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 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 Ki of an initial loose complex of 3.3 nM, a forward isomerization rate constant k2 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 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.
Riewald
M
M
Department of Vascular Biology, The Scripps Research Institute, La Jolla, California 92037, USA.
Schleef
R R
RR
D000595:Amino Acid Sequence; D001120:Arginine; D001483:Base Sequence; D001665:Binding Sites; D002918:Chymotrypsin / Q000378:metabolism*; D017931:DNA Primers; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D007700:Kinetics; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011485:Protein Binding; D014176:Protein Biosynthesis; D011506:Proteins / Q000737:chemistry / Q000494:pharmacology*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000494:pharmacology; D012044:Regression Analysis; D012697:Serine Endopeptidases / Q000378:metabolism; D015842:Serine Proteinase Inhibitors / Q000494:pharmacology*; D014357:Trypsin / Q000378:metabolism*
D017931:DNA Primers; D047908:Intracellular Signaling Peptides and Proteins; D011506:Proteins; D011994:Recombinant Proteins; D015842:Serine Proteinase Inhibitors; C086428:cytoplasmic antiproteinase; D001120:Arginine; D012697:Serine Endopeptidases; D002918:Chymotrypsin; D014357:Trypsin