MAPPING THE SERPIN-PROTEINASE COMPLEX USING SINGLE CYSTEINE VARIANTS OF ALPHA(1)-PROTEINASE INHIBITOR PITTSBURGH

To probe the covalent serpin-proteinase complex, we used wild-type and 4 new single cysteine variants (T85C, S121C, D159C, and D298C) of alp ha(1)-proteinase inhibitor Pittsburgh. Cysteines in each variant could be labeled both in native and proteinase-complexed alpha(1)-proteinas e inhibitors. Pre-reaction with 7-nitrobenz-2-oxa-1,3-diazole-chloride or fluorescein prevented complex formation only with the D298C varian t. Label at Cys(121) greatly increased the stoichiometry of inhibition for thrombin and gave an emission spectrum that discriminated between native, cleaved, and proteinase-complexed serpin and between complexe s with trypsin and thrombin, whereas fluorophore at residue 159 ore he lix F was almost insensitive to complex formation. Fluorescence resona nce energy transfer measurements for covalent and non-covalent complex es were consistent with a location of the proteinase at the end of the serpin distal from the original location of the reactive center loop, Taken together, these findings are consistent with a serpin-proteinas e complex in which the reactive center loop is fully inserted into bet a-sheet A, and the proteinase is at the far end of the serpin from its initial site of docking with the reactive center loop close to, but n ot obscuring, residue 121.