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Deuterium solvent isotope effect and proton-inventory studies of factor Xa-catalyzed reactions.

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.

SUBMITTER: Zhang D

PROVIDER: S-EPMC2535812 | biostudies-literature | 2006 Nov

REPOSITORIES: biostudies-literature

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Deuterium solvent isotope effect and proton-inventory studies of factor Xa-catalyzed reactions.

Zhang Daoning D Kovach Ildiko M IM

Biochemistry 20061101 47

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 ...[more]

PMID: 17115712

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Project description:1. The hydrolyses of the p-nitrophenyl esters of N-benzyloxycarbonylglycine, alpha-N-benzyloxycarbonyl-L-lysine and N-methoxycarbonyl-L-phenylalanylglycine catalysed by papain (EC 3.4.22.2) have been studied in solvents having a variable composition of 2H2O and H2O. 2. kcat., which represents deacylation in the papain-catalysed hydrolysis of reactive esters, is some 2.3-fold less in 2H2O compared with H2O. The magnitude of kcat. has been determined as a function of the 2H atom fraction of the solvent. 3. Both linear and non-linear methods of least-square regression analysis have been applied to the data in order to obtain best-fit parameter values for several three-parameter models which express kcat. in terms of the 2H atom fraction of the solvent. These models represent some possible modes of restructuring of the active site protonic configuration consequent upon transition state formation. 4. The results of curve fitting reveal an essentially linear dependence of kcat. upon the 2H atom fraction, and it may therefore be concluded that the isotope effect originates from a single proton which is in the process of transfer in the transition state. 5. It is postulated on the basis of this and other evidence that the mobile proton is transferred from an attacking water molecule to the imidazole side chain of His-159 during tetrahedral intermediate formation. This has the effect of stabilizing the transition state and promoting catalysis. The role of His-159 in deacylation is therefore to provide general base catalysis. 6. Models that involve two or more protons, such as a two-proton relay system analogous to that proposed for the serine proteinases, or a multiproton 'medium' effect, are considered unlikely on the basis of the data reported in this paper. 7. A more detailed examination of possible transition state structures reveals that the only structure compatible with available experimental data and consistent with certain theoretical predictions is one in which the proton translocated in concern with reorganization of the heavy atom framework. In addition, the transition state vibrations of the mobile proton are strongly coupled to those of the heavy atoms. These properties of the transition state are also manifest in the transition state for the deacylation of serine proteinases.

Dataset Information

Deuterium solvent isotope effect and proton-inventory studies of factor Xa-catalyzed reactions.

Publications

Deuterium solvent isotope effect and proton-inventory studies of factor Xa-catalyzed reactions.

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