Abstract
The specificity and reactivity of human alpha 1-proteinase inhibitor has been investigated by in vitro mutagenesis of the reactive site P1 methionine 358 residue to alanine 358 and cysteine 358. A comparison of the second-order association rates of both uncharged mutants with 9 serine proteinases indicated that each reacted similarly to either the normal plasma inhibitor or to a mutant containing valine in this position (Travis, J., Owen, M., George, P., Carrell, R., Rosenberg, S., Hallewell, R. A., and Barr, P. J. (1985) J. Biol. Chem. 260, 4384-4389) when tested against either neutrophil or pancreatic elastase. However, oxidation, carboxymethylation, or aminoethylation of the cysteine mutant to yield a charged P1 residue resulted in a significant decrease in association rates with both elastolytic enzymes, and aminoethylation created an excellent trypsin and plasmin inhibitor. These results indicate that the specificity of alpha 1-proteinase inhibitor is determined in a general manner by the class of amino acid residue in the P1 position. Substitution within the same category, such as from valine to alanine or cysteine among the aliphatic hydrophobic residues, has little effect on association rates with the elastolytic enzymes tested. However, alteration from an uncharged to a charged residue may cause considerable changes in both inhibitor specificity and reactivity as noted here with the cysteine derivatives and also previously with a natural variant in which methionine 358 to arginine 358 conversion resulted in the production of a potent thrombin inhibitor (Owen, M. C., Brennan, S. O., Lewis, J. H., and Carrell, R. W. (1983) N. Engl. J. Med. 309, 694-698).
Highlights
From the $Department of Biochemistly, University of Georgia, Athens, Georgia 30602 and the JChiron Corporation, Emeryuille, California 94608
For construction of al-PI mutants containing alanine and cysteine residues in place of methionine a t position 358, we introduced the appropriate DNA sequence expression of boththealanineand cysteine mutants was achieved in yeast
Since it seems well established that: (a)the best inhibitors of serineproteinases are excellent substrates, the difference being in the fact that dissociation ratesareextremely slow [23];( b ) human cyI-PI reacts most rapidly with neutrophil and pancreatic elastase [2]; and (e)both enzymes cleave peptide bonds after methionine, valine, and alanine residues [24, 25] but not after methionine sulfoxide residues [26], the introduction of valine
Summary
Synthesis of Inhibitor Mutants in Yeast”M13 site-specific mutagenesis was performed as described previously using the natural q PI cDNA [10]. Complex formation with the V8 proteinase was not detected (Fig. 4,lane 2) and the inhibitorwas, instead, converted to a modified form of lower Mr Since a glutamic acid residue is known to occur in the Ps position it is possible that the enzyme cleaved the modified mutant inhibitor either at this position or at themodified Pl position, inactivating the inhibitor. After incubation with either poreciitnheer human neutrophil elastase or porcine pancreatic elastrypsin or plasmin (latter not shownc)o,mplex formation was tase while oxidation of thecysteinevariantdestroyedits readily detected with either the reduced cysteine or the ami- inhibitory activity againsatll proteinases tested except human noethylcysteine mutant (Fig. 5, lanes and 5 ). Porcine pancreatic elastase Bovine chymotrypsin Porcine trypsin Human cathepsin G Human plasmin
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