Substitution of Tyrosine for Phenylalanine in Fibrinopeptide A Results in Preferential Thrombin Cleavage of Fibrinopeptide B from Fibrinogen
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- 作者：Michael M. Rooney ; Jennifer L. Mullin ; and Susan T. Lord
- 刊名：Biochemistry
- 出版年：1998
- 出版时间：September 29, 1998
- 年：1998
- 卷：37
- 期：39
- 页码：13704 - 13709
- 全文大小：69K
- 年卷期：v.37,no.39(September 29, 1998)
- ISSN：1520-4995
文摘
Phenylalanine at residue 8 in the A
chain of fibrinogen is a highly conserved amino acidthat is believed to be critical for binding and catalysis by the serine protease thrombin. We have examinedthe requirement for Phe at this position by constructing a variant recombinant fibrinogen with a conservativesubstitution of tyrosine for phenylalanine, A F8Y fibrinogen. We found that the variant fibrinopeptideA (F8Y 1-16) was cleaved by thrombin, in contrast to the lack of cleavage of an A 1-23 peptide andan A 1-50 fusion protein with the same substitution. This result indicates that fibrinogen residuesother than A 1-50 participate in thrombin binding and fibrinogen proteolysis. We found, for the firsttime, that thrombin-catalyzed lysis of the fibrinogen B
ta2.gif" BORDER=0 ALIGN="middle"> chain preceded lysis of the A chain, such thatfibrinopeptide B (FpB) was released prior to F8Y 1-16. Kinetic analysis demonstrated that F8Y 1-16was a very poor substrate for thrombin, with a specificity constant 280-fold lower than normal fibrinopeptideA. FpB was also a poor substrate, but the specificity constant for FpB was only 4-fold lower than normal.Consequently, FpB was preferentially released from A F8Y fibrinogen. This "role reversal" had a dramaticeffect on polymerization, such that the rate of A F8Y fibrinogen polymerization was 13% of the rate ofnormal recombinant fibrinogen. These results confirm the importance of phenylalanine at A chain residue8 for efficient thrombin-catalyzed proteolysis of fibrinogen, and further demonstrate that sequentialfibrinopeptide release has an important role in normal polymerization.
chain of fibrinogen is a highly conserved amino acidthat is believed to be critical for binding and catalysis by the serine protease thrombin. We have examinedthe requirement for Phe at this position by constructing a variant recombinant fibrinogen with a conservativesubstitution of tyrosine for phenylalanine, A F8Y fibrinogen. We found that the variant fibrinopeptideA (F8Y 1-16) was cleaved by thrombin, in contrast to the lack of cleavage of an A 1-23 peptide andan A 1-50 fusion protein with the same substitution. This result indicates that fibrinogen residuesother than A 1-50 participate in thrombin binding and fibrinogen proteolysis. We found, for the firsttime, that thrombin-catalyzed lysis of the fibrinogen Bta2.gif" BORDER=0 ALIGN="middle"> chain preceded lysis of the A chain, such thatfibrinopeptide B (FpB) was released prior to F8Y 1-16. Kinetic analysis demonstrated that F8Y 1-16was a very poor substrate for thrombin, with a specificity constant 280-fold lower than normal fibrinopeptideA. FpB was also a poor substrate, but the specificity constant for FpB was only 4-fold lower than normal.Consequently, FpB was preferentially released from A F8Y fibrinogen. This "role reversal" had a dramaticeffect on polymerization, such that the rate of A F8Y fibrinogen polymerization was 13% of the rate ofnormal recombinant fibrinogen. These results confirm the importance of phenylalanine at A chain residue8 for efficient thrombin-catalyzed proteolysis of fibrinogen, and further demonstrate that sequentialfibrinopeptide release has an important role in normal polymerization.