Conversion of alpha 1-antichymotrypsin into a human neutrophil elastase inhibitor: demonstration of variants with different association rate constants, stoichiometries of inhibition, and complex stabilities.
Despite the homology with alpha 1-protease inhibitor (alpha 1PI), wild-type antichymotrypsin (ACT) is a substrate for HNE rather than an inhibitor of the enzyme. In order to investigate the nature of the specificity between serpins and serine proteases, the reactions of human neutrophil elastase (HNE) with wild-type recombinant ACT and recombinant variants of ACT were studied. ACT variants were generated where (1) the primary interaction site, the P1 position, was replaced with the P1 residue of alpha 1PI, (2) the residues corresponding to P3-P3' were replaced with those of alpha 1PI, and (3) the residues corresponding to the canonical recognition sequence as well as flanking residues encompassing the exposed reactive loop of the inhibitor were replaced with the corresponding residues of alpha 1PI. Each variant was analyzed to determine the effect of the replacements on reactions with human neutrophil elastase and chymotrypsin with regard to (1) the second-order rate constant for enzyme-serpin complex formation, (2) the number of moles of serpin required to completely inhibit 1 mol of enzyme (the stoichiometry of inhibition, SI), and (3) the stability of the enzyme-serpin complex. Replacing Leu with Met in the P1 position (rACT-L358M) was sufficient to convert rACT into an inhibitor of HNE with an apparent second-order rate constant (k'/[I]) of 4 x 10(4) M-1 s-1 and an SI of 5. The high SI was due to a concurrent hydrolytic reaction at sites in the reactive loop.(ABSTRACT TRUNCATED AT 250 WORDS)
Department of Medicine and Microbiology, University of Pennsylvania, Philadelphia 19104.
H
H
Rubin
M
M
Plotnick
Z M
ZM
Wang
X
X
Liu
Q
Q
Zhong
N M
NM
Schechter
B S
BS
Cooperman
D000595:Amino Acid Sequence; D002918:Chymotrypsin / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D019272:Leukocyte Elastase; D008969:Molecular Sequence Data; D010196:Pancreatic Elastase / Q000037:antagonists & inhibitors* / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D013329:Structure-Activity Relationship; D000514:alpha 1-Antichymotrypsin / Q000737:chemistry* / Q000378:metabolism / Q000494:pharmacology
D010446:Peptide Fragments; D011994:Recombinant Proteins; D000514:alpha 1-Antichymotrypsin; D002918:Chymotrypsin; D010196:Pancreatic Elastase; D019272:Leukocyte Elastase