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 and thus a target of drug design for control of its activity. The most efficient non-physiologic inhibitor of thrombin is hirudin, a naturally occurring small protein. Hirudin and its synthetic mimics employ a range of hydrogen bonding, salt bridging and hydrophobic interactions with thrombin to achieve tight binding with Ki values in the nano- to femtomolar range. The one-dimensional 1H NMR spectrum carried out at 600 MHz reveals a resonance at 15.33 ppm downfield from silanes in complexes between human α-thrombin and r-hirudin in pH 5.6-8.8 buffers and between 5 and 35 °C. There is also a resonance between 15.17 and 15.54 ppm seen in human α-thrombin complexes with hirunorm IV, hirunorm V, an Nα(Me)Arg-peptide, RGD-hirudin and Nα-2-naphthylsulfonyl-glycyl-DL-4-amidinophenylalanyl-piperidide acetate salt (NAPAP), while there is no such low-field resonance observed in a complex of porcine trypsin and NAPAP. The chemical shifts suggest that these resonances represented H-bonded environments. H-donor acceptor distances in the corresponding H-bonds are estimated to be <2.7 Å. Addition of Phe-Pro-Arg-Chloromethylketone (PPACK) to a complex of human α-thrombin with r-hirudin results in an additional signal at 18.03 ppm, which is 0.10 ppm upfield from one observed (Kovach, I. M. et al. Biochemistry 2009, 48, 7296-7304) for thrombin covalently modified with PPACK. In contrast, the peak at 15.33 ppm remains unchanged. The fractionation factors for the thrombin-hirudin type complexes are near 1.0 within 20% error. The most likely site of the short H-bond in thrombin complexes with the hirudin family of inhibitors is in the hydrophobic patch of the C-terminus of hirudin where Glu57' and Glu58' are embedded and interact with Arg75 and Arg77 and their solvate water (on thrombin). Glu57' and Glu58' present in the hirudin family of inhibitors is a key binding epitope of fibrinogen, thrombin's prime substrate, which lends substantial interest to the SHB as a binding element at the fibrinogen recognition site.Biochemistry 03/2013; · 3.38 Impact Factor
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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: 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