THE REACTIVE-SITE LOOP OF THE SERPIN SCCA1 IS ESSENTIAL FOR CYSTEINE PROTEINASE INHIBITION

The high-moleculer-weight serine proteinase inhibitors (serpins) are r estricted, generally, to inhibiting proteinases of the serine mechanis tic class. However, the viral serpin, cytokine response modifier A, an d the human serpins, antichymotrypsin and squamous cell carcinoma anti gen 1 (SCCA1), inhibit different members of the cysteine proteinase cl ass. Although serpins employ a mobile reactive site loop (RSL) to bait and trap their target serine proteinases, the mechanism by which they inactivate cysteine proteinases is unknown. Our previous studies sugg est that SCCA1 inhibits papain-like cysteine proteinases in a manner s imilar to that observed for serpin-serine proteinase interactions. How ever, we could not preclude the possibility of an inhibitory mechanism that did not require the serpin RSL. To test this possibility, we emp loyed site-directed mutagenesis to alter the different residues within the RSL. Mutations to either the hinge or the variable region of the RSL abolished inhibitory activity. Moreover, RSL swaps between SCCA1 a nd the nearly identical serpin, SCCA2 (an inhibitor of chymotrypsin-li ke serine proteinases), reversed their target specificities. Thus, the re were no unique motifs within the framework of SCCA1 that independen tly accounted for cysteine proteinase inhibitory activity. Collectivel y these data suggested that the sequence and mobility of the RSL of SC CA1 are essential for cysteine proteinase inhibition and that serpins are likely to utilize a common RSL-dependent mechanism to inhibit both serine and cysteine proteinases.