The high-molecular-weight serine proteinase inhibitors (serpins) are restricted, generally, to inhibiting proteinases of the serine mechanistic class. However, the viral serpin, cytokine response modifier A, and the human serpins, antichymotrypsin and squamous cell carcinoma antigen 1 (SCCA1), inhibit different members of the cysteine proteinase class. 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 suggest that SCCA1 inhibits papain-like cysteine proteinases in a manner similar to that observed for serpin-serine proteinase interactions. However, we could not preclude the possibility of an inhibitory mechanism that did not require the serpin RSL. To test this possibility, we employed 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 and the nearly identical serpin, SCCA2 (an inhibitor of chymotrypsin-like serine proteinases), reversed their target specificities. Thus, there were no unique motifs within the framework of SCCA1 that independently accounted for cysteine proteinase inhibitory activity. Collectively, these data suggested that the sequence and mobility of the RSL of SCCA1 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.The high-molecular-weight serine proteinase inhibitors (serpins) comprise a superfamily of structurally well conserved proteins present in plants, animals, fungi, and viruses (1). In higher vertebrates, serpins regulate proteolytic events associated with coagulation, fibrinolysis, apoptosis, and inflammation (reviewed in ref.2). Unlike small-molecular-weight serine proteinase inhibitors, such as those of the Kazal and Kunitz families, serpins inhibit serine proteinases via a nonstandard, suicide substrate-like mechanism (3-5). Although the sequence of events are not known precisely, this mechanism involves exposure of the reactive site loop (RSL) of the serpin to the active site of the proteinase (3, 5). After the RSL is bound by the active site of the proteinase, the serpin undergoes a major conformational rearrangement characterized by partial or full insertion of the RSL into -sheet A (5), RSL cleavage, and formation of a covalent serpin-enzyme complex. In addition, this conformational change deforms the active site of the enzyme, thereby impeding deacylation and contributing to the stability of the covalent complex (6). However, if the rate of the loop insertion is retarded, or if stabilizing interactions between the serpin and the proteinase are lost, then the enzyme completes the deacylation step and escapes inhibition (7). In this latter case, the complex dissociates into an inactivated, cleaved serpin and an active proteinase. Thus, a serpin can serve ...
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