Synthesis and expression of the Erythrina trypsin/tissue plasminogen activator (tPA) inhibitor encoding-gene : genetic dissection to correlate the interaction of Erythrina and Soybean trypsin inhibitors with tPA

Doctoral Thesis


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University of Cape Town

A trypsin inhibitor had previously been isolated from Erythrina caffra, a member of the Leguminosae family. The inhibitor, Erythrina trypsin inhibitor (ETI), is unique among plantderived inhibitors, in that in addition to trypsin, it inhibits chymotrypsin and tissue plasminogen activator (tPA). ETI was previously sequenced and its crystal structure determined from which it could be seen that ETI has good homology to soybean trypsin inhibitor (STI). However, STI does not inhibit tPA. From the three-dimensional structure of ETI it was known that the amino-terminus of the molecule forms a finger-like structure stabilized by hydrogen bonds and hydrophobic interactions. In addition, the N-terminal finger region is located in close proximity to the reactive site loop and the Nterminal residue (Val) is bound up in the finger region. In STI the N-terminal region is located in close proximity to the reactive site loop and is folded into a structure similar to that in ETI. While the crystal structure of STI was not detailed enough to determine secondary interactions such as hydrogen bonds it was hypothesized that the N-terminal region is stabilized as in ETI. It was further hypothesized that the N-terminal residue of STI (Asp), because of its hydrophilic nature, is not involved in the structured N-terminal finger region of this protein. This leaves this Asp residue of STI free to form an ion pair with Lys at position 60 in trypsin when STI and trypsin interact.

Bibliography: pages 229-241.