Thrombin is the pivotal serine protease enzyme in the blood cascade system. Phe-Pro-Arg-chloromethylketone (PPACK), phosphate, and phosphonate ester inhibitors form a covalent bond with the active-site Ser of thrombin. PPACK, a mechanism-based inhibitor, and the phosphate/phosphonate esters form adducts that mimic intermediates formed in reactions catalyzed by thrombin. Therefore, the dependence of the inhibition of human alpha-thrombin on the concentration of these inhibitors, pH, and temperature was investigated. The second-order rate constant (ki/Ki) and the inhibition constant (Ki) for inhibition of human alpha-thrombin by PPACK are (1.1 +/- 0.2) x 10(7) M(-1) s(-1) and (2.4 +/- 1.3) x 10(-8) M, respectively, at pH 7.00 in 0.05 M phosphate buffer and 0.15 M NaCl at 25.0 +/- 0.1 degrees C, in good agreement with previous reports. The activation parameters at pH 7.00 in 0.05 M phosphate buffer and 0.15 M NaCl are as follows: DeltaH = 10.6 +/- 0.7 kcal/mol, and DeltaS = 9 +/- 2 cal mol(-1) degrees C(-1). The pH dependence of the second-order rate constants of inhibition is bell-shaped. Values of pKa1 and pKa2 are 7.3 +/- 0.2 and 8.8 +/- 0.3, respectively, at 25.0 +/- 0.1 degrees C. A phosphate and a phosphonate ester inhibitor gave higher values, 7.8 and 8.0 for pKa1 and 9.3 and 8.6 for pKa2, respectively. They inhibit thrombin more than 6 orders of magnitude less efficiently than PPACK does. The deuterium solvent isotope effect for the second-order rate constant at pH 7.0 and 8.3 at 25.0 +/- 0.1 degrees C is unity within experimental error in all three cases, indicating the absence of proton transfer in the rate-determining step for the association of thrombin with the inhibitors, but in a 600 MHz 1H NMR spectrum of the inhibition adduct at pH 6.7 and 30 degrees C, a peak at 18.10 ppm with respect to TSP appears with PPACK, which is absent in the 1H NMR spectrum of a solution of the enzyme between pH 5.3 and 8.5. The peak at low field is an indication of the presence of a short-strong hydrogen bond (SSHB) at the active site in the adduct. The deuterium isotope effect on this hydrogen bridge is 2.2 +/- 0.2 (phi = 0.45). The presence of an SSHB is also established with a signal at 17.34 ppm for a dealkylated phosphate adduct of thrombin.