Deuterium Solvent Isotope Effect and Proton-Inventory Studies of Factor Xa-Catalyzed Reactions

Abstract

Kinetic solvent isotope effects (KSIE) for the Factor Xa (FXa)-catalyzed activation of prothrombin in the presence and absence of Factor Va (FVa) and 5.0 ×10 −5 M phospholipid vesicles, are slightly inverse, 0.82−0.93, when substrate concentrations are at 0.2 K m . This is consistent with ratedetermining association of the enzyme-prothrombin assembly, rather than 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 K m the KSIE is 1.6 ± 0.3, when FXa is in 1 : 1 ratio with FVa, but becomes increasingly inverse, 0.30 ± 0.05 and 0.19 ± 0.04, when FXa : FVa 1:4, with increasing FXa concentration 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-α-Z-D-Arg-Gly-ArgpNA·2HCl (S-2765) and H-D-Ile-L-Pro-L-Arg-pNA.HCl (S-2288) is most consistent with twoproton bridges forming at the transition state between Ser 195 OγH and His 57 Nε2 and His 57 Nδ1 and Asp 102 COOβ-at the active site, with transition state fractionation factors of ϕ 1 = ϕ 2 = 0.57 ± 0.07 and ϕ S = 0.78 ± 0.16 for solvent rearrangement for S-2765, and ϕ 1 = ϕ 2 = 0.674 ± 0.001 for S-2288 under enzyme saturation with substrate at pH 8.40 and 25.0 ± 0.1 °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.Factor Xa (FXa) is one of the best characterized of the serine proteases in the cardiovascular system.(1-6) It catalyzes the efficient activation of a large precursor protein, prothrombin, to form α-thrombin.(1;7;8) Although several proteolytic enzymes can bring about this reaction, it is FXa that performs this key function under physiological conditions. On the basis of extensive investigations, the prothrombin complex is known to consist of phospholipid surface, prothrombin, Ca 2+ and factor Va (FVa). (8)(9)(10)(11)(12) This construct facilitates the activation of prothrombin by ∼3 × 10 5 -fold over catalysis by FXa alone.(13;14) The activation of human prothrombin involves the hydrolysis of two peptide bonds, Arg 322 -Ile 323 followed by Arg 273 -Thr 274 . In contrast, when FXa acts alone, bond cleavage occurs in the opposite order. Different intermediates are formed in the two pathways that have also been studied independently.(9; 14) More in-depth kinetic studies lately revealed that an appreciable fraction of prothrombin is channeled directly to thrombin product without releasing intermediate products. (14)

Proteolysis inside the Membrane Is a Rate-Governed Reaction Not Driven by Substrate Affinity

“…A decrease in rate in deuterium oxide is commonly used to identify a rate-limiting hydrolysis step in a reaction (Fersht, 1999). Surprisingly, hydrolysis itself was not rate-limiting in membranes, since the ratio of the water/deuterium oxide proteolysis rates for GlpG was only a modest 1.26±0.32 (Figure 5A) instead of the expected ≥2 for serine proteases (Elrod et al, 1980; Zhang and Kovach, 2006). In contrast, such mild effects are often caused by deuterium exchange with protein groups, and indeed we observed a gradual effect on proteolysis when we titrated deuterium oxide in the presence of water (Figure 5B).…”

The use of solvent isotope effects in the pursuit of enzyme mechanisms

“…Some examples [22][23][24][25] of applications of solvent isotope effects to mechanistic enzymology, [2006][2007] Simultaneous general-acid-general-base catalysis of nucleotidyl transfer in the action of nucleic-acid polymerases (Castro et al…”

Locating the rate-determining step(s) for three-step hydrolase-catalyzed reactions with dynafit

“…Michaelis-Menten parameters were also calculated by fitting the integrated Michaelis-Menten equation: [16] (1)…”

“…The experimental details of the proton inventory studies with of the FXa-catalyzed hydrolysis of N-α-Z-D-Arg-Gly-Arg-pNA·2HCl have been described previously. [16] Stock solutions for enzymes were prepared by dilution with Tris buffer to 2.90 × 10 −7 M thrombin, 7.70 × 10 −7 M and 20.8 × 10 −7 M APC. Substrate solutions were prepared in DMSO at 2.5 × 10 −3 M S-2238 and 0.01 M S-2366.…”

“…4 A comparison of Michaelis Menten parameters calculated from the individual rate constants obtained with DYNAFIT in Table 6 with those calculated using eq 1 for the integrated Michaelis-Menten equation. The nine sets of 2000 data pairs were obtained for proton inventory study [16] …”