STRUCTURAL MAPPING OF THE ACTIVE-SITE SPECIFICITY DETERMINANTS OF HUMAN TISSUE-TYPE PLASMINOGEN-ACTIVATOR - IMPLICATIONS FOR THE DESIGN OF LOW-MOLECULAR-WEIGHT SUBSTRATES AND INHIBITORS

The recent structure determination of the catalytic domain of tissue-t ype plasminogen activator (tPA) suggested residue Arg(174) could play a role in P3/P4 substrate specificity, Six synthetic chromogenic tPA s ubstrates of the type R-Xaa-Gly-Arg-p-nitroanilide, in which R is an N -terminal protection group, were synthesized to test this property, Al though changing the residue Xaa (in its L or D form) at position P3 fr om the hydrophobic Phe to an acidic residue, Asp or Glu, gave no impro vement in catalytic efficiency, comparative analysis of the substrates indicated a preference for an acidic substituent occupying the S3 sit e when the S4 site contains a hydrophobic or basic moiety, The 2.9 Ang strom structure determination of the catalytic domain of human tPA in complex with the bis-benzamidine inhibitor 2,7-bis (4-amidinobenzylide ne)-cycloheptan-1-one reveals a three-site interaction, salt bridge fo rmation of the proximal amidino group of the inhibitor with Asp(189) i n the primary specificity pocket, extensive hydrophobic surface burial , and a weak electrostatic interaction between the distal amidino grou p 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 pla y a major role in the S4 specificity of tPA, On the other hand, this r esidue would modulate S3 specificity, and may be exploited to fine tun e the specificity and selectivity of tPA substrates and inhibitors.