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Volume 271, Number 24, Issue of June 14, 1996 pp. 14526-14532
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.

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Human Cytoplasmic Antiproteinase Neutralizes Rapidly and Efficiently Chymotrypsin and Trypsin-like Proteases Utilizing Distinct Reactive Site Residues

(Received for publication, February 20, 1996, and in revised form, March 15, 1996)

From the Department of Vascular Biology, The Scripps Research Institute, La Jolla, California 92037

Human cytoplasmic antiproteinase (CAP) is an intracellular serpin that has been reported to utilize Arg³⁴¹ as the reactive site P₁ 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 ¹²⁵I-thrombin and ¹²⁵I-urokinase, but also with ¹²⁵I-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 k₂ to the tight complex of 0.014 s¹, and an overall second order association rate constant of 6 × 10⁶ M¹ s¹, 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 Met³⁴⁰, in contrast to thrombin, which interacts as expected at Arg³⁴¹. 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.

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