Structure-function relationships of proteinase inhibitors from soybean (Bowman-Birk) and lima bean. Modification by N-acetylimidazole.

Contributions of tyrosyl residues to trypsinand chymotrypsin-inhibitory activities in two homologous proteinase inhibitors were investigated by modifying them with N-acetylimidazole under various conditions. In Bowman-Birk soybean proteinase inhibitor, Tyr 55, immediately following the antichymotryptic site, Leu 53-Ser 54, is relatively inaccessible to N-acetylimidazole and can only be acetylated in the presence of 6 M guanidine hydrochloride but not in 8 M urea. The acetylation of Tyr 55 is accompanied by 60% loss in antichymotryptic activity. Deacetylation with hydroxylamine restores the activity to the original level. Tyr 69, located in the antitrypsin portion of the inhibitor, is exposed relatively to N-acetylimidazole and can be acetylated without denaturing agent. The acetylation of Tyr 69 parallels decrease in antitryptic activity. The inhibitor acetylated at Tyr 69 is fully active toward chymotrypsin and has 30 to 40% antitryptic activity of the native. The original level of antitryptic activity is restored upon deacetylation. Tyr 69 of lima bean proteinase inhibitor is relatively inaccessible to N-acetylimidazole: 75% acetylation in the presence of 6 M guanidine hydrochloride and 17% without the denaturing agent. The acetylated inhibitor is fully active toward chymotrypsin but retains only 29% (acetylated without guanidine hydrochloride) and 17% (acetylated with guanidine hydrochloride) of the original antitryptic activity. Deacetylation partially restores the lost antitryptic activity in the inhibitor acetylated without the denaturing agent. The total and irreversible loss of antitryptic activity in samples acetylated in the presence of 8 M urea or 6 M guanidine hydrochloride is attributed to the acetylation at the c-amino group of Lys 26 at the trypsininhibitory site.