Rapid Kinetics of Na+ Binding to Thrombin

Abstract

The kinetic mechanism of Na ؉ binding to thrombin was resolved by stopped-flow measurements of intrinsic fluorescence. Na ؉ binds to thrombin in a two-step mechanism with a rapid phase occurring within the dead time of the spectrometer (<0.5 ms) followed by a single-exponential slow phase whose k obs decreases hyperbolically with increasing [Na ؉ ]. The rapid phase is due to Na ؉ binding to the enzyme E to generate the E:Na ؉ form. The slow phase is due to the interconversion between E* and E, where E* is a form that cannot bind NaTemperature studies in the range from 5 to 35°C show significant enthalpy, entropy, and heat capacity changes associated with both Na ؉ binding and the E to E* transition. As a result, under conditions of physiologic temperature and salt concentrations, the E* form is negligibly populated (<1%) and thrombin is almost equally partitioned between the E (40%) and E:Na ؉ (60%) forms. Single-site Phe mutations of all nine Trp residues of thrombin enabled assignment of the fluorescence changes induced by Na ؉ binding mainly to Trp-141 and Trp-215, and to a lesser extent to Trp-148, Trp-207, and Trp-237. However, the fast phase of fluorescence increase is influenced to different extents by all Trp residues. The distribution of these residues over the entire thrombin surface demonstrates that Na ؉ binding induces long-range effects on the structure of the enzyme as a whole, contrary to the conclusions drawn from recent structural studies. These findings elucidate the mechanism of Na ؉ binding to thrombin and are relevant to other clotting factors and enzymes allosterically activated by monovalent cations.

Role of the A chain in thrombin function

“…This kinetic response to Na + binding is unprecedented for a thrombin mutant and vouch for a drastic perturbation of the structure of the enzyme. Mutation of some Trp residues to Phe abrogates the fast phase, but not the slow phase [16]. In no case has Na + binding been associated with a decrease rather than an increase in intrinsic fluorescence.…”

“…We specifically sought to explain the molecular nature of the perturbation affecting a residue located >20 Å away from residues of the catalytic triad, the S1 pocket or the Na + site. Binding of Na + to thrombin elicits a 10-15% increase in intrinsic fluorescence [13,16,21,22] that is contributed by changes in the environment of all nine Trp residues of the enzyme distributed over the entire surface up to 35 Å away from the bound cation [16]. The fluorescence increase can be decomposed into a fast phase due to Na + binding to the E form to generate E:Na + and a slow phase due to the interconversion of the Na + -free forms E* and E [16,23].…”

“…Binding of Na + to thrombin elicits a 10-15% increase in intrinsic fluorescence [13,16,21,22] that is contributed by changes in the environment of all nine Trp residues of the enzyme distributed over the entire surface up to 35 Å away from the bound cation [16]. The fluorescence increase can be decomposed into a fast phase due to Na + binding to the E form to generate E:Na + and a slow phase due to the interconversion of the Na + -free forms E* and E [16,23]. The biphasic response was observed for all mutants reported in this study except R4A, for which no slow phase was detected but only a fast transition to decreasing levels of fluorescence without appreciable saturation (Fig.…”

“…1), but is also in van der Waals interaction with Trp29 (3.8 Å) and Trp207 (3.6 Å) in the B chain. Both the E*-E and E-E:Na + interconversions affect the environment of all nine Trp residues of thrombin, including Trp29 and Trp207 that behave as a paired fluorophore due to their proximity [16]. Reciprocity of linkage demands perturbation of the A chain around Arg4 to propagate long-range to the Na + site in the B chain via Trp29 and Trp207.…”

“…Stopped-flow fluorescence measurements were carried out with an Applied Photophysics SX20 spectrometer, with excitation at 280 nm and a cut-off filter at 305 nm [16]. Samples of thrombin at a final concentration of 100 nM in 5 mM Tris, 0.1% PEG8000, pH 8.0 at 15 °C were mixed 1:1 with 60 µl of the same buffer containing variable amounts of NaCl (up to 400 mM) kept at constant ionic strength of 400 mM with choline chloride.…”