When amino acids Pro60B, Pro60C, and Trp60D are deleted from thrombin, the resulting mutant (des-PPW) exhibits (compared to the wild-type enzyme): a similar second order rate constant of inhibition (k(on)) for diisopropyl fluorophosphate, and a comparable inhibition constant (K(i)) for benzamidine, suggesting that the charge stabilizing system and the primary binding pocket are little altered, if at all, by the mutation. As predicted from the x-ray structure, des-PPW is remarkably sensitive to the bovine pancreatic trypsin inhibitor, with a K(i) over 3 x 10(3) times tighter relative to thrombin, but des-PPW is also markedly less susceptible to inactivation by antithrombin III, with a k(on) that is over 100-fold lower. The catalytic constant (kcat) for most p-nitroanilide substrates tested is preserved or even increased, but the Michaelis constant (Km) increases. In contrast, the Km for the fibrinogen A alpha-chain is essentially unchanged, whereas kcat decreases approximately 50-fold. Unlike thrombin, the rate of fibrinopeptide B release becomes, following a lag phase, comparable to that of fibrinopeptide A. Inasmuch as des-PPW cleaves an additional peptide bond in the bovine fibrin alpha-chain, it remains a highly specific serine protease, which releases a single peptide from denatured casein (versus two with thrombin). Protein C activation by des-PPW is approximately 30 times slower than by thrombin in the absence, as well as in the presence, of calcium and thrombomodulin. Although this study confirms that the B-insertion restricts access to the active site cleft, it also suggests that other motifs and/or discrete amino acids are mainly responsible for the narrow specificity of thrombin.