Alpha 1-proteinase inhibitor variant T345R. Influence of P14 residue on substrate and inhibitory pathways.
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.
Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee 37232.
D B
DB
Hood
J A
JA
Huntington
P G
PG
Gettins
D000818:Animals; D001120:Arginine; D001483:Base Sequence; D002942:Circular Dichroism; D004355:Drug Stability; D004563:Electrochemistry; D006801:Humans; D007700:Kinetics; D019272:Leukocyte Elastase; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010196:Pancreatic Elastase / Q000378:metabolism; D017433:Protein Structure, Secondary; D013329:Structure-Activity Relationship; D013552:Swine; D013912:Threonine; D014357:Trypsin / Q000378:metabolism; D000515:alpha 1-Antitrypsin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
D000515:alpha 1-Antitrypsin; D013912:Threonine; D001120:Arginine; D010196:Pancreatic Elastase; D019272:Leukocyte Elastase; D014357:Trypsin