Human cytoplasmic antiproteinase neutralizes rapidly and efficiently chymotrypsin and trypsin-like proteases utilizing distinct reactive site residues

Human cytoplasmic antiproteinase (CAP) is an intracellular serpin that has been reported to utilize Arg(341) as the reactive site P-1 residue to neutralize a broad variety of extracellular serine proteases with trypsin-like specificity. Both native CAP and recombinant CAP purified from Escherichia coli were observed to form SDS-stable complexes not only with I-125-thrombin and I-125-urokinase, but also with I-125-chymotrypsin. Kinetic studies indicated that the amidolytic activity of chymotrypsin is inhibited efficiently and rapidly by CAP in a two-step process with a dissociation constant K-i of an initial loose complex of 3.3 nM, a forward isomerization rate constant k(2) to the tight complex of 0.014 s(-1), and an overall second order association rate constant of 6 x 10(6) M(-1) s(-1) similar to the kinetic constants obtained for the formation of the trypsin-CAP complex. N-terminal amino acid sequencing and mass spectrometry indicated that chymotrypsin interacts with CAP at Met(340), in contrast to thrombin, which interacts as expected at Arg(341). Thus, CAP is the first serpin that has been shown to be capable to inhibit efficiently and with similar association rate constants different proteases at distinct reactive site residues, strongly supporting the notion of a highly mobile and flexible serpin reactive site loop and suggesting that this inhibitor may have evolved separate reactive sites for the specific regulation of different proteolytic activities.