Distinguishing the specificities of closely related proteases

Elucidating subtle specificity differences between closely related enzymes is a fundamental challenge for both enzymology and drug design. We have addressed this issue for two closely related serine proteases, tissue-type plasminogen activator (t-PA) and urokinase-type plasminogen activator (u-PA). Although these two proteases possess an extremely high degree of structural similarity, recognize the same primary endogenous substrate, and share common, physiological inhibitors, we have identified small, highly selective substrates whose relative reactivities towards the two enzymes vary by a factor of more than 9000. Discovery of selective substrates establishes a basis for the rational design of specific, small molecule inhibitors of each enzyme and provides new insight into details of molecular recognition by proteins.