Interaction of human α-1-antitrypsin with porcine trypsin

Abstract

enzyme-inhibitor complex can be dissociated in alkaline solutions, with the release of a large fragment of a-1-antitrypsin which has an amino-terminal threonine residue and a molecular weight between 46 000 and 50 000, and a small peptide. During this base-catalyzed hydrolysis, a single new carboxyl-terminal lysine residue is formed. This residue must be the carboxyl-terminal residue of the amino-terminal peptide of a-1-antitrypsin. If the trypsin inhibitor, phenylmethanesulfonyl fluoride, is not added, catalytically active trypsin can be measured after a-l-antitrypsin-trypsin complexes are dissociated at high pH. These findings support the hypothesis that trypsin reacts with a-1-antitrypsin at a Lys-Thr bond and that no peptides of the reactants have been lost from the intact complex. Available evidence suggests that the a-l-antitrypsin-trypsin complex is analogous to the tetrahedral or acyl intermediates which form only transiently between trypsin and its substrates.Attempts to elucidate the amino acids at the inhibitory site in a-1-antitrypsin by modification of specific amino acids have produced ambiguous results. Reagents which are reported to a-1-ANTITRYPSININTERACTION WITH TRYPSIN VOL.1 7, no. 3, 19 7 8 393 supports an alternative hypothesis which explains all of the existing data.

Trypsin complexed with α1‐proteinase inhibitor has an increased structural flexibility

“…Model experiments to exclude or confirm this possibility are now being performed in our laboratory. Our failure to form a stable complex between c(1-PI and mutant trypsin His57Ala, Asp~°2Asn further confirms the general notion that the catalytic apparatus of the proteinase is essential for complex formation with serpins [18,19,21,22].…”

“…One striking difference is that the structure of serpins unlike that of canonical inhibitors, while reacting with the proteinases, undergo a dramatic change from a stressed, labile conformation to a relatively ordered, heat-stable form [18,19,20]. Furthermore, serpins unlike canonical proteinase inhibitors form kinetically stable complexes with proteinases [21,22]. The question if the general structure of such complexes corresponds to a tetrahedral [22] or an acyl-enzyme intermediate [19,21] is still debated.…”

“…Furthermore, serpins unlike canonical proteinase inhibitors form kinetically stable complexes with proteinases [21,22]. The question if the general structure of such complexes corresponds to a tetrahedral [22] or an acyl-enzyme intermediate [19,21] is still debated.…”

Mechanism of Inhibition of Porcine Elastase by Human Alpha-1-Antitrypsin

“…Carboxy-terminal analysis of alpha-l-antitrypsin, inactive alpha-l-antitrypsin, and alpha-1-antitrypsin* It was considered important to the present studies to redetermine the carboxy-terminal residue of the inhibitor preparation being used, particularly as there are conflicting reports in the literature as to the carboxyterminal residue of human alpha-l-antitrypsin (5,(41)(42)(43)(44). Thus, alpha-l-antitrypsin (20-nmol quantities) was reacted with carboxypeptidase Y at an enzyme:substrate ratio of 1:100 (weight/weight) for periods of Thus it was concluded that lys was the carboxynl)…”

“…These enzymes usually cleave proteins or amide or ester substrates at amino acids with small aliphatic side-chains (4). Trypsin (5) has been shown to attack a lys-x peptide bond in alpha-l-antitrypsin; however, it then forms a stable tetrahedral or acyl complex with the inhibitor which does not hydrolyze as do similar bonds in other proteins. If elastases are inhibited by a similar mechanism, then they probably attack alpha-l-antitrypsin at a specific residue at the active site for elastase, and become tightly bound to the inhibitor.…”