.alpha.1-Proteinase Inhibitor Variant T345R. Influence of P14 Residue on Substrate and Inhibitory Pathways

Abstract

To test whether the presence of charged residues at position P14 of the reactive center region of noninhibitory members of the serpin family of protein proteinase inhibitors is responsible for their lack of proteinase inhibitory properties, we expressed a variant of the alpha 1-proteinase inhibitor (alpha 1-PI) with arginine substituted for threonine at this position (T345R) and characterized its functional properties. Although the T345R variant reacted with proteinases principally as a substrate, it was still capable of forming stable complexes with the three serine proteinases examined, human neutrophil elastase (HNE), porcine pancreatic elastase (PPE), and trypsin. The fraction of T345R alpha 1-PI that formed a complex with proteinase was quantitated by autoradiography of SDS gels of the variant incubated with 125I-labeled proteinase. The stoichiometry of inhibition (S.I.) (number of mol of alpha 1-PI required to completely inhibit 1 mol of proteinase), which was 1 for both plasma alpha 1-PI and wild-type recombinant alpha 1-PI interacting with each of the proteinases, was very much greater than 1 for T345R variant alpha 1-PI. Values of 9.5, 45, and about 70 were estimated for variant alpha 1-PI inhibition of trypsin, HNE, and PPE, respectively. An inverse relationship between the apparent second-order rate constant and the S.I. for inhibition of PPE by T345R alpha 1-PI suggested that the mutation did not affect the rate-determining step of formation of a transient intermediate complex. Following cleavage of the reactive center loop, there was a large increase in protein stability and changes in the CD spectrum, both consistent with insertion of the reactive center loop into beta-sheet A. This behavior is similar to that of wild-type alpha 1-PI. We conclude that the presence of a charged residue at P14 does not prevent reactive center loop insertion or the functioning of alpha 1-PI as an inhibitor of serine proteinases but does significantly alter the relative rates of the substrate and inhibitory pathways in favor of the former, probably by reducing the rate of the latter reaction.

Conformational Changes of the Reactive‐Centre Loop and β‐Strand 5A Accompany Temperature‐Dependent Inhibitor‐Substrate Transition of Plasminogen‐Activator Inhibitor 1

“…This idea originally stems from the observation that the non-inhibitory ovalbumin has an arginine residue in the P,, position (Wright et al, 1990), the insertion of the charged and bulky arginine residue into the interior of the protein believed to be energetically unfavorable. Furthermore, substrate behaviour is exhibited by variants containing substitions with charged amino acids or proline in the hinge region (Caso et al, 1991;Skriver et al, 1991;Hopkins et al, 1993;Audenaert et al, 1994;Hood et al, 1994;Lawrence et al, 1994), and by binary complexes between a serpin and peptides corresponding to P,-P,, Schulze et al, 1990;Bjiirk et al, 1992). Two possible mechanisms for explaining the need for insertion have been discussed (Potempa et al, 1994).…”

The isoniazid-NAD adduct is a slow, tight-binding inhibitor of InhA, the Mycobacterium tuberculosis enoyl reductase: Adduct affinity and drug resistance

“…The following equations (29) were used to obtain the equilibrium and kinetic constants for the inhibition.…”

“…Because inhibition is competitive with respect to both DD- (29). Kinetic parameters for WT and mutant InhA enzymes obtained through regular steady-state kinetics were similar to the values reported earlier (20,27); the K mDD-CoA values used were 46, 70, 36, 104, and 307 M for WT, I21V, I47T, S94A, and M161V, respectively, and the K mNADH values were 66, 120, 152, and 250 M and 1.3 mM, respectively.…”

39 Structure function relationships of plasminogen activator inhibitor type-2 (PAI-2)

“…Some of these molecules still exhibited increased thermodynamic stability following cleavage, indicating that at least partial RCL insertion was occurring. It is likely that the observed conversion of these inhibitors to substrates results from a decreased rate of RCL insertion rather than complete blocking of insertion (Hood et al 1994;Tucker et al 1995;Gils etal. 1996).…”

“…Surprisingly, PI3 does not appear to play a critical role in this process in PAI-2. Previous studies have identified constraints on the size and charge of residues in the proximal hinge region (particularl PI4) of various serpins imposed by the tightly packed, hydrophobic nature of the protein core underlying p-sheet A (Stein et al 1989;Wright et al 1990;Hood et al 1994;Tucker et al 1995;Gils et al 1996;Lukacs et al 1996;Huntington et al 1997). Interactions around the proximal hinge region (particularly those involving P14) most likely influence inhibitory activity by determining the rate o RCL insertion into p-sheet A, and hence partitioning between the inhibitory and substrate pathways (Wright & Scarsdale 1995).…”