Recombinant Q53E- and Q53N--chicken egg white cystatin variants inhibit papain, actinidin and cathepsin B.
A cloned synthetic gene coding for (AEF S1M M29I M89L) chicken egg white cystatin was modified site-specifically at position Q53 by cassette mutagenesis. Two recombinant variants were isolated from a pIN-III-ompA E. coli expression system and purified by Cm-papain affinity chromatography. The mutations at the position 53 were confirmed by amino acid composition and amino acid sequence analysis of the appropriate tryptic peptides. The complexes of both cystatin variants, the Q53E- and Q53N-variant with papain, display Ki values similar to those determined with native chicken cystatin. However, the Ki values of the complexes with actinidin are hundredfold and with cathepsin B three hundredfold higher than with the native chicken cystatin. The different inhibition kinetics of these variants compared to wild type chicken cystatin emphasizes the specificity of single amino acid substitutions for optimal contacts between the binding segments of enzyme and inhibitor.
Genenger
G
G
Abteilung für Klinische Chemie, Universität München, Germany.
Lenzen
S
S
Mentele
R
R
Assfalg-Machleidt
I
I
Auerswald
E A
EA
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002401:Cathepsin B / Q000037:antagonists & inhibitors*; D002645:Chickens; D015891:Cystatins / Q000235:genetics / Q000494:pharmacology*; D003546:Cysteine Endopeptidases / Q000378:metabolism*; D005260:Female; D066298:In Vitro Techniques; D007700:Kinetics; D008969:Molecular Sequence Data; D010063:Ovum; D010206:Papain / Q000037:antagonists & inhibitors*; D011995:Recombination, Genetic
D015891:Cystatins; D003546:Cysteine Endopeptidases; D002401:Cathepsin B; C018745:actinidain; D010206:Papain