Mutagenesis studies toward understanding the mechanism of the cofactor function of thrombomodulin.
Thrombomodulin (TM) is as essential cofactor in protein C activation by thrombin. To investigate the cofactor effect of TM on the P3-P3' binding specificity of thrombin, we prepared a Gla-domainless protein C (GDPC) and an antithrombin (AT) mutant in which the P3-P3' residues of both molecules were replaced with the corresponding residues of the factor Xa cleavage site in prethrombin-2. TM is known to interact with GDPC, but not AT in the complex. Thrombin did not react with either mutant in the absence of a cofactor. While the thrombin-TM complex also did not react with the AT mutant, it activated the GDPC mutant with a normal k(cat), but an approximately 4-fold impaired K(m) value. Further studies revealed that the active-site directed inhibitor p-aminobenzamidine acts as a competitive inhibitor of both wild-type and GDPC mutant in reaction with the thrombin-TM complex. These results suggest that the interaction of the P3-P3' residues of GDPC with the active-site pocket of the thrombin-TM complex makes a dominant contribution to the binding specificity of the reaction. Moreover, the observation that the GDPC mutant, but not the AT mutant, functions as an effective substrate for the thrombin-TM complex suggests that GDPC interaction with the thrombin-TM complex may be associated with the alteration of the conformation of the P3-P3' residues of the substrate.
Rezaie
Alireza R
AR
Edward A. Doisy Department of Biochemistry and Molecular Biology, Saint Louis University School of Medicine, 1402 S. Grand Blvd. Saint Louis, MO 63104, USA. rezaiear@slu.edu
Yang
Likui
L
D000991:Antithrombins / Q000235:genetics / Q000378:metabolism; D004792:Enzyme Precursors / Q000378:metabolism; D015951:Factor Xa / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D016296:Mutagenesis / Q000235:genetics*; D011486:Protein C / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011516:Prothrombin / Q000378:metabolism; D013917:Thrombin / Q000378:metabolism; D018180:Thrombomodulin / Q000378:metabolism*
D000991:Antithrombins; D004792:Enzyme Precursors; D011486:Protein C; D018180:Thrombomodulin; D011516:Prothrombin; C012007:prethrombins; D013917:Thrombin; D015951:Factor Xa