CONVERSION OF ALPHA(1)-ANTICHYMOTRYPSIN INTO A HUMAN NEUTROPHIL ELASTASE INHIBITOR - DEMONSTRATION OF VARIANTS WITH DIFFERENT ASSOCIATION RATE CONSTANTS, STOICHIOMETRIES OF INHIBITION, AND COMPLEX STABILITIES

Despite the homology with al-protease inhibitor (alpha 1PI), wild-type antichymotrypsin (ACT) is a substrate for HNE rather than an inhibito r of the enzyme. In order to investigate the nature of the specificity between serpins and serine proteases, the reactions of human neutroph il elastase (HNE) with wild-type recombinant ACT and recombinant varia nts of ACT were studied. ACT variants were generated where (1) the pri mary interaction site, the P1 position, was replaced with the P1 resid ue of alpha 1PI, (2) the residues corresponding to P3-P3' were replace d with those of alpha 1PI, and (3) the residues corresponding to the c anonical recognition sequence as well as flanking residues encompassin g the exposed reactive loop of the inhibitor were replaced with the co rresponding residues of alpha 1PI. Each variant was analyzed to determ ine the effect of the replacements on reactions with human neutrophil elastase and chymotrypsin with regard to (1) the second-order rate con stant for enzyme-serpin complex formation, (2) the number of moles of serpin required to completely inhibit 1 mol of enzyme (the stoichiomet ry of inhibition, SI), and (3) the stability of the enzyme-serpin comp lex. Replacing Leu with Met in the P1 position (rACT-L358M) was suffic ient to convert rACT into an inhibitor of HNE with an apparent second- order rate constant (k'/[I]) of 4 x 10(4) M(-1) s(-1) and an SI of 5. The high SI was due to a concurrent hydrolytic reaction at sites in th e reactive loop. N-Terminal sequence analysis of HNE cleavage products demonstrated a pattern similar to that of HNE cleavage of ACT, except that P1-P1' cleavage was more highly represented in the rACT-L358M/HN E reaction. The complex of rACT-L358M and HNE was not long-lived, with return of almost complete enzyme activity in approximately 4 h. Repla cement of six residues around the reactive center of ACT with those of alpha 1PI (rACT-P3P3') created an improved inhibitor of HNE. This var iant had an apparent second-order rate constant of 1 x 10(5) M(-1) s(- 1) and an SI of approximately 1. The HNE-rACT-P3P3' complex demonstrat ed increased stability compared to rACT-L358M, with return of HNE acti vity occurring over a 20-h period. Larger loop switches of 15 and 20 r esidues resulted in variants (rACT-P10P5' and rACT-P10P10') that were substrates for HNE, hydrolyzed predominantly at P1-P1' yet retained ch ymotrypsin inhibitory activity. These results suggest that the P1 posi tion as well as residues surrounding this site are important for serpi n function and specificity and that loop switches are not necessarily sufficient for transferring the structural elements responsible for bi ochemical activity.