Characterization of the Two-step Pathway for Inhibition of Thrombin by α-Ketoamide Transition State Analogs
1998; Elsevier BV; Volume: 273; Issue: 9 Linguagem: Inglês
10.1074/jbc.273.9.4843
ISSN1083-351X
AutoresSidney D. Lewis, Bobby J. Lucas, Stephen F. Brady, John T. Sisko, Kellie J. Cutrona, Philip E.J. Sanderson, Roger Freidinger, Shi‐Shan Mao, Stephen J. Gardell, Jules A. Shafer,
Tópico(s)Coagulation, Bradykinin, Polyphosphates, and Angioedema
ResumoThe interaction of thrombin with several potent and selective α-ketoamide transition state analogs was characterized. L-370,518 (H-N-Me-d-Phe-Pro-t-4-aminocyclohexylglycylN-methylcarboxamide) a potent (Ki = 90 pm) and selective (>104-foldversus trypsin) ketoamide thrombin inhibitor was shown to bind thrombin via a two-step reaction wherein the initially formed thrombin-inhibitor complex (EI1) rearranges to a more stable, final complex (EI2). A novel sequential stopped-flow analysis showed thatk−1, the rate constant for dissociation of EI1, was comparable tok2, the rate constant for conversion of EI1 to EI2 (0.049 and 0.035 s−1, respectively) indicating that formation of the initial complex EI1 is partially rate controlling. Replacement of the N-terminal methylamino group in L-370,518 with a hydrogen (L-372,051) resulted in a 44-fold loss in potency (Ki = 4 nm) largely due to an increase in k−1. Consequently in the reaction of L-372,051 with thrombin formation of EI1 was not rate controlling. Replacement of the P1′N-methylcarboxamide group of L-370,518 with an azetidylcarboxamido (L-372,228) produced a 58-fold increase in the value of the equilibrium constant (K−1) for dissociation of EI1. Nevertheless, L-372,228 was a 2-fold more potent thrombin inhibitor (Ki = 40 pm) than L-370,518 due to its 16-fold higherk2 and 10-fold lowerk−2 values. The desketoamide analogs of L-370,518 and L-372,051, namely L-371,912 and L-372,011, inhibited thrombin via a one-step process. The Ki value for L-371,912 and the K−1 value for its α-ketoamide analog, L-370,518, were similar (5 and 14 nm, respectively). Likewise, the Ki value for L-372,011 and the K−1 value for its α-ketoamide analog, L-372,051, were similar (330 and 285 nm, respectively). These observations are consistent with the view that the α-ketoamides L-370,518 and L-372,051 form initial complexes with thrombin that are similar to the complexes formed by their desketoamide analogs, and in a second step the α-ketoamides react with the active site serine residue of thrombin to form a more stable hemiketal adduct. The interaction of thrombin with several potent and selective α-ketoamide transition state analogs was characterized. L-370,518 (H-N-Me-d-Phe-Pro-t-4-aminocyclohexylglycylN-methylcarboxamide) a potent (Ki = 90 pm) and selective (>104-foldversus trypsin) ketoamide thrombin inhibitor was shown to bind thrombin via a two-step reaction wherein the initially formed thrombin-inhibitor complex (EI1) rearranges to a more stable, final complex (EI2). A novel sequential stopped-flow analysis showed thatk−1, the rate constant for dissociation of EI1, was comparable tok2, the rate constant for conversion of EI1 to EI2 (0.049 and 0.035 s−1, respectively) indicating that formation of the initial complex EI1 is partially rate controlling. Replacement of the N-terminal methylamino group in L-370,518 with a hydrogen (L-372,051) resulted in a 44-fold loss in potency (Ki = 4 nm) largely due to an increase in k−1. Consequently in the reaction of L-372,051 with thrombin formation of EI1 was not rate controlling. Replacement of the P1′N-methylcarboxamide group of L-370,518 with an azetidylcarboxamido (L-372,228) produced a 58-fold increase in the value of the equilibrium constant (K−1) for dissociation of EI1. Nevertheless, L-372,228 was a 2-fold more potent thrombin inhibitor (Ki = 40 pm) than L-370,518 due to its 16-fold higherk2 and 10-fold lowerk−2 values. The desketoamide analogs of L-370,518 and L-372,051, namely L-371,912 and L-372,011, inhibited thrombin via a one-step process. The Ki value for L-371,912 and the K−1 value for its α-ketoamide analog, L-370,518, were similar (5 and 14 nm, respectively). Likewise, the Ki value for L-372,011 and the K−1 value for its α-ketoamide analog, L-372,051, were similar (330 and 285 nm, respectively). These observations are consistent with the view that the α-ketoamides L-370,518 and L-372,051 form initial complexes with thrombin that are similar to the complexes formed by their desketoamide analogs, and in a second step the α-ketoamides react with the active site serine residue of thrombin to form a more stable hemiketal adduct. Our pursuit of novel antithrombotic agents has focused on direct inhibitors of thrombin, a trypsin-like serine proteinase that plays a key role in thrombosis. One strategy for the design of potent thrombin inhibitors is to replace the substrate P1 1The nomenclature of inhibitor residues and the corresponding enzyme pockets follow that of Schechter and Berger (23Schechter I. Berger A. Biochem. Biophys. Res. Commun. 1967; 27: 157-162Google Scholar).1The nomenclature of inhibitor residues and the corresponding enzyme pockets follow that of Schechter and Berger (23Schechter I. Berger A. Biochem. Biophys. Res. Commun. 1967; 27: 157-162Google Scholar). carboxamido group with an electrophilic keto or aldehyde group (1Mehdi S. Bioorg. Chem. 1993; 21: 249-259Google Scholar). Cyclotheonamide A is a naturally occurring proteinase inhibitor from a marine sponge (Theonella) that contains such a potency-enhancing keto group (2Fusetani N. Matsunaga S. Matsumoto H. Takebayashi Y. J. Am. Chem. Soc. 1990; 112: 7053-7054Google Scholar, 3Hagihara M. Schreiber S.L. J. Am. Chem. Soc. 1992; 114: 6570-6571Google Scholar, 4Wipf P. Kim H. J. Org. Chem. 1993; 58: 5592-5594Google Scholar). Cyclotheonamide A is a potent (Ki = 1 nm) reversible inhibitor of thrombin (5Lewis S.D. Ng A.S. Baldwin J.J. Fusetani N. Naylor A.M. Shafer J.A. Thromb. Res. 1993; 70: 173-190Google Scholar, 6Marynoff B.E. Greco M.N. Zhang H.-C. Andrade-Gordon P. Kauffman J.A. Nicolaou K.C. Liu A. Brungs P.H. J. Am. Chem. Soc. 1995; 117: 1225-1239Google Scholar) and several other trypsin-like proteinases (5Lewis S.D. Ng A.S. Baldwin J.J. Fusetani N. Naylor A.M. Shafer J.A. Thromb. Res. 1993; 70: 173-190Google Scholar, 7Maryanoff B.E. Qiu X. Padmanabhan K.P. Tulinsky A. Almond Jr., H.R. Andrade-Gordon P. Greco M.N. Kauffman J.A. Nicolaou K.C. Liu A. Brungs P.H. Fusetani N. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 8048-8052Google Scholar). Various α-ketoamide derivatives of tripeptide substrates of thrombin have been prepared in an attempt to identify selective, potent, reversible inhibitors of thrombin (8Lewis S.D. Ng A.S. Lyle E.A. Mellott M.J. Appleby S.D. Brady S.F. Stauffer K.J. Sisko J.T. Mao S.-S. Veber D.F. Nutt R.F. Lynch J.J. Cook J.J. Gardell S.J. Shafer J.A. Thromb. Haemostasis. 1995; 74: 1107-1113Google Scholar, 9Brady S.F. Sisko J.T. Stauffer K.J. Colton C.D. Qiu H. Lewis S.D. Ng A.S. Shafer J.A. Bogusky M.J. Veber D.F. Nutt R.F. Bioorg. & Med. Chem. Lett. 1995; 3: 1063-1078Google Scholar). Placement of at-4-AChxGly 2The abbreviations used are:t-4-AChxGly, trans-4-aminocyclohexylglycine; Z, benzyloxycarbonyl; GPR, Gly-Pro-Arg; afc, 7-amino-4-trifluoromethylcoumarin; DAPA, dansylarginine-N-(3-ethyl-1,5-pentanediyl)amide.2The abbreviations used are:t-4-AChxGly, trans-4-aminocyclohexylglycine; Z, benzyloxycarbonyl; GPR, Gly-Pro-Arg; afc, 7-amino-4-trifluoromethylcoumarin; DAPA, dansylarginine-N-(3-ethyl-1,5-pentanediyl)amide.residue at the P1 position in α-ketoamide thrombin inhibitors provided excellent selectivity for thrombin, relative to tryspin (10Brady S.F. Lewis S.D. Colton C.D. Stauffer K.J. Sisko J.T. Ng A.S. Homnick C.F. Bogusky M.J. Shafer J.A. Veber D.F. Nutt R.F. Kaumaya P.T.P. Hodges R.S. Peptides: Chemistry, Structure and Biology. Mayflower Scientific, Ltd., London1995: 331-333Google Scholar, 11Cutrona K.J. Sanderson P.E.J. Tetrahedron Lett. 1996; 37: 5045-5048Google Scholar, 12Lyle T.A. Chen Z. Appleby S.D. Freidinger R.M. Gardell S.J. Lewis S.D. Li Y. Lyle E.A. Lynch Jr., J.J. Mulichak A.M. Ng A.S. Naylor-Olsen A.M. Sanders W.M. Bioorg. & Med. Chem. Lett. 1997; 7: 67-72Google Scholar), probably because the S1 specificity pocket of thrombin is larger than that of trypsin (13Bode W. Turk D. Karshikov A. Protein Sci. 1992; 1: 426-471Google Scholar, 14Chen Z. Li Y. Mulichak A.M. Lewis S.D. Shafer J.A. Arch. Biochem. Biophys. 1995; 322: 198-203Google Scholar). In the present study, we show that α-ketoamide inhibitors containingt-4-AChxGly inactivate thrombin via a two-step reaction, wherein an initially formed weak complex (EI1) rearranges to a more stable thrombin-inhibitor complex (EI2) (Scheme FSI). Inhibitors that inactivate enzymes via a two-step pathway are usually assumed to formEI1 rapidly in a preequilibrium reaction wherek−1 is assumed to be much greater thank2 (15Morrison J.F. Trends Biochem. Sci. 1982; 7: 102-105Google Scholar). Sculley et al. (16Sculley M.J. Morrison J.F. Cleland W.W. Biochim. Biophys. Acta. 1996; 1298: 78-86Google Scholar) recently discussed the difficulty in distinguishing cases wherek−1 ≫ k92 from those where k−1 ∼ k2. We now describe a novel sequential stopped-flow analysis that circumvents this difficulty and allowed us to rigorously evaluate kinetic pathways for inactivation of thrombin by a family of active site-directed thrombin inhibitors. Methods for the synthesis of the α-ketoamide and desketoamide derivatives of H-N-Me-d-Phe-Pro-4-t-AChxGly have been reported elsewhere (Table I and Refs. 9Brady S.F. Sisko J.T. Stauffer K.J. Colton C.D. Qiu H. Lewis S.D. Ng A.S. Shafer J.A. Bogusky M.J. Veber D.F. Nutt R.F. Bioorg. & Med. Chem. Lett. 1995; 3: 1063-1078Google Scholar, 10Brady S.F. Lewis S.D. Colton C.D. Stauffer K.J. Sisko J.T. Ng A.S. Homnick C.F. Bogusky M.J. Shafer J.A. Veber D.F. Nutt R.F. Kaumaya P.T.P. Hodges R.S. Peptides: Chemistry, Structure and Biology. Mayflower Scientific, Ltd., London1995: 331-333Google Scholar, 11Cutrona K.J. Sanderson P.E.J. Tetrahedron Lett. 1996; 37: 5045-5048Google Scholar, 12Lyle T.A. Chen Z. Appleby S.D. Freidinger R.M. Gardell S.J. Lewis S.D. Li Y. Lyle E.A. Lynch Jr., J.J. Mulichak A.M. Ng A.S. Naylor-Olsen A.M. Sanders W.M. Bioorg. & Med. Chem. Lett. 1997; 7: 67-72Google Scholar). The inhibitor concentrations were determined from titration with a known amount of thrombin as described previously (5Lewis S.D. Ng A.S. Baldwin J.J. Fusetani N. Naylor A.M. Shafer J.A. Thromb. Res. 1993; 70: 173-190Google Scholar). DansylarginineN-(3-ethyl-1,5-pentanediyl)amide (DAPA) was obtained from American Diagnostica. Concentrations of DAPA were determined from measurements of absorbance at 330 nm using an extinction coefficient of 4.01 cm−1 mm−1 (17Nesheim M.E. Prendergast F.G. Mann K.G. Biochemistry. 1979; 18: 996-1003Google Scholar). The sources of other materials were described previously (5Lewis S.D. Ng A.S. Baldwin J.J. Fusetani N. Naylor A.M. Shafer J.A. Thromb. Res. 1993; 70: 173-190Google Scholar, 8Lewis S.D. Ng A.S. Lyle E.A. Mellott M.J. Appleby S.D. Brady S.F. Stauffer K.J. Sisko J.T. Mao S.-S. Veber D.F. Nutt R.F. Lynch J.J. Cook J.J. Gardell S.J. Shafer J.A. Thromb. Haemostasis. 1995; 74: 1107-1113Google Scholar). Proteinase assays were performed at room temperature in 50 mm Tris, pH 7.5, 150 mm NaCl, and 0.1% polyethylene glycol 8000 unless otherwise indicated.Table IStructures of thrombin inhibitors Open table in a new tab The inhibition constants were determined as described previously (5Lewis S.D. Ng A.S. Baldwin J.J. Fusetani N. Naylor A.M. Shafer J.A. Thromb. Res. 1993; 70: 173-190Google Scholar, 8Lewis S.D. Ng A.S. Lyle E.A. Mellott M.J. Appleby S.D. Brady S.F. Stauffer K.J. Sisko J.T. Mao S.-S. Veber D.F. Nutt R.F. Lynch J.J. Cook J.J. Gardell S.J. Shafer J.A. Thromb. Haemostasis. 1995; 74: 1107-1113Google Scholar, 9Brady S.F. Sisko J.T. Stauffer K.J. Colton C.D. Qiu H. Lewis S.D. Ng A.S. Shafer J.A. Bogusky M.J. Veber D.F. Nutt R.F. Bioorg. & Med. Chem. Lett. 1995; 3: 1063-1078Google Scholar). When total inhibitor ([It]) and enzyme ([Et]) concentrations were comparable, the quadratic equation (18Williams J.W. Morrison J.F. Methods Enzymol. 1979; 63: 437-467Google Scholar, 19Henderson P.J.F. Biochem. J. 1972; 127: 321-333Google Scholar, 20Cha S. Biochem. Pharmacol. 1975; 24: 2177-2185Google Scholar) for tight-binding inhibitors (Equation 1) was used to calculate the apparent inhibition constant (Ki*) from the dependence of substrate hydrolysis on [It] and [Et], where Vi and Vo represent the initial rates of substrate hydrolysis in the presence and absence of inhibitor, respectively.Vi/Vo={([Et]−[It]−Ki*)+[([It]+Ki*−[Et])2+4Ki*[Et]]1/2}/2[Et]Equation 1 When [It] ≫ [Et], the variation of Vi with [It] is described by Equation 2.Vo/Vi=1+[It]/Ki*Equation 2 In assays where the substrate fully equilibrates with inhibitor and enzyme (i.e. analysis of progress curves, see below), Equation 3 relates the apparent inhibition constant,Ki*, to the intrinsic, final inhibition constantKi.Ki=Ki*/(1+[S]/Km)Equation 3 In assays where the inhibitor and enzyme were preequilibrated (i.e. before addition of substrate), and the rate of dissociation of inhibitor from enzyme was slow, and didn't occur during the time of the activity assay, the use of Equation 3 is inappropriate and the apparent inhibition constant,Ki*, is equal to Ki. Activity assays for the determination of Ki were routinely performed at [S] < Km; hence,Ki ≅ Ki* and the decision of when it was appropriate to use Equation 3 were circumvented. The overall dissociation constant (Ki) is defined by Equations 4 and 5 for the one- and two-step pathways, respectively (Scheme FSI).Ki=[E][I][EI]=K−1=k−1/k1Equation 4 Ki=[E][I][EI1]+[EI2]=K−1k−2k−2+k2.Equation 5 The rate of inhibitor binding to thrombin was followed by displacement of the fluorescent probe p-aminobenzamidine from the active site of thrombin. The decrease in fluorescence (F) was monitored using an Applied Photophysics stopped-flow spectrometer (DX.17MV) interfaced with an Archimedes 420/I computer as described previously (5Lewis S.D. Ng A.S. Baldwin J.J. Fusetani N. Naylor A.M. Shafer J.A. Thromb. Res. 1993; 70: 173-190Google Scholar, 8Lewis S.D. Ng A.S. Lyle E.A. Mellott M.J. Appleby S.D. Brady S.F. Stauffer K.J. Sisko J.T. Mao S.-S. Veber D.F. Nutt R.F. Lynch J.J. Cook J.J. Gardell S.J. Shafer J.A. Thromb. Haemostasis. 1995; 74: 1107-1113Google Scholar). Rate constants were derived from analysis of the average of 4–7 replicate traces with 1000–4000 data points per trace. Typically,p-aminobenzamidine (100–600 μm) was mixed with 0.25–1 μm thrombin prior to reaction with an equal volume of inhibitor (2.5–80 μm). When decay of the fluorescent signal was monophasic, Equation 6 (“single exponential with floating end point,” Applied Photophysics software) was used to evaluate the kinetic parameter kobs.F=(Fo−Ff)e(−kobst)+FfEquation 6 In Equation 6, F is the measured fluorescence at timet, kobs is the apparent first-order rate constant for the approach of F to its final value Ff, and Fo corresponds to the fluorescence at t = 0. The dependence of the pseudo-first-order rate constant (kobs) for displacement of p-aminobenzamidine is a linear function of [It]. Equation 7 was used to obtain k1, where [I]eff = [It]/(1 + [P]/Kp) and Kp (the equilibrium constant for dissociation of the enzyme-p-aminobenzamide complex) is equal to 47 μm (5Lewis S.D. Ng A.S. Baldwin J.J. Fusetani N. Naylor A.M. Shafer J.A. Thromb. Res. 1993; 70: 173-190Google Scholar).kobs=k−1+k1[I]effEquation 7 When the decay of fluorescence accompanying probe displacement was a biphasic process, Equation 8 (“double exponential with floating end point,” Applied Photophysics software) was used to yield the kinetic parameters kobs1 and kobs2.F=Ae(−kobs1t)+Be(−kobs2t)+FfEquation 8 In Equation 8 kobs1 and kobs2 are the apparent first-order rate constants for the approach of F to its final value, and A and B are amplitude terms associated with their corresponding first-order processes. The dependence on [It] of the pseudo-first-order rate constants, kobs1and kobs2, for displacement of p-aminobenzamidine was a linear and hyperbolic function of [I]eff, respectively, and was fit by Equations 9 and 10for evaluation of k1, k2, and Ki, init.kobs1=k−1+k1[I]effEquation 9 kobs2=k−2+k2[I]effKi,init+[I]effEquation 10 Stopped-flow experiments also yielded progress curves for thrombin-mediated hydrolysis of Z-GPR-afc (400-nm excitation with a 455-nm emission block) in the presence of inhibitor (5Lewis S.D. Ng A.S. Baldwin J.J. Fusetani N. Naylor A.M. Shafer J.A. Thromb. Res. 1993; 70: 173-190Google Scholar, 8Lewis S.D. Ng A.S. Lyle E.A. Mellott M.J. Appleby S.D. Brady S.F. Stauffer K.J. Sisko J.T. Mao S.-S. Veber D.F. Nutt R.F. Lynch J.J. Cook J.J. Gardell S.J. Shafer J.A. Thromb. Haemostasis. 1995; 74: 1107-1113Google Scholar). Briefly, inhibitor was mixed with Z-GPR-afc (22 μm) prior to reaction with an equal volume of 5–20 nm thrombin. Substrate depletion was less than 10% during the run. Rates were measured under pseudo-first-order conditions (i.e.[inhibitor] ≫ [thrombin], [Z-GPR-afc] ≫ [thrombin], and [Z-GPR-afc] < Km). The best fit for a single exponential decay was “single exponential with steady state” (from the kinetic software package supplied by Applied Photophysics), or alternatively, the data were transferred to Kaleidagraph (version 3.0.5 Abelbeck Software) and fitted using Equation 11. Kaleidagraph utilizes the Levenberg-Marquardt algorithmF=Vst+(Vi,init−Vs)[1−exp(−kobst)]/kobs+FoEquation 11 for nonlinear least-squares regression. Equation 11 as developed by Williams and Morrison (18Williams J.W. Morrison J.F. Methods Enzymol. 1979; 63: 437-467Google Scholar) and by Cha (20Cha S. Biochem. Pharmacol. 1975; 24: 2177-2185Google Scholar) is commonly used in the analysis of monophasic progress curves. In Equation 11, F is the measured fluorescence defined as a function of the initial (Vi, init) and final (Vs) steady state velocities (change in fluorescence per unit time due to thrombin-catalyzed substrate hydrolysis) and the apparent first-order rate constant (kobs) for the approach of enzymic activity to its final value. To monitor directly the time-dependent inhibition of thrombin in the presence of a substrate, the following analysis was employed. At long times (kobs t ≫ 1) where Equation11 reduces to Equation 12, F is a linear function of time. Extrapolation of the linear time dependenceF=Vst+(Vi,init−Vs)/kobs+FoEquation 12 of F at long times to zero time and determination of the difference (Δ) between the values of F on the extrapolated linear plot (Equation 12) and those on the plot describing the time dependence of the experimentally determined values of F (Equation 11) should provide an indication of the time-dependent approach of enzymic activity to its final value as indicated by Equation 13 where Δo is the value of Δ at t = 0.Δ/Δo=exp(−kobst)Equation 13 When the time-dependent decrease in the parameter Δ/Δo was biphasic, the dependence of F on time was fit by Equation 14 where C and A are constants.F=Vst+C[1−Aexp(−kobs1t)−(1−A)exp(−kobs2t)]+FoEquation 14 As in the case of the monophasic process described above, the dependence of F on time should become linear at long times (Equation15). Extrapolation of the linear portionF=Vst+C+FoEquation 15 of the F versus time plot to zero time and determination of the differences (Δ) between the values of F on the extrapolated plot and the plot defined by the observed time dependence of F yields a biphasic time dependence for the parameter Δ/Δo as indicated by Equation 16.Δ/Δo=Aexp(−kobs1t)+(1−A)exp(−kobs2t)Equation 16 It is important to note that the relative magnitude of the amplitude factors A and 1 − A reflect the relative amount of hydrolysis products formed in the fast and slow phases and not the relative amount of enzyme inactivated in the slow and fast phases. When the time-dependent approach of enzymic activity to its final value was monophasic, the dependence of kobs on [I]eff (where [I]eff = [It]/(1 + [S]/Km)) was fit by Equation 7 to obtaink1. When the approach of enzymic activity to its final value was biphasic, the time dependence of fluorescence was fit by Equations 14 or 16 to determine kobs1,kobs2, and A (the fraction of the biphasic reaction described by kobs1). The dependence of kobs1 and kobs2 on [I]eff was fit by Equations Equation 9, Equation 10 to obtain the parameters k1,k2, and Ki, init. The values for k−1 and k−2 could not always be determined accurately from the fit of the data by Equations7, 9, and 10. In such cases, other methods were used to determine their values (see below). The value of k−2 was determined from the time-dependent regeneration of free enzyme as measured by the hydrolysis of a fluorogenic substrate (Scheme FSII). Thrombin was preincubated with inhibitor at a concentration much greater thanKi and for sufficient amount of time to ensure complete formation of EI2. After preincubation the enzyme-inhibitor complex was diluted into a solution containing 60 μm d-Phe-Pro-Arg-afc ([S] ≫Km, Km = 0.3 μm). The resulting progress curve was fit to Equation 11 to obtainkobs which is equivalent tokoff. Equation 17 was used to relatekoff to the rate constants for the two-step pathway depicted in Schemes FSI and FSII.koff=k−2k−1k−1+k2Equation 17 For very potent inhibitors, k−2 was exceedingly small and the long times required for complete regeneration of enzyme compromised enzyme stability. In these casesVs was determined in a separate experiment from an identical dilution of enzyme with substrate. The experimentally determined value of Vs was fixed in Equation 11, and the initial data from the progress curve (∼3–5 half-lives) was fitted by nonlinear regression to Equation 11 to determinekoff. To determine the value of k−1, a sequential stopped-flow method was used. Equal volumes of thrombin and inhibitor ([I]t ≫Ki, init) were aged long enough (first mix) to ensure complete formation of EI1 but with minimal formation of EI2 (see Scheme FSI). After preincubation (first mix), EI1 was diluted (second mix) with an equal volume of DAPA (Scheme FSIII). There is an increase in fluorescence when DAPA binds to the active site of thrombin (excitation 280 nm, emission block 420 nm (17Nesheim M.E. Prendergast F.G. Mann K.G. Biochemistry. 1979; 18: 996-1003Google Scholar, 21Nesheim M.E. J. Biol. Chem. 1983; 258: 14708-14717Google Scholar)). A high concentration of DAPA (≥40 μm) ensured that the pseudo-first-order rate constant (kDAPA[DAPA]) for the reaction rate of E with DAPA was much greater thank−1 + k2 and k1[I]; hence, the reaction of thrombin with DAPA is not rate-limiting. In Scheme FSIII the pseudo-first-order rate constant, kobs, for regeneration of Efrom EI1 is described by Equation 18.kobs=k−1+k2Equation 18 The value of kobs was estimated using Equation 11 to fit the time-dependent increase of fluorescence associated with DAPA binding to the active site of thrombin concomitant with inhibitor displacement. Equation 11 was used to correct for the small but detectable steady state drift due to photo-bleaching of DAPA. In a typical experiment for the determination of k−1 + k2 for the reaction of thrombin with L-370,518, 0.8 μm thrombin was aged with an equal volume of 4 μm L-370,518 (first mix). After 1 s, the aged solution was mixed (second mix) with an equal volume of 80 μm DAPA. Treatment of thrombin with L-371,912 or the corresponding α-ketoamide analog L-370,518 inhibited thrombin-catalyzed hydrolysis of a fluorogenic substrate (Fig. 1). The equilibrium constants (Ki) for dissociation of the complexes between thrombin and the desketoamide L-371,912 and the ketoamide L-370,518 were 5 ± 0.5 nm and 90 ± 10 pm, respectively. The titrations shown in the figure insets established that these inhibitors form 1:1 complexes with thrombin that are devoid of catalytic activity. Kinetic analysis of the binding of the desketoamide L-371,912 and ketoamide L-370,518 to thrombin suggested that they bind to thrombin via different kinetic pathways. Fig. 2shows the monophasic and biphasic time-dependent inhibition of thrombin obtained upon mixing thrombin with desketoamide or ketoamide, respectively, and a fluorogenic substrate. The data in Fig. 2 were fit by a monoexponential (desketoamide, Equation 13) and biexponential (ketoamide, Equation 16) decay equations to yield pseudo-first-order rate constants for desketoamide L-371,912, (kobs), and ketoamide L-370,518 (kobs1, kobs2) of 2.9, 1.36, and 0.034 s−1, respectively. Figs. 3 and4 A illustrate the linear dependence of kobs (desketoamide) and kobs1 (ketoamide) on the inhibitor concentration. In contrast the dependence of kobs2 on the concentration of ketoamide was hyperbolic (Fig. 4 B). Both the monophasic time-dependent inhibition of thrombin by the desketoamide L-371,912 and the linear dependence of the corresponding pseudo-first-order rate constant on the inhibitor concentration suggest that the desketoamide binds to thrombin via a single one-step process. A two-step pathway (Scheme FSI) is required to account for the biphasic time-dependent inhibition of thrombin by the ketoamide L-370,518 and the linear and hyperbolic dependence of kobs1 and kobs2 on inhibitor concentration. A two-step pathway (Scheme FSI) involves formation of an initial complex (EI1) that subsequently rearranges to a more stable complex (EI2). The best fit of kobs (Fig. 3) and kobs1(Fig. 4 A) by Equations 7 and 9 yieldedk1 values of 9.4 ± 0.5 and 3.6 ± 0.3 μm−1 s−1 for the desketoamide and ketoamide, respectively. The dependence of kobs2 on the effective inhibitor concentration can be represented by Equation 10, whereinKi, init is equivalent tok−1/k1 whenk−1 ≫ k2. The best fit of kobs2 for the ketoamide L-370,518 to Equation 10 (Fig. 4 B) yielded k2 and Ki, init values of 0.035 ± 0.003 s−1 and 15 ± 5 nm, respectively. The transient accumulation and decay of EI1 gives rise to the biphasic ligand binding kinetics associated with a two-step pathway. At lower inhibitor concentrations, EI1accumulation decreases and the biphasic binding process becomes monophasic. This situation made it difficult to determinekobs2 at inhibitor concentrations <Ki, init and is responsible for the uncertainty in the value of Ki, initdetermined from the fit of the data in Fig. 4 B to Equation10.Figure 4Dependence of the rate constantskobs1 (A) and kobs2 (B) on L-370,518 concentration under stopped-flow conditions. Thrombin (2.5–50 nm) was mixed with an equal volume of 23 μmZ-GPR-afc and L-370,518 (final concentaration, 0.025–1 μm). A, the solid line represents the best fit of Equation 9 to the data. The solid circlesare the pseudo-first-order rate constants (kobs) for displacement (as derived from stopped-flow) of 300 μm p-aminobenzamidine from 0.5 μm thrombin by 2.5 and 5 μm L-370,518. To compare the different experimental procedures, the concentration of L-370,518 was adjusted to yield [L-370,518]eff as described under “Experimental Procedures.” B, the solid line represents the best nonlinear least squares fit of Equation 10 to the data.View Large Image Figure ViewerDownload (PPT) To characterize further the reaction pathway for the binding of the desketoamide L-371,912 and ketoamide L-370,518 to thrombin, the fluorescent probep-aminobenzamidine was employed to monitor ligand binding to thrombin. In the concentration ranges studied,p-aminobenzamidine displacement by L-371,912 or L-370,518 was a monophasic first-order process (Fig. 5, inset). The linear dependence of the pseudo-first-order rate constant (kobs) for p-aminobenzamidine displacement (Equation 7) on the concentration of L-371,912 and L-370,518 (Fig. 5) yielded k1 values of 8.2 ± 0.3 and 3.4 ± 0.2 μm−1s−1 for binding of the desketoamide L-371,912 and ketoamide L-370,518 to thrombin, respectively. The similarity of thesek1 values to those obtained from the plots depicted in Fig. 3 and 4 A (Table II) indicates that these second-order rate constants are truly equivalent tok1.Table IIKinetic constants for the binding of inhibitors to thrombinCompoundKi2-aData were determined from the inhibitory effect of inhibitor on the steady state velocity for thrombin-catalyzed hydrolysis of a fluorogenic substrate.k1k−12-bData were determined from sequential stopped-flow unless otherwise stated.k2k−2K−12-cK−1 was determined from the ratiok−1/k1.Ki,init2-dKi,init was determined from (k−1 +k2)/k1.k2/Ki,initnmμm−1s−1s−110−4s−1nmμm−1s−1L-370,3102.82.40.007L-372,112120028342-eDetermined from the equation k−1 =Ki × k1.L-370,5180.093.50.0490.0353.614241.5L-371,91259.40.054L-372,05144.71.340.05482852970.18L-372,01133072.4L-372,2280.040.520.420.560.3580819000.292-a Data were determined from the inhibitory effect of inhibitor on the steady state velocity for thrombin-catalyzed hydrolysis of a fluorogenic substrate.2-b Data were determined from sequential stopped-flow unless otherwise stated.2-c K−1 was determined from the ratiok−1/k1.2-d Ki,init was determined from (k−1 +k2)/k1.2-e Determined from the equation k−1 =Ki × k1. Open table in a new tab The apparent discrepancy between the biphasic inhibition of thrombin-catalyzed substrate hydrolysis and the monophasicp-aminobenzamidine displacement observed in the case of L-370,518 stems from the differences in the two experimental methods. With the elevated L-370,518 concentrations used for the p-aminobenzamidine displacement studies (2.5–25 μm), only a small amount of free enzyme is present at the end of the first phase (Ki, init ∼15 nm, see Fig. 4 B); hence, the binding of E to I to form EI1 is the dominant reaction. The amplitude of the second phase, which is governed by the conversion of residual E to EI1 and EI2, is too small to measure by the p-aminobenzamidine displacement method. On the other hand, the activity assay can detect the small amount of active enzyme present at the end of the first phase and measure its subsequent decay during the second phase. According to Equation 7, the y intercept of the linear plots of kobs versus inhibitor concentration (Figs. 3, 4 A, a
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