also studied at I 0.025, pH 6.0. The rates of the pentasaccharide-catalyzed and full-length heparin-catalyzed reactions of the variants with the two proteinases were only measured at I 0.025, pH 6.0, and I0.05, pH 7.4, respectively. Reaction mixtures contained 100–2000 nm antithrombin and 5–10 nm proteinase, with or without 0.1–700 nm pentasaccharide or full-length heparin. The concentration of the saccharides was in most cases ≤10% of the antithrombin concentration. However, in the analyses of the pentasaccharide-catalyzed thrombin inhibition by the N135A and K114A/N135A variants, the pentasaccharide concentration approached and exceeded the antithrombin concentration, due to the small accelerating effect. After different reaction times, aliquots were diluted 10-fold in I 0.15, pH 7.4 buffer, containing 100 μmS-2238 for thrombin or Spectrozyme FXa (American Diagnostica, Greenwich, CT) for factor Xa, and the residual proteinase activity was determined. Observed pseudo-first order rate constants,kobs, were obtained by fitting the decrease of this activity with time to a single exponential decay function with an end point of zero activity (25Olson S.T. Björk I. Shore J.D. Methods Enzymol. 1993; 222: 525-560Crossref PubMed Scopus (267) Google Scholar). Second order rate constants for uncatalyzed reactions were obtained by dividing kobs with the antithrombin concentration. Most such rate constants for pentasaccharide- or full-length heparin-catalyzed reactions were derived from the least-squares slope of the linear dependence of kobs on the concentration of the antithrombin-saccharide complex, calculated from measured dissociation constants (11Olson S.T. Björk I. Sheffer R. Craig P.A. Shore J.D. Choay J. J. Biol. Chem. 1992; 267: 12528-12538Abstract Full Text PDF PubMed Google Scholar, 22Turk B. Brieditis I. Bock S.C. Olson S.T. Björk I. Biochemistry. 1997; 36: 6682-6691Crossref PubMed Scopus (104) Google Scholar, 31Desai U.R. Petitou M. Björk I. Olson S.T. J. Biol. Chem. 1998; 273: 7478-7487Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar, 33Björk I. Ylinenjärvi K. Olson S.T. Hermentin P. Conradt H.S. Zettlmeissl G. Biochem. J. 1992; 286: 793-800Crossref PubMed Scopus (53) Google Scholar). Alternatively, the rate constants for some catalyzed reactions were calculated from kobs measured at a single saccharide concentration by first subtracting kobs for the uncatalyzed reaction and then dividing by the calculated concentration of the antithrombin-saccharide complex (22Turk B. Brieditis I. Bock S.C. Olson S.T. Björk I. Biochemistry. 1997; 36: 6682-6691Crossref PubMed Scopus (104) Google Scholar, 31Desai U.R. Petitou M. Björk I. Olson S.T. J. Biol. Chem. 1998; 273: 7478-7487Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar, 33Björk I. Ylinenjärvi K. Olson S.T. Hermentin P. Conradt H.S. Zettlmeissl G. Biochem. J. 1992; 286: 793-800Crossref PubMed Scopus (53) Google Scholar), with several such values averaged. Uncatalyzed rate constants for thrombin inhibition by the N135A and K114A/N135A variants at I 0.025, pH 6.0, and I 0.05, pH 7.4, were obtained from the intercepts on the ordinate of the plots of kobs versusthe concentration of the antithrombin-heparin complex from which also the heparin-catalyzed rate constants were derived. K114A and K114M antithrombin variants were expressed on an N135A background in a baculovirus system, as in our previous studies of the roles of Arg47 and Arg129 of the inhibitor in heparin binding (19Arocas V. Bock S.C. Olson S.T. Björk I. Biochemistry. 1999; 38: 10196-10204Crossref PubMed Scopus (46) Google Scholar, 20Desai U. Swanson R. Bock S.C. Björk I. Olson S.T. J. Biol. Chem. 2000; 275: 18976-18984Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). In addition, a K114M variant was expressed on an N135Q background in a baby hamster kidney cell system. The N135A or N135Q substitutions produce antithrombin forms, corresponding to β-antithrombin in plasma, which have high heparin affinity due to the absence of an oligosaccharide side chain on Asn135 (21Ersdal-Badju E. Lu A. Peng X. Picard V. Zendehrouh P. Turk B. Björk I. Olson S.T. Bock S

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Lysine 114 of Antithrombin Is of Crucial Importance for the Affinity and Kinetics of Heparin Pentasaccharide Binding