Revised Manuscript Received November 2, 1992 abstract: A major feature of the structure of ai-antitrypsin is a five-stranded A-sheet into which the reactive center loop inserts after cleavage. We describe here the effect of the Z mutation (342Glu to Lys) at the head of the fifth strand of the A-sheet on the mobility of the reactive center loop and hence on the physical properties of the antitrypsin molecule. The mutant Z but notthe normal M antitrypsin spontaneously polymerizes at 37 C by a mechanism involving the insertion of the reactive center loop of one molecule into the A-sheet of a second. It is demonstrated that Z antitrypsin polymerized after incubation with 1.0 M guanidinium chloride at 37 C at the same rate as M antitrypsin. Reducing the temperature to 4 C favored the formation of the L-state in M antitrypsin in which the loop is stably incorporatedinto the A-sheet, but resulted in loop-sheet polymerization in Z antitrypsin. Z, like M antitrypsin, undergoes the 5 to R transition, but we show that the accompanying change in thermal stability results from loop-sheet polymerization (S) which can be prevented by the insertion of the cleaved strand of the reactive center loop into the A-sheet (R). Z antitrypsin has a reduced association rate constant with neutrophil elastase [(5.3 ą 0.06) X 107 and (1.2 ą 0.02) X 107 M_1 s-1 for M and Z, respectively], but both M and Z antitrypsin had K\values of less than 5 pM. Residues P9-P10 of the reactive center loop can be enzymatically cleaved in native M antitrypsin, but these residues are inaccessible in the binary complex ofM antitrypsin with a reactive center loop peptide and in native as well as binary complexed Z antitrypsin. This together with the CD spectrum of Z antitrypsin suggests a rearrangement of the loop with opening of the A-sheet to allow spontaneous intermolecular loop-sheet polymerization. ai-Antitrypsin is the archetypal member of the serine proteinase inhibitor (serpin) superfamily. Members of this family each have a unique inhibitory specificity but share a similar molecularstructure (Huber & Carrell, 1989). The major feature of this structure is the A-sheet, a five-stranded/3-pleated sheet into which the reactive center loop inserts following cleavage. The structure of the cleaved form is known (Loebermann et al., 1984; Baumann et al., 1991), but that of the native inhibitor has yet to be determined. The only intact model is that of the noninhibitor ovalbumin (Stein et al., 1990) in which the reactive center loop is in the form of an extended three-turn helix. As such this is an unsatisfactory conformation for inhibition, and predictably the helix will require opening in the inhibitory serpins by the partial insertion of its N-terminal stalk into theA-sheet (Stein et al., 1991). It is now apparent that the serpin reactive center loop is mobile and able to adopt varying conformations (Carrell et al., 1991; Mottonen et al., 1992). For example, under mild denaturing conditions at 4 C the reactive center loop of antitrypsin can be locked into the A-sheet, forming a thermostable, inactive protein, the L-state (Carrell et al., 1991). In contrast, if the temperature is elevated to 37 C, these same denaturing conditions favor the insertion of the reactive center loop of one antitrypsin molecule into the A-sheet of a second, so-called, loop-sheet polymerization (Evans, 1991; Mast et al., 1992; Lomas et al., 1992). Most northern Europeans have only the normal M form of antitrypsin, but some 4% are heterozygotesfor the Z deficiency variant (Laurell & Eriksson, 1963). This variant results in f This work was supported by the Medical Research Council and the Wellcome Trust. DAL is an MRC Training Fellow, and D. L1. E. is an Elmore Fellow.