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

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Abstract
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-Val66-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. 263,7655–7669]. 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→uPro mutant is completely inactive and the Gln→Leu mutant has an approximately 150 times higher K1 value than wild-type oryzacystatin. On the other hand, mutations at the central Val residue of the Gln-Val-Val-Ala-Gly region result in moderate effects on activity: the Val→Gly mutant is as active as wild oryzacystatin, and the Val→Asp mutant has a 40 times higher K1 value than the wild type. (4) The completely inactive Gln→Pro mutant is unable to bind to a Cm-papain column, suggesting that the affinity site of oryzacystatin is also its reactive site. These results clearly indicate that the Gln-Val-Val-Ala-Gly sequence of the cystatin molecule is the primary region of interaction with the cysteine proteinase and is thus responsible for the inhibitory activity.

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