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

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, Ca2+ and factor Va (FVa).(8-12) This construct facilitates the activation of prothrombin by ∼3 × 105-fold over catalysis by FXa alone.(13;14) The activation of human prothrombin involves the hydrolysis of two peptide bonds, Arg322-Ile323 followed by Arg273-Thr274. 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) FXa activation of prothrombin without FVa is inadequate for survival. FVa itself is processed from FV by cleavage by (human) thrombin at Arg709 into a heavy and a light chain.(10) Both chains are required for the interaction with FXa, but only the heavy chain binds prothrombin without Ca2+.(11) The binding sites for FXa and prothrombin have been located in the middle section(11) and the COOH terminus(12) of the heavy chain of FVa, respectively.(15) In fact, an exosite of FXa, which is exposed upon interaction with FVa for prothrombin tethering, seems to contain amino acids near the active site of the enzyme. This can serve to provide optimal docking of the scissile bonds of prothrombin. The crystal structures of meizothrombin desF1 and prothrombin-2 show the two prothrombin cleavage sites 36 A apart.(16) For prothrombin to be cleaved at Arg322, a rotation around the Gly319 and Gly324 hinge points has to occur. The rate of cleavage at the second site is not affected much by the presence of FVa.(11) Like other proteases,(17) FXa uses a general-base catalytic apparatus (the catalytic triad), in addition to an oxyanion hole and an elaborate binding site for prothrombin. Deuterium kinetic solvent isotope effects (KSIEs)(18-22) have been used to establish the occurrence of proton bridges at the rate-determining transitions state(s) (TSs) in biological general acid base-catalyzed reactions.(23) An extension of the KSIE studies to partial solvent isotope effects or proton inventories has provided additional information on the number and nature of proton bridges at the rate-determining TSs. Proton inventories are measured in mixtures of buffered water and heavy water.(18-22;24-29) A close relative of FXa is α-thrombin, the product of FXa catalysis and the central enzyme in the blood cascade. We have in the recent past presented conclusions from the analysis of proton inventory data obtained on the human α-thrombin-catalyzed hydrolysis of natural substrates and oligopeptide mimics of them.(30;31) In thrombin-catalyzed reactions, as in the pancreatic proteases, multi-proton catalysis pertains to proton bridges between Ne2 of His57 and the OγH of Ser195 and Nδ1 of His57 and COOβ- of Asp102 at the active site.(23;31) We carried out proton inventory experiments for the human α-thrombin-catalyzed activation of fibrinogen into fibrinopeptide A, the formation of fibrin colloid and the human α-thrombin-catalyzed activation of protein C (PC) to activated protein C (APC).(30) The proton inventories for fibrinogen activation are bowl shaped and are most consistent with two proton bridges forming at the TS of the chemical process at all substrate concentrations. In contrast, the activation of PC gives KSIEs slightly inverse at substrate concentrations below Km, indicating the dominance of physical rather than chemical steps limiting the rate under these conditions. The effect of P site interactions with S binding sites on protease enzymes has been established by a variety of studies including proton inventories. The last of these was our study of thrombin-catalyzed reactions of oligopeptide substrates.(31) When the requisite number and kind of P-S site interactions are present, the H-bonding distance between proton acceptors and donors of the catalytic triad contracts at the rate-determining TS. We have also shown that the hallmark of the effect of P' residues in leaving group departure is a conformational change associated with a major rearrangement of solvating water. In thrombin-catalyzed reactions, a net increase occurs in the isotopic fractionation factors for hydrogen bonds at solvation sites in the rate-determining step. This effect is elicited by interactions at the leaving groups’ binding site or substrate specificity site, which observation is well supported by other studies of P' site interactions in catalysis(32-34) by and inhibition(35) of proteases. The proton inventory technique has been particularly revealing about the importance of solvent reorganization in substrate binding and leaving group release in thrombin-catalyzed reactions. The premise of this endeavor has been that association and rearrangement of the FXa-prothrombin assembly is likely to limit the rate at the aqueous-lipid interface, as the very role of activation on heterogeneous surfaces is the facilitation of chemical transformation by juxtaposing and binding reactant partners. The process is associated with solvent restructuring and, thus, the physiological reaction catalyzed by FXa is likely to show a strong contribution from solvent rearrangement. Indeed, the activation of prothrombin by FXa gave inverse KSIEs that approached 0.2 − 0.3 in the presence of FXa : FVa in 1 : 4 ratio and 50 μM phospholipid vesicles (LUV), as the substrate concentrations were raised above Km, indicating the dominance of physical rather than chemical steps limiting the rate at the aqueous-lipid interface. In the presence of FXa : FVa = 1 : 1 and at enzyme saturation with substrate, however, the KSIEs became small normal, i.e. greater than 1.0. To obtain an unmasked picture of the rate-determining events in peptide bond hydrolysis catalyzed by FXa, we also studied proven mimics of the natural substrates with the proton inventory technique. FXa-catalyzed reactions of chromogenic substrates behaved similarly to the thrombin-catalyzed reactions of oligopeptide p-nitroanilides or 4-methyl umbelliferones, i.e. showed two-proton bridges at the rate-determining TS. However, N-α-Z-D-Arg-Gly-Arg-pNA·2HCl, a very efficient substrate of FXa, hydrolyzed with a contribution from solvent rearrangement at the rate-determining TS while saturated the enzyme. Solvent restructuring is likely to accompany leaving group departure, which then is part of the rate-determining process. Once more, the results of KSIE and proton inventory studies bring to light the critical importance of the participation of proton bridges and water structure in catalysis of another key serine protease, FXa.