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: The pH-independent hydrolyses of 4-nitrophenyl chloroformate, NPCF and 4-nitrophenyl heptafluorobutyrate, NPFB in aqueous acetonitrile were studied spectrophotometrically from 15 to 45 °C. The binary solvent composition covers water concentrations from 2.349 to 53.207 and from 2.745 to 53.333 mol L−1 for NPCF and NPFB, respectively. For both esters, the dependence of log (kobs), the observed rate constant, on log [water] is sigmoidal. The approximate kinetic orders with respect to water were found to be 2 and 3 for NPCF and NPFB, respectively. ΔG≠ gradually decreases as a function of increasing [water], due to a complex, quasi-mirror image compensation of ΔH≠ and ΔS≠; both parameters increase. The structures of the transition states were probed by a proton inventory study, carried out in the presence of L2O mole fractions (L = H or D) of 0.190, 0.540, 0.890 and 0.180, 0.529, 0.890, for NPCF and NPFB, respectively. Plots of observed rate constants versus the atom fraction of deuterium in the solvent curve downward. Cyclic transition state models were fitted to the kinetic data; these models contain the ester and two water molecules (NPCF) or three water molecules (NPFB). Thus, the sigmoidal dependences of log (kobs) on log [water] are not due to a change in the number of water molecules in the transition states as a function of increasing [water]. The binary solvent mixture is micro-heterogeneous; there exists two “micro-domains,” one consists predominantly of coordinated water molecules, the other consists mostly of acetonitrile hydrogen-bonded to water molecules. NPCF is 232 times more soluble in water than NPFB. That is, the former ester is dissolved in the outer, more polar periphery of these micro-domains whereas the more hydrophobic NPFB is dissolved in their inner, less polar interiors. This conclusion is corroborated by comparing the dependence on log [water] of log [kobs], and of ET, the empirical solvent polarity parameter, as measured by solvatochromic probes of increasing hydrophobicity. Copyright © 2006 John Wiley & Sons, Ltd.Journal of Physical Organic Chemistry 11/2006; 19(11):793-802. DOI:10.1002/poc.1081 · 1.23 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 09/2011; 6(9):e24545. DOI:10.1371/journal.pone.0024545 · 3.53 Impact Factor
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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; 52(14). DOI:10.1021/bi301625a · 3.19 Impact Factor