Papain-inhibitory activity of oryzacystatin, a rice seed cysteine proteinase inhibitor, depends on the central Gln-Val-Val-Ala-Gly region conserved among cystatin superfamily members.
Oryzacystatin, a cysteine proteinase inhibitor occurring in rice seeds, contains a particular glycine residue (Gly5) near the NH2-terminal position, and the sequence Gln53-Val54-Val55-Ala56-Gly57 in a central part of the molecule. Both are conserved among most members of the cystatin superfamily. We have found from Escherichia coli expression studies that the NH2-terminal 21 residues of oryzacystatin are not essential for its papain-inhibitory activity, and that the conserved pentapeptide region may be indispensable [Abe, K., Emori, Y., Kondo, H., Arai, S., & Suzuki, K. (1988) J. Biol. Chem. 263, 7655-7659]. Here we present more detailed data based on quantitative analyses of the inhibitory activities of NH2- and COOH-terminally truncated oryzacystatin and site-directed mutants at the Gln-Val-Val-Ala-Gly region. The data indicate the following results. (1) The truncated mutants lacking the NH2-terminal 21 residues or the COOH-terminal 11 residues exhibit potent papain-inhibitory activity equivalent to the activity of wild oryzacystatin. (2) However, neither the mutant lacking the NH2-terminal 38 residues nor that lacking the COOH-terminal 35 residues is completely able to inhibit papain. (3) Site-directed mutants at the Gln residue of the Gln-Val-Val-Ala-Gly region have drastically reduced papain-inhibitory activities: the Gln----Pro mutant is completely inactive and the Gln----Leu mutant has an approximately 150 times higher Ki value than wild-type oryzacystatin.(ABSTRACT TRUNCATED AT 250 WORDS)
Arai
S
S
Department of Agricultural Chemistry, Faculty of Agriculture, University of Tokyo.
Watanabe
H
H
Kondo
H
H
Emori
Y
Y
Abe
K
K
D000595:Amino Acid Sequence; D001483:Base Sequence; D002846:Chromatography, Affinity; D002942:Circular Dichroism; D015891:Cystatins / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D012275:Oryza / Q000201:enzymology*; D010206:Papain / Q000378:metabolism*; D012639:Seeds / Q000201:enzymology*
D015891:Cystatins; D010206:Papain