Bromelain isoinhibitor (bromein), a cysteine proteinase inhibitor from pineapple stem, has a unique double-chain structure. The bromein precursor protein includes three homologous inhibitor domains, each containing an interchain peptide between the light and heavy chains. The interchain peptide in the single-chain precursor is immediately processed by bromelain, a target proteinase. In the present study, to clarify the essential inhibitory site of bromein, we constructed 44 kinds of site-directed and deletion mutants and investigated the inhibitory activity of each toward bromelain. As a result, the complete chemical structure of Leu 13 in the light chain was revealed to be essential for inhibition. Pro 12 prior to the leucine residue was also involved in the inhibitory activity and would control the location of the leucine side chain by the fixed dihedral angle of proline. Furthermore, the five-residue length of the interchain peptide was strictly required for the inhibitory activity. On the other hand, no inhibitory activity against bromelain was observed by the substitution of proline for the N terminus residue Thr 15 of the interchain peptide. In summary, these mutational analyses of bromein demonstrated that the appropriate position and conformation of Leu 13 are absolutely crucial for bromelain inhibition.Cysteine proteinases are involved in specific processing or more general degradation of proteins in a wide variety of organisms, including viruses, fungi, plants, and animals (1). Their activity is regulated by limited proteolysis of inactive precursors, by the pH of the surroundings (2), and by tight binding with proteinaceous inhibitors (3). With regard to proteinaceous inhibitors, six structurally different families of cysteine proteinase inhibitors have been reported so far: bromelain isoinhibitors (bromein) 2 (4), cystatins (5), soybean trypsin inhibitor-like inhibitors (6), thyropins (7), inhibitors homologous to the propeptide regions of cysteine proteinases (8), and clitocypins (9). Bromelain is known as cysteine proteinases in the stem and fruit of Ananas comosas, while the inhibitors exist only in the stem and have been classified into eight isoforms based on their amino acid sequences (10). The major component of bromelains, stem bromelain, has been sequenced (11) and shown to be a member of the papain superfamily (12). On the other hand, the presence of inhibitory fractions has also been confirmed in pineapple stem (13), and the amino acid sequence of the seventh bromein (bromein-7) was the first sequence determined among the inhibitory fractions (4). Hatano et al. (14) revealed the complete primary structures of all eight bromein isoforms: each isoform is composed of a light chain (10 -11 residues) and a heavy chain (40 -41 residues), which are cross-linked by five disulfide bridges.The three-dimensional solution structure of the sixth bromein with the two chains (bromein-6 N ) is characterized by inhibitory and stabilizing domains, each of which is formed by a three-stranded antiparallel -...