Hairpin loop mutations of chicken cystatin have different effects on the inhibition of cathepsin B, cathepsin L and papain.
Five recombinant hairpin loop variants of chicken cystatin (delta V55, delta V55-S56, delta P103-L105, delta I102-Q107, loop2-KD2) were constructed by cassette mutagenesis, expressed in E. coli, purified to homogeneity, characterized by protein-chemical means and by their inhibitory properties. The variant forms, modified in two of the three postulated cysteine proteinase binding regions, were inhibitorily active. However, the equilibrium dissociation constants of the complexes between papain as well as human cathepsin B or L and the cystatin variants show a weaker affinity for all three enzymes compared with recombinant chicken cystatin. These results prove the contribution of both hairpin loops to complex formation with the three enzymes. Furthermore, the kinetic constants indicate discrete differences in the molecular mechanism of interaction between chicken cystatin and papain, cathepsin B, and cathepsin L. Inhibition of cathepsin L was much less affected than inhibition of papain or cathepsin B by the modifications achieved in the five variants. Remarkably, at high enzyme concentration (above 0.5 nM) inhibition of papain by these variants was 'temporary', that means, active papain was released from the enzyme-inhibitor complex within minutes to hours (compare [1]).
Auerswald
E A
EA
Abteilung für Klinische Chemie und Klinische Biochemie, Klinikum Innenstadt, LMU München, Germany.
Nägler
D K
DK
Assfalg-Machleidt
I
I
Stubbs
M T
MT
Machleidt
W
W
Fritz
H
H
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002401:Cathepsin B / Q000037:antagonists & inhibitors*; D056668:Cathepsin L; D002403:Cathepsins / Q000037:antagonists & inhibitors*; D002645:Chickens; D003001:Cloning, Molecular; D015891:Cystatins / Q000096:biosynthesis / Q000737:chemistry* / Q000494:pharmacology*; D003546:Cysteine Endopeptidases; D010450:Endopeptidases; D014644:Genetic Variation; D007700:Kinetics; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D009838:Oligodeoxyribonucleotides; D010206:Papain / Q000037:antagonists & inhibitors*; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000494:pharmacology
D015891:Cystatins; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D002403:Cathepsins; D010450:Endopeptidases; D003546:Cysteine Endopeptidases; D002401:Cathepsin B; C534281:CTSL protein, human; D056668:Cathepsin L; D010206:Papain