2. Thrombin is a serine protease that functions in the blood clotting cascade. W

Question

2. Thrombin is a serine protease that functions in the blood clotting cascade. When thrombin is activated by upstream proteases, it becomes enzymatically active and is able to cleave fibrinogen. Fibrinogen is so named because it was discovered early in the 20th century that fibrinogen, when cleaved by thrombin (and is then called fibrin), polymerizes into a fibrous mesh that forms the blood clot, and which is visualized below in an electron micrograph A. Potent inhibitors of thrombin were isolated by chemists, and one of these, NAPAP, is shown below. The benzamidine moiety on the bottom right, in orange, was found to bind to the specificity pocket of thrombin with high affinity. The pKa of benzamidine is approximately 11.5 Given the structure of this benzamidine, after which residue(s) do you suppose thrombin cuts in proteins like fibrinogen, if it is cleaving the protein at physiological pH? How does this compare to trypsin and chymotrypsin? What residues might be in the specificity pocket of thrombin to give it this specificity? NAPAP NH NH B. Tosyl arginine methyl ester, (TAME) shown below, was discovered to be a synthetic substrate of thrombin. Given what you know about the serine protease mechanism, draw the products of the reaction of TAME with thrombin and state the other reactant that must be present in this reaction. You can draw the products using the compound on the right which has the word 'tosyl' instead of its chemical structure, if you please. Does TAME being a synthetic substrate of thrombin confirm or refute your supposition in A about the specificity of thrombin? NH CH3 NH CH3 H2N N HN Tosyl Tosyl arginine methyl ester CH3 Tosyl arginine methyl ester

Explanation / Answer

Thrombin is a serine protease which plays crucial role in the cascade of reactions in blood coagulation. These proteases play pivotal role in various processes due to their ability to hydrolyze protein substrates. There are three domains in thrombin which are involved in the interaction with fibrinogen, namely active site, a polar specificity pocket and a fibrinogen recognition site.

Thrombin recongizes the consensus sequence Leu-Val-Pro-Arg-Gly-Ser, while selectively cleaves the bond between Arg-Gly residues.

The enzyme Trypsin, most commonly used protease in Mass Spectrometry, cleaves at the C-terminal end of Lys-Arg residues. However, the presence of Pro residue at the C-terminus inhibits the cleavage to occur.

Chymotrypsin cleaves the proteins at the C-terminus of aromatic amino acid residues, Phenylalanine, Tryptophan and Tyrosin due to the presence of hydrophobic pocket.

As per the usage, high specificity makes trypsin and chymotrypsin apt for mass spectrometric applications, while, thrombin is potentially useful in cleavage of fusion tag from recombinant proteins and peptide sequences due to its ability to recognize a consensus sequence.

Studies have revelaed the presence of three residues Val (P2), Phe (P9) and Asp (P10) crucial for ?-thrombin catalyzed release of fibrinopeptide A.

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