Inhibition of α-Lytic Protease by Pro Region C-Terminal Steric Occlusion of the Active Site

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Abstract
α-Lytic protease, a chymotrypsin-like serine protease, is synthesized with an N-terminal 166 amino acid pro region which is absolutely required for folding of the protease. The pro region is also the most potent inhibitor of the protease known with a Ki of ∼10-10 M. Compared to its role in the folding reaction, relatively little is known about the mechanism by which the pro region inhibits the mature protease. While proteinaceous protease inhibitors generally function by occluding the active sites of their respective targets [Bode, W., & Huber, R. (1992) Eur. J. Biochem.204, 433−451], the pro region of α-lytic protease with its dual roles in folding and inhibition might be expected to show a novel mechanism of inhibition. However, experiments that probe both the structural and enzymatic consequences of pro region binding indicate that the pro region does not measurably distort the protease active site. Instead, the catalytic site is fully functional in the complex. Pro region inhibition of the protease is due to simple steric obstruction; the pro region C-terminus lies in the substrate binding sites of the protease. The implications of these results are discussed with regard to α-lytic protease maturation and folding. In addition, the proposed mechanism of α-lytic protease pro region inhibition is discussed with respect to data from other pro region families.