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Mechanistic Studies on Thrombin Catalysis Stuart R. Stone,* Andreas Betz, and Jan Hofsteenge Friedrich Miescher- Institut, P.O. Box 2543, CH-4002 Basel, Switzerland Received May 13, 1991; Revised Manuscript Received July 23, 1991
of the cleavage of four p-nitroanilide (pNA) substrates by human a-thrombin has been investigated by using a number of steady-state kinetic techniques. Solvent isotope and viscosity effects were used to determine the stickiness of the substrates a t the pH optimum of the reaction; a sticky substrate is defined as one that undergoes catalysis faster than it dissociates from the Michaelis complex. Whereas benzoyl-Arg-pNA could be classified as a nonsticky substrate, D-Phe-pipecolyl-Arg-pNA was very sticky. The other two substrates (tosyl-Gly-Pro-Arg-pNA and acetyl-D-Phe-pipecolyl-Arg-pNA) were slightly sticky. The pH profiles of k,,/K, were bell-shaped for all substrates. The pKa values determined from the pH dependence of k,,/K, for benzoyl-Arg-pNA were about 7.5 and 9.1. Similar pKa values were determined from the pH profiles of k,,/K, for tosyl-Gly-Pro-Arg-pNA and acetyl-D-Phe-pipecolyl-Arg-pNA amide. The groups and for the binding of the competitive inhibitor Na-dansyl-~-arginine-4-methylpiperidine responsible for the observed pK, values were proposed to be His57 and the a-amino group of Ile16. The temperature dependence of the pKa values was consistent with this assignment. The pK, values of 6.7 and 8.6 observed in the pH profile of k,,/K, for D-Phe-pipecolyl-Arg-pNA were displaced to lower values than those observed for the other substrates. The displacement of the acidic pKa value could be attributed to the stickiness of this substrate. The basic pK, value of 8.6 was caused by preferential binding of substrate molecules with protonated a-amino groups. The pH dependence of k,, for benzoyl-Arg-pNA was consistent with the binding of the substrate decreasing the pKa of His57 by more than 1 unit. The p H dependence of k,, for the other three substrates was more complicated and suggested that the rate of catalysis was influenced by the ionization of groups not directly involved in catalysis. ABSTRACT: The kinetic mechanism
Z r o m b i n is a serine protease with considerable homology to chymotrypsin in trypsin in terms of both primary and tertiary structure (Bode et al., 1989). Like trypsin, thrombin shows a preference for cleaving substrates C-terminal to a basic Correspondence should be addressed to this author at his present address: Department of Haematology, University of Cambridge, MRC Centre, Hills Road, Cambridge CB2 2QH, U.K.
0006-2960/91/0430-9841$02.50/0
residue, but the substrate specificity of thrombin is more restricted than that of trypsin (Chang, 1986; Lottenberg et al., 1981). In addition, thrombin appears to use sites that are distant from the active site in order to achieve specific interactions with macromolecular substrates, cofactors, and inhibitors (Fenton, 1981; Fenton & Bing, 1986). The recently determined crystal structures of D-Phe-Pro-ArgCH2-thrombin (Bode et al., 1989) and hirudin-thrombin complexes (Rydel 0 1991 American Chemical Society
9842 Biochemistry, Vol. 30, No. 41, 1991 et al., 1990; Griitter et al., 1990) provide some idea of the structural basis for thrombin's specificity. An insertion loop (Tyr60A-Trp60D)' of thrombin restricts the active site and this probably explains why so few macromolecular substrates and inhibitors of serine proteases bind well to thrombin. In addition, hydrophobic regions within the active-site cleft would appear to be important for the binding of substrates and inhibitors (Bode et al., 1989). The inhibitor hirudin also uses binding sites distant from the catalytic center. The C-terminal region of hirudin is bound to a surface groove that has been termed the anion-binding exosite, and it seems likely that this region of thrombin also interacts with the substrate fibrinogen and the cell-surface protein thrombomodulin (Rydel et al., 1990; Griitter et al., 1990). The kinetic mechanism of cleavage of p-nitroanilide substrates by thrombin has not been extensively investigated. The careful studies by Lottenberg et al. (1981, 1983) have documented the substrate specificity of the enzyme, and a recent study has investigated the pH and salt dependence of the kinetic parameters (De Cristofaro & Di Cera, 1990). However, a number of points remain to be established; for example, which catalytic step is rate limiting in the cleavage of p nitroanilide substrate (acylation or deacylation) and to what extent the binding step preceding the catalytic step comes to equilibrium with different substrates. In the present paper, we report the results of investigations designed to answer such questions. A range of steady-state kinetic techniques, including the study of viscosity, solvent isotope, and pH effects, has been used to determine the kinetic mechanism of the thrombincatalyzed cleavage of four p-nitroanilide substrates.
Stone et al. product was recovered by lyophilization. Yield: 1.1 g (49%). The final product gave one spot (Rf= 0.58) on TLC with butanol-acetic acid-water (3: 1:1) which was fluorescent under long-wavelength UV light. The absorption spectrum showed maxima at 246 and 330 nm. Amidolytic assays were performed in a three-buffer system containing 0.1 M MES, 0.05 M Tris, 0.05 M ethanolamine, 0.2 M NaCI, 0.2% (w/v) poly(ethy1ene glycol) of M, 6000, and substrate. The pH of the assay buffer was adjusted to the desired pH at about the temperature of the assay. The precise pH of the assay was measured later at the temperature used with a Radiometer PHM83 pH meter. In experiments in which assays were conducted in D20, the pD was taken to equal the pH meter reading plus 0.4 (Fersht, 1985). The three-buffer system was chosen because it provided adequate buffering capacity over the pH range used (pH 6-10) and the ionic strength varied only slightly over this range (Ellis & Morrison, 1982). The release of pnitroaniline due to cleavage of the substrates was monitored at 400-410 nm by using a Hewlett-Packard 8452A spectrophotometer. The extinction change for the reaction under the conditions of the assay was determined to be 9300 M-' cm-'. The concentrations of the substrates were determined from their absorbance at 342 nm as described by Lottenberg and Jackson (1983). For the determination of k,,, and K, values, six different substrate concentrations that varied over an 1 1-fold range were used. Dilutions of substrate were made as recommended by Fromm (1979, and at least one concentration was above and one below the K, value. At pH values above 9, a significant nonenzymatic rate of hydrolysis of the substrates tos-GlyPro-Arg-pNA and Bz-Arg-pNA was observed and this rate was subtracted from the enzyme-catalyzed rate. The concentration of thrombin used was chosen such that measurable initial rates would be obtained and less than 10% of the substrate would be utilized in the first 5 min of the assay. The concentration of thrombin used varied from 10 pM for assays with D-Phe-Pip-Arg-pNA to 0.2 pM for those with Bz-ArgpNA. The inhibition of thrombin by the substrate analogue DAMPA was studied in the presence of 100 pM D-Phe-PipArg-pNA and with 50 pM enzyme. For experiments examining the effect of viscosity on the kinetic parameters, the viscosity of the assay was determined at 37 OC by using an Ubbelodhe viscometer.
EXPERIMENTAL PROCEDURES Materials. The substrates Bz-Arg-pNA,2 tos-Gly-ProArg-pNA (Chromozym TH), and D-Phe-Pip-Arg-pNA (S2238) were obtained from Bachem (Bubendorf, Switzerland), Boehringer-Mannheim (Mannheim, Germany) and KabiVitrum (Molndal, Sweden), respectively. Ac-D-Phe-PipArg-pNA was prepared from D-Phe-Pip-Arg-pNA by acetylation with p-nitrophenyl acetate followed by purification by reversed phase HPLC with a C I 8column. Human a-trombin was prepared and characterized as described previously (Stone & Hofsteenge, 1986). The preparation used was fully active as determined by active-site titration with 4-methylumbelliferyl pguanidinobenzoate (Jameson et al., 1973) and was essentially free from degraded forms as assessed by gel electrophoresis THEORY AND DATAANALYSIS and Edman degradation. Synthesis of Na-Dansyl-~-arginine-4-methylpiperidine The reaction catalyzed by thrombin can be represented by Amide ( D A M P A ) . The mixed anhydride of N-t-Boc-diScheme I, where E is the enzyme, S is the substrate, ES is the Cbz-L-arginine (3.68 mmol) was prepared by standard Michaelis complex, EP2 is the acyl-enzyme complex, and PI methods and reacted with 4-methylpiperidine (4.78 mmol). and P2 are the hydrolysis products [p-nitroaniline and peptide The neutral fraction was purified by chromatography on a (or Bz-ArgOH), respectively]. column of silica gel and the t-Boc protecting group was reScheme I moved by reaction with trifluoroacetic acid. The product (3.02 k kl k3 g) was reacted with dansyl chloride (4.39 mmol) for 4 h at E S & E S pI + E P ~ E + p2 0 "C in 15 mL of chloroform containing 11 mmol of trik-1 ethylamine. The neutral dansylated product was purified on Under steady-state conditions, the dependence of velocity a column of silica. The Cbz groups were removed by reduction of the reaction (u) on the substrate concentration can be dein 30 mL of methanol containing 2.5 mL of acetic acid, 340 scribed by the Michaelis-Menten equation: mg of ammonium formate and 150 mg of Pd (10%)on active carbon. The catalyst was removed by filtration and the -U- - kCa,[Sl (1) [ E O ~ K m + [SI I The sequence numbering for thrombin is that of Bode et al. (1989) where [E,] is the enzyme concentration. For the reaction and is based on chymotrypsin numbering. mechanism presented in Scheme I, k,, (catalytic constant) * Abbreviations: Ac, acetyl; Bz, benzoyl; Cbz, benzyloxycarbonyl; and K, (Michaelis constant) can be defined by eqs 2a and 3a. amide; Pip, pipecolyl; DAMPA, Ne-dansyl-~-arginine-4-methylpiperidine pNA, p-nitroanilide; t-Boc, terr-butyloxycarbonyl; tos, 4-toluenesulfonyl. For p-nitroanilide substrates, it is generally accepted that
+
-
-
Biochemistry, Vol. 30, No. 41, 1991 9843
Mechanistic Studies on Thrombin Catalysis acylation is rate limiting (k, >> k2),and if this is also the case for thrombin, the expressions for k,, and K, can be simplified to eqs 2b and 3b. If deacylation is rate limiting (k2 >> k3), the expressions for k,, and K, are given by eqs 2c and 3c (Polgiir, 1987). kcat = k2k3 / (k2 + k3)
(2a)
If k3 >> k2, then k,, = k2
(2b)
If k2 >> k,, then kcat = k3
(2c)
Km = [(k-, + k,)k,l/[k,(k,
+ k3)I
(3a)
If k3 >> k2, then K, = (k-,
+ k2)/kl
(3b)
If k2 >> k3, then K, = [(k-,
+ k2)k3]/k,k2
Dk,t = k3/Dk3
(9)
In this case, the value of D(k,t/Km) will equal 1.0 for a sticky substrate and it will be significantly greater than 1.O but will not necessarily equal Dk,t when the substrate is not sticky. p H Dependence of the Interaction with Substrates and for p-nitroanilide Inhibitors. The pH profiles of k,,/K, substrates with thrombin were bell-shaped and could be described by
(3c)
Viscosity Effects. A sticky substrate is defined as one that dissociates from the Michaelis complex more slowly than this complex undergoes catalysis, Le., k2 > k-, (Cleland, 1977). For a nonsticky substrate, dissociation from the Michaelis complex is faster than reaction (k-, > k2) and the binding step comes to equilibrium. The stickiness of a substrate can be determined from the effect of viscosity on k,,/K, (Brouwer & Kirsch, 1982; Cleland, 1986). For the mechanism presented in Scheme I, the value of kca,/K, will be given by (4) This equation applies irrespective of whether acylation (k2 > k3) is rate limiting. The magnitudes of both k, and k-, will be dependent on the viscosity of the assay and it has been shown that the dependence of k,,/K, on the relative viscosity of the solution (qrcl) is given by (Brouwer & Kirsch, 1982)
where qrelis the viscosity of the assay with added viscogenic reagent relative to that of the assay without any such reagent. When eq 5 is written in the double-reciprocal form (eq 6 ) , it is apparent that a plot of K,/kcat against qrclwill be linear with a slope of I l k , and a horizontal intercept of -k-,/k2:
Initial velocity data obtained by varying the substrate at a particular viscosity were fitted to eq 1 by using weighted, robust linear regression (Cornish-Bowden & Endrenyi, 1981). The resulting values of kca,/K, were weighted according to the inverse of their variances and fitted to eq 6 to obtain estimates for kl and k2/k-,. Solvent Isotope Effects. The general base catalysis involving the catalytic histidine of thrombin is expected to proceed more slowly in D 2 0 (PolgPr, 1987). If acylation is rate limiting (k, > k,), Dkcatwill be given by eq 9, while eq 8 will still describe D(k,,/K,):
(7)
where Dk2is the rate of acylation in the presence of D20. When the substrate is not sticky (k-, >> k2), D(k,,/K,) will equal Dk,t. In contrast, there will be no solvent isotope effect on k,,/K, for a sticky substrate (k-,
