Interaction between chicken cystatin and the cysteine proteinases actinidin, chymopapain A, and ficin.
The cysteine proteinase inhibitor cystatin, from chicken egg white, bound with equimolar stoichiometry to the cysteine proteinases actinidin, chymopapain A, and ficin. The changes of near-ultraviolet absorption and fluorescence induced by the binding differed appreciably for the three enzymes, indicating that these spectral changes arise predominantly from aromatic residues in the proteinases. In contrast, the near-ultraviolet circular dichroism changes were similar for all three enzymes, supporting previous evidence that these changes originate mainly from the single tryptophan residue in cystatin, Trp-104. The pseudo-first-order rate constant for the binding increased linearly with the inhibitor concentration up to as high concentrations as could be measured for the three proteinases. This behavior is consistent with the complexes being formed by simple, bimolecular reactions, as was concluded previously for the reaction of cystatin with active and inactivated forms of papain. The second-order association rate constant varied only about 4-fold, from 2.2 X 10(6) to 9.6 X 10(6) M-1.s-1, for the three enzymes, the higher of these values being similar to that measured previously for the reaction with papain. These observations are consistent with the association rate being governed mainly by the frequency of collision between the binding areas of enzyme and inhibitor. All three cystatin-proteinase complexes dissociated to intact inhibitor, demonstrating reversibility. The dissociation rate constants varied about 20000-fold, from 4.6 X 10(-7) s-1 for ficin to 1.1 X 10(-2) s-1 for actinidin, reflecting substantial differences between the enzymes in the nature of the interactions with the inhibitor.(ABSTRACT TRUNCATED AT 250 WORDS)
Björk
I
I
Department of Veterinary Medical Chemistry, Swedish University of Agricultural Sciences, Uppsala.
Ylinenjärvi
K
K
D000818:Animals; D002645:Chickens; D002917:Chymopapain / Q000037:antagonists & inhibitors / Q000378:metabolism*; D015891:Cystatins / Q000378:metabolism*; D003546:Cysteine Endopeptidases / Q000378:metabolism*; D015853:Cysteine Proteinase Inhibitors; D005361:Ficain / Q000037:antagonists & inhibitors / Q000378:metabolism*; D007700:Kinetics; D011485:Protein Binding; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet
D015891:Cystatins; D015853:Cysteine Proteinase Inhibitors; D003546:Cysteine Endopeptidases; C018745:actinidain; D005361:Ficain; D002917:Chymopapain