Overlapping binding sites for trypsin and papain on a Kunitz‐type proteinase inhibitor from Prosopis juliflora

Abstract

Proteinase inhibitors are among the most promising candidates for expression by transgenic plants and consequent protection against insect predation. However, some insects can respond to the threat of the proteinase inhibitor by the production of enzymes insensitive to inhibition. Inhibitors combining more than one favorable activity are therefore strongly favored. Recently, a known small Kunitz trypsin inhibitor from Prosopis juliflora (PTPKI) has been shown to possess unexpected potent cysteine proteinase inhibitory activity. Here we show, by enzyme assay and gel filtration, that, unlike other Kunitz inhibitors with dual activities, this inhibitor is incapable of simultaneous inhibition of trypsin and papain. These data are most readily interpreted by proposing overlapping binding sites for the two enzymes. Molecular modeling and docking experiments favor an interaction mode in which the same inhibitor loop that interacts in a canonical fashion with trypsin can also bind into the papain catalytic site cleft. Unusual residue substitutions at the proposed interface can explain the relative rarity of twin trypsin/papain inhibition. Other changes seem responsible for the relative low affinity of PTPKI for trypsin. The predicted coincidence of trypsin and papain binding sites, once confirmed, would facilitate the search, by phage display for example, for mutants highly active against both proteinases.

Effect of trypsin inhibitor from Crotalaria pallida seeds on Callosobruchus maculatus (cowpea weevil) and Ceratitis capitata (fruit fly)

“…The use of proteinaceous inhibitors in insect control strategies has good potential, because insect digestive proteinases are promising targets in the control of various insects, including lepidopterans such as Manduca sexta [3], Heliothis zea [4], Spodoptera litura [5], and Lucilia cuprina [6], and also various coleopterans [7][8][9][10][11]. Despite several suggested physiological functions in plants [12][13][14], the inhibitors are known for their role in response to abiotic [15,16] biotic stresses, especially in plant defense processes against insect pest attack [9,[17][18][19][20]. These plant proteinaceous inhibitors are generally small, stable and abundant proteins [21] showing specificity for serine proteinase, cysteine proteinase, aspartic proteinase or metallo-proteinases [17].…”

Affinity Chromatography as a Key Tool to Purify Protein Protease Inhibitors from Plants

“…Protease inhibitor purified from Prosopis juliflora seeds (PjTKI) presents a competitive inhibition mechanism directly interacting between its reactive site (Arg 64) and the catalytic site in target trypsin (Ser 195). Moreover, PjTKI also possesses an inhibitory activity against papain and a cysteine protease present in the digestive system of several phytophagous insect-pests Franco et al, 2002). This bifunctional property was also observed for the Kunitz inhibitor from Adenanthera pavonina seeds (ApTKI).…”

Genomic and cDNA Cloning, Expression, Purification, and Characterization of Chymotrypsin-Trypsin Inhibitor from Winged Bean Seeds

“…In this respect, the two proteins were indistinguishable. Although there are reports of inhibitory properties against cysteine proteases in trypsin inhibitors, 39) WbCTI and rWbCTI had no inhibitory activities against cysteine proteases or amylases (data not shown). Inhibition of the two proteinases by WbCTI might occur due to the overlapping binding sites for the two enzymes.…”