Full and partial deuterium solvent isotope effect studies of alpha-thrombin-catalyzed reactions of natural substrates.
ABSTRACT Proton inventory studies of the thrombin-catalyzed fibrinogen activation to fibrinopeptide A are most consistent with a two-proton bridge forming at the transition state probably between Ser195 OgammaH and His57 Nepsilon2 and His57 Ndelta1 and Asp102 COObeta- at the active site, with fractionation factors 0.66 +/- 0.03 under enzyme saturation with substrate and 0.64 +/- 0.03 at fibrinogen concentration at 0.2 Km, at pH 8.0, pD 8.6, and 25.0 +/- 0.1 degrees C. Strongly inverse solvent isotope effects (SIEs) result from inverse lag times and maximal slopes of blood clotting plots, which are also anion and cation dependent. The blood clot is much coarser in D2O, as indicated in clotting curves with 3-9 times shorter lag time and steeper slopes with respect to H2O. The finer the particles, the weaker the H-bonds interlocking the fibrin mesh and/or in water structure around fibrin. Proton inventories of inverse lag times and maximal slopes of blood clotting curves in buffers containing Na+ and Cl- ions give the best fit to an exponential dependence on deuterium content in the buffer and give fractionation factors 5.6 +/- 0.5 and 7.8 +/- 0.6 at pH 8.0 and 25.0 +/- 0.1 degrees C. The thrombin-catalyzed activation of protein C (PC) to APC is associated with inverse kinetic SIEs (KSIEs) of 0.75 +/- 0.09 and 1.02 +/- 0.06 in 0.3 M NaCl and 0.3 M choline chloride, respectively, at substrate concentrations = 0.2 Km. In comparison, thrombin-catalyzed hydrolysis of chromogenic substrates gives greater KSIEs (Enyedy, E. I.; Kovach. I. M J. Am. Chem. Soc. 2004, 126, 6017-6024) and more complex proton inventories than the ones reported here for the first time for natural substrates. The present study illuminates differences in the character of the rate-determining transition state for the initial phase of the two physiological reactions catalyzed by thrombin.
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ABSTRACT: Thrombin is the pivotal serine protease enzyme in the blood cascade system. Phe-Pro-Arg-chloromethylketone (PPACK), phosphate, and phosphonate ester inhibitors form a covalent bond with the active-site Ser of thrombin. PPACK, a mechanism-based inhibitor, and the phosphate/phosphonate esters form adducts that mimic intermediates formed in reactions catalyzed by thrombin. Therefore, the dependence of the inhibition of human alpha-thrombin on the concentration of these inhibitors, pH, and temperature was investigated. The second-order rate constant (ki/Ki) and the inhibition constant (Ki) for inhibition of human alpha-thrombin by PPACK are (1.1 +/- 0.2) x 10(7) M(-1) s(-1) and (2.4 +/- 1.3) x 10(-8) M, respectively, at pH 7.00 in 0.05 M phosphate buffer and 0.15 M NaCl at 25.0 +/- 0.1 degrees C, in good agreement with previous reports. The activation parameters at pH 7.00 in 0.05 M phosphate buffer and 0.15 M NaCl are as follows: DeltaH = 10.6 +/- 0.7 kcal/mol, and DeltaS = 9 +/- 2 cal mol(-1) degrees C(-1). The pH dependence of the second-order rate constants of inhibition is bell-shaped. Values of pKa1 and pKa2 are 7.3 +/- 0.2 and 8.8 +/- 0.3, respectively, at 25.0 +/- 0.1 degrees C. A phosphate and a phosphonate ester inhibitor gave higher values, 7.8 and 8.0 for pKa1 and 9.3 and 8.6 for pKa2, respectively. They inhibit thrombin more than 6 orders of magnitude less efficiently than PPACK does. The deuterium solvent isotope effect for the second-order rate constant at pH 7.0 and 8.3 at 25.0 +/- 0.1 degrees C is unity within experimental error in all three cases, indicating the absence of proton transfer in the rate-determining step for the association of thrombin with the inhibitors, but in a 600 MHz 1H NMR spectrum of the inhibition adduct at pH 6.7 and 30 degrees C, a peak at 18.10 ppm with respect to TSP appears with PPACK, which is absent in the 1H NMR spectrum of a solution of the enzyme between pH 5.3 and 8.5. The peak at low field is an indication of the presence of a short-strong hydrogen bond (SSHB) at the active site in the adduct. The deuterium isotope effect on this hydrogen bridge is 2.2 +/- 0.2 (phi = 0.45). The presence of an SSHB is also established with a signal at 17.34 ppm for a dealkylated phosphate adduct of thrombin.Biochemistry 07/2009; 48(30):7296-304. · 3.38 Impact Factor
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ABSTRACT: Hydrolytic reactions of oligopeptide 4-nitroanilides catalyzed by human-alpha-thrombin, human activated protein C and human factor Xa were studied at pH 8.0-8.4 and 25.0+/-0.1 degrees C by the progress curve method and individual rate constants were calculated mostly within 10% internal error using DYNAFITV. A systematic strategy has been developed for fitting a three-step consecutive mechanism to eighteen hundred to six thousand time-course data points polled from two to four independent kinetic experiments. Enzyme and substrate concentrations were also calculated. Individual rate constants well reproduce published values obtained under comparable conditions and the Michaelis-Menten kinetic parameters calculated from these elementary rate constants are also within reasonable limits of published values. For comparison, the integrated Michaelis-Menten equation was also fitted to data from twelve sets. Both the k(cat) and k(cat)/K(m) values are within 15% agreement with those calculated using the elementary rate constants obtained with DYNAFITV. Rate constants for the second and third consecutive steps are within 3-4 fold indicating that both determine the overall rate. The Factor Xa-catalyzed hydrolysis of N-alpha-Z-D-Arg-Gly-Arg-pNA.2HCl at pH 8.4 in a series of buffers containing increasing fractions of deuterium at 25.0+/-0.1 degrees C shows a very strong dependence of k(3) and a moderate dependence of k(2) on D content in the buffer: the fractionation factors are: 0.49+/-0.03 for K(1,) 0.70+/-0.05 for k(2), and (0.32+/-0.03)(2) for k(3).Biochimica et Biophysica Acta 06/2008; 1784(5):827-33. · 4.66 Impact Factor
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ABSTRACT: The proprotein convertases (PCs) are calcium-dependent proteases responsible for processing precursor proteins into their active forms in eukariotes. The PC1/3 is a pivotal enzyme of this family that participates in the proteolytic maturation of prohormones and neuropeptides inside the regulated secretory pathway. In this paper we demonstrate that mouse proprotein convertase 1/3 (mPC1/3) has a lag phase of activation by substrates that can be interpreted as a hysteretic behavior of the enzyme for their hydrolysis. This is an unprecedented observation in peptidases, but is frequent in regulatory enzymes with physiological relevance. The lag phase of mPC1/3 is dependent on substrate, calcium concentration and pH. This hysteretic behavior may have implications in the physiological processes in which PC1/3 participates and could be considered an additional control step in the peptide hormone maturation processes as for instance in the transformation of proinsulin to insulin.PLoS ONE 01/2011; 6(9):e24545. · 3.73 Impact Factor