An examination of the inhibitory mechanism of serpins by analysing the interaction of trypsin and chymotrypsin with alpha 2-antiplasmin.
Human alpha 2-antiplasmin (alpha 2-AP) has previously been shown to possess overlapping inhibitory sites for trypsin and chymotrypsin [Potempa, Shieh and Travis (1988) Science 241, 699-700]. Since this is currently unique among active-site-directed inhibitors of proteinases, and difficult to explain in terms of accepted inhibitory mechanisms, we re-examined the claim. Initial characterization of purified alpha 2-AP revealed an additional 12 residues preceding the published N-terminus, prompting us to revise the previous numbering. We found that trypsin caused cleavage of the Arg376-Met377 bond in the reactive-site loop of the inhibitor, whereas chymotrypsin caused cleavage at two sites in approx. equal amounts at 37 degrees C: Met374-Ser375 (site 1) and Met377-Ser378 (site 2). At 0 degrees C alpha 2-AP became a more efficient inhibitor of chymotrypsin, and the proportion of cleavage at site 1 declined, indicating that chymotrypsin prefers to react with site 2 at 0 degrees C. Inhibitors of the alpha 2-AP type are inactivated when cleaved in their reactive-site loops by proteinases that they do not inhibit, so we conclude that site 1 is treated as a substrate by chymotrypsin. Site 2 is the inhibitory site for chymotrypsin. We confirm that alpha 2-AP does indeed have overlapping reactive sites for trypsin and chymotrypsin, and since the locations of chymotrypsin-interaction sites vary with temperature, we suggest that alpha 2-AP cannot have rigid reactive-site geometry. More likely, it has a mobile reactive-site loop of the type that has been recently demonstrated for eglin C.
Enghild
J J
JJ
Pathology Department, Duke University Medical Center, Durham, NC 27710.
Valnickova
Z
Z
Thøgersen
I B
IB
Pizzo
S V
SV
Salvesen
G
G
D000595:Amino Acid Sequence; D001665:Binding Sites; D002918:Chymotrypsin / Q000378:metabolism*; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry; D014357:Trypsin / Q000378:metabolism*; D014361:Trypsin Inhibitors / Q000737:chemistry / Q000378:metabolism; D000979:alpha-2-Antiplasmin / Q000737:chemistry / Q000378:metabolism*
D010446:Peptide Fragments; D014361:Trypsin Inhibitors; D000979:alpha-2-Antiplasmin; D002918:Chymotrypsin; D014357:Trypsin