The recent structure determination of the catalytic domain of tissue-type plasminogen activator (tPA) suggested residue Arg 174 could play a role in P3/P4 substrate specificity. Six synthetic chromogenic tPA substrates of the type R-Xaa-Gly-Arg-p-nitroanilide, in which R is an N-terminal protection group, were synthesized to test this property. Although changing the residue Xaa (in its L or D form) at position P3 from the hydrophobic Phe to an acidic residue, Asp or Glu, gave no improvement in catalytic efficiency, comparative analysis of the substrates indicated a preference for an acidic substituent occupying the S3 site when the S4 site contains a hydrophobic or basic moiety. The 2.9 Å structure determination of the catalytic domain of human tPA in complex with the bis-benzamidine inhibitor 2,7-bis-(4-amidinobenzylidene)-cycloheptan-1-one reveals a three-site interaction, salt bridge formation of the proximal amidino group of the inhibitor with Asp 189 in the primary specificity pocket, extensive hydrophobic surface burial, and a weak electrostatic interaction between the distal amidino group of the inhibitor and two carbonyl oxygens of the protein. The latter position was previously occupied by the guanidino group of Arg 174 , which swings out to form the western edge of the S3 pocket. These data suggest that the side chain of Arg 174 is flexible, and does not play a major role in the S4 specificity of tPA. On the other hand, this residue would modulate S3 specificity, and may be exploited to fine tune the specificity and selectivity of tPA substrates and inhibitors.The 60-kDa multidomain tissue-type plasminogen activator (tPA) 1 catalyzes the conversion of the zymogen plasminogen into the active enzyme plasmin, the rate-limiting step in the endogenous fibrinolytic cascade (1). Recombinant tPA is used therapeutically as a fibrinolytic agent in the treatment of acute myocardial infarction and pulmonary embolism. Its fibrin dependent activity has attracted particular interest as it allows targeting of enzymatic activity to its natural substrate plasminogen (2, 3).The recent structure determination of the catalytic domain of tPA (4) in complex with benzamidine (hereafter termed b-tPA) revealed a strong structural similarity to other trypsin-like serine proteases. 187 residues of tPA have topological equivalents in ␣-chymotrypsin, forming the basis for the chymotrypsinogen numbering of the tPA catalytic domain used in this report (4). Compared with chymotrypsin, tPA contains a deletion of three residues at the C terminus of the A-chain, two single residue deletions in the B-chain, and insertions at six different positions totaling 24 residues. Five of these insertions are noteworthy: one forms a helix, the "intermediate helix" (Pro 164 -Leu 171 ), 2 and four form surface loops, referred to as the 37-loop (Lys 36 -Arg 39 ), 60-loop (Phe 59 -His 62 ), 110-loop (Ser 110 -Cys 111 ), and 186-loop (Asp 185 -Ala 186G ) (see Fig. 1). Most of these surface loops cluster around the active site cleft and are involved in sp...