Interaction of the cysteine proteinase inhibitor chicken cystatin with papain

Abstract

The two forms of chicken cystatin, with different isoelectric points, that have been described previously were indistinguishable in analyses of amino- and carboxy-terminal residues, amino acid composition, and peptide maps. The two forms thus are highly similar and most likely differ only in an amide group or in a small charged substituent. The binding of either cystatin form to highly purified, active papain was accompanied by the same pronounced changes in near-ultraviolet circular dichroism, ultraviolet absorption, and fluorescence emission. These changes were compatible with perturbations of the environment of aromatic residues in one or both proteins of the complex, arising from local interactions or from a conformational change. Modification of the single tryptophan residue of cystatin, at position 104, with N-bromosuccinimide resulted in considerably smaller spectroscopic changes on binding of the inhibitor to papain, indicating that the environment of this residue is affected by the binding. Analogous modification of Trp-69 and Trp-177 of papain markedly affected the fluorescence changes observed on binding of cystatin to the enzyme, similarly suggesting that these two residues of papain are involved in the interaction. The fluorescence increase of papain at alkaline pH, arising from Trp-177 and due to deprotonization of the adjacent His-159, was abolished on binding of cystatin to the enzyme, further supporting the proposal that this region of papain participates in the interaction with the inhibitor. A stoichiometry of binding of either cystatin form to papain of 1:1 and a lower limit for the binding constant of 10(9) M-1 were determined by titrations monitored by either the ultraviolet absorption or fluorescence changes induced by the interaction.

Interaction of human cathepsin C with chicken cystatin

“…The excitation and emission bandwidths were 5 and 10 nm, respectively. Titration of cathepsin C with chicken cystatin was performed as described previously for other cysteine proteinase-cystatin interactions [21,22] by monitoring the decrease in fluorescence emission intensity accompanying the interaction at 330 nm, where the maximum fluorescence change was observed (see Section 3). The final concentration of cathepsin C was 11.5 nM.…”

“…From previous studies it is known that Trp 104 of chicken cystatin and Trp 26, Trp 60 and Trp 177 of papain [21,27,28] are involved in the enzyme-inhibitor interaction. However, the fluorescence intensity accompanying the interaction decreased only by 28% (at 330 nm), which is substantially less than for the chicken cystatin/papain interaction [21]. Nevertheless, the value is comparable to those of other chicken cystatin/cathepsin interactions (B. Turk, unpublished results).…”

Interaction of chicken cystatin with inactivated papains

“…The reaction between this inhibitor and papain has been studied as a convenient model for the general mechanism of action of cysteine proteinase inhibitors. Chicken cystatin forms a tight (K, -60 fM) equimolar complex with papain, blocking the active site of the enzyme (Anastasi et al, 1983;Nicklin & Barrett, 1984;Lindahl et al, 1988;Bjork et al, 1989). Complex formation is accompanied by pronounced spectroscopic changes, most likely reflecting local perturbations of the environment of aromatic residues in both enzyme and inhibitor (Lindahl et al, 1988;Bjork et al, 1989).…”

“…Chicken cystatin forms a tight (K, -60 fM) equimolar complex with papain, blocking the active site of the enzyme (Anastasi et al, 1983;Nicklin & Barrett, 1984;Lindahl et al, 1988;Bjork et al, 1989). Complex formation is accompanied by pronounced spectroscopic changes, most likely reflecting local perturbations of the environment of aromatic residues in both enzyme and inhibitor (Lindahl et al, 1988;Bjork et al, 1989). The kinetics of the interaction are compatible with the complex being formed in a simple, reversible bimolecular reaction with an association rate constant approaching the limit expected for a rate controlled by macromolecular diffusion (Nicklin & Barrett, 1984;Bjork et al, 1989).…”

“…The kinetics of the interaction are compatible with the complex being formed in a simple, reversible bimolecular reaction with an association rate constant approaching the limit expected for a rate controlled by macromolecular diffusion (Nicklin & Barrett, 1984;Bjork et al, 1989). The inhibitory reactive site has been suggested to include the peptide bond between Gly-9 and Ala-10, the Gln-Leu-Val-Ser-Gly sequence at residues 53-57 and the region around Trp-104 of the inhibitor (Ohkubo et al, 1984;Barrett et al, 1986;Abrahamson et al, 1987;Lindahl et al, 1988). The recently published X-ray crystal structure of a variant of chicken cystatin shows that these regions form a contiguous hydrophobic, wedge-shaped edge of the molecule (Bode et al, 1988).…”