Influence of the P5 Residue on α1-Proteinase Inhibitor Mechanism

Abstract

The reactive center loop of native ␣ 1 -proteinase inhibitor has been reported to be in a helical conformation and in a ␤-strand conformation by two different studies. In the ␤-strand loop structure the P 5 glutamic acid plays a unique role by stabilizing the loop in the predicted optimal conformation for the interaction with target proteinases and insertion into ␤-sheet A. We hypothesize here that disrupting the interactions that stabilize the ␤-strand conformation of the loop would result in changes in the inhibitory properties of the serpin. In addition, our earlier studies on reactive center loop mutants of ␣ 1 -proteinase inhibitor suggested that the P 5 residue was important in stabilizing the ␣ 1 -proteinase inhibitor-proteinase complexes. To address these issues we made mutants of ␣ 1 -proteinase inhibitor with glycine, glutamine, or lysine at the P 5 position and measured the rates and stoichiometries of inhibition with trypsin and human neutrophil elastase and the stabilities of the resulting complexes. In most cases the rate of inhibition was reduced by about half and the stoichiometry increased between 2-and 4-fold. The only exception was for trypsin with the lysine variant where the P 5 was now the favored site of cleavage. These data show that the P 5 Glu is important in maintaining the reactive center loop in a conformation optimal for interaction with the proteinase and for a fast rate of loop insertion. The complexes formed with trypsin and the variant serpins were less stable than that formed with wild-type serpin and resulted in up to 33% regeneration of trypsin activity over a period of 6 days, compared with 17% with wild type. Thus, the P 5 residue of ␣ 1 -proteinase inhibitor is important in all steps of the inhibitory mechanism in a manner consistent with the structural role played by this residue in the ␤-strand loop structure of native ␣ 1 -proteinase inhibitor.One of the best studied members of the serpin family of serine proteinase inhibitors is ␣ 1 -proteinase inhibitor (␣ 1 -PI), 1 which is an important inhibitor of neutrophil elastase and is deficient in emphysema (1-3). ␣ 1 -PI inhibits its target serine proteinases by the branched suicide-substrate pathway (1, 4) characteristic of inhibitory serpins (Fig. 1). The structure of native ␣ 1 -PI has been solved by two groups (5, 6). From one study (6), the reactive center loop, which contains the scissile P 1 -P 1 Ј bond, 2 was found to be in a helical conformation similar to other native serpins, namely ovalbumin and an ␣ 1 -antichymotrypsin variant (8, 9). In the other, more detailed, structure of native ␣ 1 -PI (5) it was found that the reactive center loop is in an extended ␤-strand conformation similar to the canonical conformation found for other classes of protein inhibitors of serine proteinases (10). In this structure the P 5 glutamic acid residue plays a crucial role in the stabilization of the ␤-strand conformation of the reactive center loop by forming hydrogen bonds with Arg-196 and the backbone amide of Met-226. These res...

Importance of the P4′ Residue in Human Granzyme B Inhibitors and Substrates Revealed by Scanning Mutagenesis of the Proteinase Inhibitor 9 Reactive Center Loop

“…The higher the k ass , the faster the reaction and the less likely it is to follow the substrate pathway. Multiplication of the k ass by the SI yields a value for the rate of formation of the nonreversible intermediate that precedes the inhibitory/substrate pathway branch point (37,38).…”

NSP4 Is Stored in Azurophil Granules and Released by Activated Neutrophils as Active Endoprotease with Restricted Specificity

“…To increase the reactivity and specificity of a1PI toward NSP4, we constructed a1PI mutants with altered specificity by mutating amino acid residues in the P4, P3, and P1 position based on the specificity profiling results. Glutamic acid at position P5 (28) and the residues on the prime side of the reactive center loop were left unchanged, as these residues are critical for the interaction with target proteases and for the conformational rearrangement of a1PI after complexation. Antithrombin was previously shown to inactivate purified NSP4, but was not suited to trap NSP4 in the presence of other neutrophil proteases.…”

Kinetic Dissection of α1-Antitrypsin Inhibition Mechanism

“…After incubation for 1 h at 25°C, the reaction mixture was diluted 10-fold with the assay buffer and the residual activity of elastase was determined using 1 mM N-succinyl-(Ala) 3 -p-nitroanilide. The association rate constant between ␣ 1 AT and the protease was determined using a continuous assay procedure (25,33). Elastase or trypsin (10 nM) was incubated with various concentrations of ␣ 1 AT (100 -300 nM) in the assay buffer containing 1 mM N-succinyl-(Ala) 3 -pnitroanilide or 0.1 mM D-Pro-Phe-Arg-p-nitroanilide, respectively.…”