Role of Lys335 in the metastability and function of inhibitory serpins

The native form of inhibitory serpins (serine protease inhibitors) is not i n the thc thermodynamically most stable, state but in a metastable staler w hich is critical to inhibitory functions. To understand structural basis an d functional roles of the native metastability of inhibitory serpins, we ha ve been characterizing stabilizing mutations of human alpha(1)-antitrypsin. a prototype inhibitory serpin. One of the sites that has been shown to be critical in stability and inhibitory activity of alpha(1)-antitrypsin is Ly s335. In the present study, detailed roles of this lysine were analyzed by assessing the effects of 13 different amino acid substitutions. Results sug gest that size and architect of the side chains at the 335 site determine t he metastability of alpha(1)-antitrypsin. Moreover, factors such as polarit y and flexibility of the side chain at this site, in addition to the metast ability, seem to be critical for the inhibitory activity. Substitutions of the lysine at equivalent positions in two Other inhibitory serpins, human a lpha(1)-antichymotrypsin and human antithrombin III. also increased stabili ty and decreased inhibitory activity toward alpha-chymotrypsin and thrombin , respectively. These results and characteristics of lysine side chain, suc h as flexibility, polarity, and the energetic cost upon burial, suggest tha t this lysine is one of the structural designs in regulating metastability and function of inhibitory serpins in general.