Abstract:
:The studies reported here were undertaken to determine whether the thrombin precursors prothrombin, prethrombin 1, prethrombin 2, and Meizo thrombin interact with the fluorescent, reversible thrombin inhibitor dansylarginine N,N-(3-ethyl-1,5-pentanediyl)amide (DAPA) [Nesheim, M. E., Prendergast, F. G., & Mann, K. G. (1979) Biochemistry 18, 996--1003]. The results indicate that prothrombin and prethrombin 1, in which the cleavage sites at Arg274-Thr275 and Arg323-Ile324 both remain intact, 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, 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 similarly was studied for comparative purposes. The binding of DAPA to either protein is accompanied by changes in the fluorescence properties of the dansyl moiety including increases in emission intensity, excited-state lifetime, polarization, and a slight blue shift in the wavelength of maximum emission intensity. Corrected excitation spectra indicate energy transfer to DAPA from one or more aromatic side chains of both proteins. Values of P0 for both complexes were extrapolated from Perrin plots of polarization vs. temperature and suggest 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 is substantially less than that of the DAPA-thrombin complex. In contrast, the intensity of the Meizo thrombin-DAPA complex is greater than that of the DAPA-thrombin complex. From measurements of intensity changes the dissociation constants and stoichiometry of DAPA binding to thrombin and prethrombin 2 were measured. Prethrombin 2 binds to DAPA with a Kd = 5.9 x 10(-7) M (n = 1) while thrombin binds about 30 times more tightly with a Kd = 2.0 x 10(-8) M (n = 1). The active site directed irreversible thrombin inhibitors diisopropyl phosphorofluoridate and D-phenylalanylprolylarginyl chloromethyl ketone displace DAPA from thrombin but not from prethrombin 2. The results of these studies indicate the binding of a presumed substrate analogue (DAPA) to an inactive zymogen, prethrombin 2. In addition, 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.
journal_name
Biochemistryjournal_title
Biochemistryauthors
Hibbard LS,Nesheim ME,Mann KGdoi
10.1021/bi00539a003subject
Has Abstractpub_date
1982-05-11 00:00:00pages
2285-92issue
10eissn
0006-2960issn
1520-4995journal_volume
21pub_type
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