Full and Partial Deuterium Solvent Isotope Effect Studies of α-Thrombin-Catalyzed Reactions of Natural Substrates
Department of Chemistry, The Catholic University of America, Washington, Washington, D.C., United States Journal of the American Chemical Society
(Impact Factor: 12.11).
04/2005; 127(11):3760-6. DOI: 10.1021/ja043258o
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.
Available from: Vitor Oliveira
- "As we performed the assays with saturating substrate concentrations, the ratio vssD2O/vssH2O represents the solvent isotope effect in the kcat constant for the hydrolysis of the assayed substrate by mPC1/3. The observed value vssD2O/vssH2O = 0.6 was approximately the same value reported for serine peptidases with deacylation as the rate-limiting step in the catalysis . Therefore this solvent deuterium effect further supports the conclusion that the lag phase is not a kcat step. "
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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 · 3.23 Impact Factor
Available from: Vernon Emmett Anderson
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ABSTRACT: The application of tools developed by physical organic chemists to enzyme reactions to ascertain how the environment of the enzyme active site affects the reactivity of oxygen nucleophiles is reviewed. The three molecular mechanisms of generating enhanced electron density in the reactant state of the nucleophile by altering the coordination and/or bonding of the nucleophile are outlined as follows: desolvation, coordination of the alcohol proton and ionization. The nucleophilic activation of alcohols by enzymes and the spectroscopic characterization of nucleophiles at enzyme active sites are also illustrated.
Chemical Reviews 09/2006; 106(8):3236-51. DOI:10.1021/cr050281z · 46.57 Impact Factor
Available from: Ildiko M Kovach
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ABSTRACT: Kinetic solvent isotope effects (KSIEs) for the factor Xa (FXa)-catalyzed activation of prothrombin in the presence and absence of factor Va (FVa) and 5.0 x 10(-5) M phospholipid vesicles are slightly inverse, 0.82-0.93, when substrate concentrations are at 0.2 Km. This is consistent with the rate-determining association of the enzyme-prothrombin assembly, rather than the rate-limiting chemical transformation. FVa is known to effect a major conformational change to expose the first scissile bond in prothrombin, which is the likely event triggering a major solvent rearrangement. At prothrombin concentrations > 5 Km, the KSIE is 1.6 +/- 0.3, when FXa is in a 1:1 ratio with FVa but becomes increasingly inverse, 0.30 +/- 0.05 and 0.19 +/- 0.04, when FXa/FVa is 1:4, with an increasing FXa and substrate concentration. The rate-determining step changes with the conditions, but the chemical step is not limiting under any circumstance. This corroborates the proposed predominance of the meizothrombin pathway when FXa is well-saturated with the prothrombin complex. In contrast, the FXa-catalyzed hydrolysis of N-alpha-Z-D-Arg-Gly-Arg-pNA.2HCl (S-2765) and H-D-Ile-L-Pro-L-Arg-pNA.HCl (S-2288) is most consistent with two-proton bridges forming at the transition state between Ser195 OgammaH and His57 N(epsilon)2 and His57 Ndelta1 and Asp102 COObeta- at the active site, with transition-state fractionation factors of phi1 = phi2 = 0.57 +/- 0.07 and phiS = 0.78 +/- 0.16 for solvent rearrangement for S-2765 and phi1 = phi2 = 0.674 +/- 0.001 for S-2288 under enzyme saturation with the substrate at pH 8.40 and 25.0 +/- 0.1 degrees C. The rate-determining step(s) in these reactions is most likely the cleavage of the C-N bond and departure of the leaving group.
Biochemistry 11/2006; 45(47):14175-82. DOI:10.1021/bi061218m · 3.02 Impact Factor
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