Studies on the reactive site of the cystatin superfamily using recombinant cystatin A mutants. Evidence that the QVVAG region is not essential for cysteine proteinase inhibitory activities.
For study of the inhibition mechanism of the cystatin superfamily, cystatin A artificial mutants were obtained in which a well-conserved QVVAG region in the cystatin superfamily was changed to KVVAG or QVTAG and these mutants were then expressed in E. coli. For this, genes with these sequences were synthesized enzymatically from 11 oligodeoxynucleotides and expressed under the tac promoter gene of the E. coli plasmids. The products expressed were then purified on Sephadex G-50 and HPLC DEAE-5PW columns. The substitutions in cystatin A were confirmed by the amino acid compositions, N-terminal amino acid sequences and elution positions on ion-exchange chromatography of the products. The Ki values of these products for the cysteine proteinases, papain and cathepsins B, H and L, were determined in comparison with those of wild type recombinant cystatin A. Results showed that the cystatin A mutants had similar inhibitory activities to those of wild type recombinant cystatin A. Namely replacement of amino acids in the QVVAG sequence of cystatin A did not significantly affect the inhibitory activities on these proteinases. The results suggest that the QVVAG region is less important than the N-terminal region of cystatin for inhibitory activities on cysteine proteinases.
Nikawa
T
T
Division of Enzyme Chemistry, University of Tokushima, Japan.
Towatari
T
T
Ike
Y
Y
Katunuma
N
N
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D015891:Cystatins / Q000235:genetics* / Q000378:metabolism; D003546:Cysteine Endopeptidases / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D005796:Genes; D008969:Molecular Sequence Data; D005810:Multigene Family; D009154:Mutation; D010957:Plasmids; D011401:Promoter Regions, Genetic; D013379:Substrate Specificity
D015891:Cystatins; D003546:Cysteine Endopeptidases