Evidence that human α-thrombin is a monovalent cation-activated enzyme

The activity of human α-thrombin (EC 3.4.21.5) on small peptide substrates was enhanced by NaCl or KCl while tetramethylammonium chloride ((CH 3) 4NCl) or choline chloride (HO(CH 2) 2N(CH 3) 3Cl) which were used as ionic strength controls were without effect. The steady-state kinetic parameters of thrombin amidolysis of several peptidyl p-nitroanilide substrates were measured. Na + enhanced thrombin activity by decreasing the K m,app (0.2 to 0.7-fold) of all substrates, as well as increasing thombin turnover (3.4 to 4.5-fold) of some substrates. The average K A for Na +for the four substrates examined was 3.5 × 10 −2 m. A comparison of the effects of Na + vs K + on thrombin hydrolysis of a single substrate indicated that both cations similarly decreased the K m,app (0.2 to 04.-fold) and increased the k cat,app (3.1 to 3.4-fold) except that higher K + concentrations ( K A = 2.8 × 10 −1M) were required. The rate of inactivation of thrombin by the active site-directed inhibitor N-p-tosyl-lysine chloromethyl ketone under pseudo-first-order conditions was enhanced 3-fold by saturating NaCl. Also, the fibrinogen clotting activity of thrombin was enhanced by NaCl compared to the choline chloride control. Spectral studies demonstrated that thrombin titration by Na + caused a positive ultraviolet difference spectrum with maxima at 281.5 and 288.5 nm (Δϵ 288.5 = +1067). The K m for Na + was 2.3 × 10 −2 m which agrees with the kinetically determined K A for Na +. The results are consistent with Na + binding to thrombin causing a conformational change in the active site. It is concluded that human α-thrombin is a monovalent cation-activated enzyme.