Progressive development of a thrombin inhibitor binding site

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Abstract
Whether the thrombin precursors prothrombin, prethrombin 1, prethrombin 2 and Meizo thrombin interact with the fluorescent, reversible thrombin inhibitor dansyl-arginine N,N-(3-ethyl-1,5-pentanediyl)amide (DAPA) was studied. Prothrombin and prethrombin 1, in which the cleavage sites at Arg274-Thr275 and Arg323-Ile324 both remain intact, apparently do not bind DAPA, while prethrombin 2 or Meizo thrombin, which results, respectively, from a single cleavage of prothrombin at Arg274-Thr275 or Arg323-Ile324, apparently do bind the inhibitor. Since prethrombin 2 is a precursor of thrombin without measurable enzymatic activity, a thorough characterization of its interaction with DAPA was undertaken. The interaction of DAPA with bovine thrombin was studied for comparative purposes. The binding of DAPA to either protein was accompanied by changes in the fluorescence properties of the dansyl moeity including increases in emission intensity, excited-state lifetime, polarization and a slight blue shift in the wavelength of maximum emission intensity. Corrected excitation spectra indicated energy transfer to DAPA from one or more aromatic side chains of both proteins. Values of Po [partial pressure of O2] for both complexes were extrapolated from Perrin plots of polarization vs. temperature and suggested that the dansyl moiety is held more rigidly in thrombin than in prethrombin 2. With excitation at either 280 or 335 nm the emission intensity of DAPA-prethrombin 2 was substantially less than that of the DAPA-thrombin complex. The intensity of the Meizo thrombin-DAPA complex is greater than that of the DAPA-thrombin complex. From measurements of intensity changes the Kd and stoichiometry of DAPA binding to thrombin and prethrombin 2 were measured. Prethrombin 2 bound DAPA with a Kd = 5.9 .times. 10-7 M (n = 1) while thrombin bound .apprx. 30 times more tightly with a Kd = 2.0 .times. 10-8 M (n = 1). The active site directed irreversible thrombin inhibitors diisopropyl phosphorofluoridate and D-phenylalanylprolylarginyl cholormethyl ketone displace DAPA from thrombin but not from prethrombin 2. The binding of a presumed substrate analog (DAPA) to an inactive zymogen, prethrombin 2 was indicated. The lack of DAPA binding to prothrombin and prethrombin 1, under conditions in which it binds to prethrombin 2, implicates events that accompany cleavage at Arg274-Arg275 in the progressive formation of an active site even though further cleavage at Arg323-Ile324 is required for expression of enzymatic activity.