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 th e reactive center region of noninhibitory members of the serpin family of protein proteinase inhibitors is responsible for their lack of pro teinase inhibitory properties, we expressed a variant of the or alpha( 1)-proteinase inhibitor (alpha(1)-PI) with arginine substituted for th reonine at this position (T345R) and characterized its functional prop erties. Although the T345R variant reacted with proteinases principall y as a substrate, it was still capable of forming stable complexes wit h the three serine proteinases examined, human neutrophil elastase (HN E), porcine pancreatic elastase (PPE), and trypsin. The fraction of T3 45R alpha(1)-PI that formed a complex with proteinase was quantitated by autoradiography of SDS gels of the variant incubated with I-125-lab eled proteinase. The stoichiometry of inhibition (S.I.) (number of mol of alpha(1)-PI required to completely inhibit 1 mol of proteinase), w hich 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, an d PPE, respectively. An inverse relationship between the apparent seco nd-order rate constant and the S.I. for inhibition of PPE by T345R alp ha(1)-PI suggested that the mutation did not affect the rate-determini ng step of formation of a transient intermediate complex. Following cl eavage of the reactive center loop, there was a large increase in prot ein stability and changes in the CD spectrum, both consistent with ins ertion 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 presen ce of a charged residue at P14 does not prevent reactive center loop i nsertion or the functioning of alpha(1)-PI as an inhibitor of serine p roteinases but does significantly alter the relative rates of the subs trate and inhibitory pathways in favor of the former, probably by redu cing the rate of the latter reaction.