Structure-function study of a plant protein proteinase inhibitor: site-directed mutagenesis of Cucurbita maxima trypsin inhibitor V

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Benavente, R.
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M. Duane Enger
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Cucurbita marima Trypsin Inhibitor-V (CMTI-V), isolated from pumpkin, is an inhibitor of trypsin and [beta]-Factor XIIa (FXIIa). CMTI-V binds to enzyme as would a substrate, but whereas substrate is cleaved at its scissile bond and the resulting fragments released, CMTI-V remains bound to its target enzyme, thus inhibiting activity. Amino acids at the amino side of the scissile bond are referred to as P1, P2, etc., away from the bond. Carboxyl-side amino acids are referred to as P1', P2', etc., away from the bond. We focused on the inhibitor amino acid positions P1' (D45) and P3 (V42). CMTI-V belongs to the Potato I inhibitor family, all members of which have a negatively-charged residue at P1' and conserve a hydrophobic (usually Val) residue at P3. We hypothesized that the P1' residue affects stabilization of the transition-state inhibitor-enzyme complex, preventing inhibitor cleavage and release. We additionally hypothesized that the P3 Val side chain is important in positioning the main chain for optimal inhibitor-enzyme contacts between P1-P4. Mutants D45L, D45E, D45N, D45V, V42G, V42M and V42S were constructed and activities against trypsin and FXIIa were compared to wildtype. Inhibitory assay data on activity against trypsin and FXIIa indicate a loss of function for mutants D45L, D45N and D45V. Little change was seen for mutants D45E, V42G, V42M and V42S. We also tested the ability of all mutants versus wildtype to bind over longer periods of time (24-48 hours) to target enzymes. We found an inability of mutants D45L and D45V to bind to both trypsin and FXIIa. D45E bound well to both enzymes. There were slighter decreases in the ability of all other mutants (D45N, V42G, V42M and V42S) to bind to target enzyme. These data suggest that the negatively-charged side chain at the P1' position is essential to CMTI-V's inhibitory function. The hydrophobic side chain at the P3 position appears to play a role in the ability of the inhibitor to bind for long periods of time to target enzyme although loss of hydrophobicity at that position does not affect inhibitory activity as measured by enzyme inhibition assays.

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Wed Jan 01 00:00:00 UTC 1997