Identification of the Reactive Site of Potato Proteinase Inhibitor II-a

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Description

Limited hydrolysis of potato proteinase inhibitor II-a with a catalytic quantity of TPCK-treated bovine trypsin [EC 3. 4. 4. 4] at pH 3 results in the selective cleavage of a single Lys-Ser bond of the inhibitor. Fragmentation of the enzymatically modified inhibitor by reduction, S-carboxymethylation, and gel filtration reveal that the Lys-Ser bond is that between residues 63 and 64 of the inhibitor. This peptide bond is also selectively split by a catalytic quantity of TLCK-treated bovine chymotrypsin [EC 3.4. 4.5] at the same pH. The modified inhibitor retains full activities against trypsin, chymotrypsin, and a bacterial proteinase, Nagarse. However, the elimination of the newly formed carboxyl-terminal lysine from the modified inhibitor by carboxypeptidase B [EC 3.4. 2.2] digestion is accompanied by virtually complete loss of activity against trypsin and also by considerable reduction of the activities against chymotrypsin and Nagarse. It was therefore concluded that the Lys-Ser bond of residues 63 and 64 in inhibitor II-a is not only the reactive site for trypsin but is also the main reactive site for chymotrypsin and Nagarse of the inhibitor.

Journal

  • J Biochem (Tokyo)

    J Biochem (Tokyo) 73 (5), 1039-1048, 1973

    The Japanese Biochemical Society

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