Studies on reactivity of human leukocyte elastase, cathepsin G, and porcine pancreatic elastase toward peptides including sequences related to the reactive site of alpha 1-protease inhibitor (alpha 1-antitrypsin)

Mcrae, B.; Nakajima, K.; Travis, J.; Powers, J.C.

Biochemistry 19(17): 3973-3978

1980


ISSN/ISBN: 0006-2960
PMID: 6967733
DOI: 10.1021/bi00558a013
Accession: 006511625

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Abstract
The reactions of human leukocyte (HL) elastase porcine pancreatic (PP) elastase and human cathepsin G were studied with peptide substrates. Most of the peptides had a prolyl residue at P2, since several serine proteases productively bound such substrates only with the proline at the subsite (S2) adjacent to the primary substrate binding site (S1). HL elastase prefers a valyl residue over Ala or Met at S1, while PP elastase prefers an Ala. With both elastases, extension of the peptide chain results in significant increases in kcat/KM. With the appropriate substrates, HL elastase is as reactive as PP elastase. Cathepsin G shows a preference for Phe over Met at S1 and a preference for Phe over Ala or Leu at S1'. Extension of the peptide chain yields little increase in rate; the kcat/KM values observed with cathepsin G are not as large as those of the other enzymes. The .alpha.1-protease inhibitor (.alpha.1-PI) reactive site was the sequence-Ala-Ile-Pro-Met*Ser-Ile-Pro-Pro-, where the asterisk indicates the bond cleaved (P1.sbd.P1') when .alpha.1-PI.cntdot.protease complexes are split. The octapeptide Ac-Ala-Ile-Pro-Met-Ser-Ile-Pro-Pro-NH2 and the analogue with a P1' Thr instead of Ser were synthesized. All 3 enzymes bound to and cleaved the peptides at the P1 Met bond. The KM values were in the millimolar range, showing that this particular sequence alone does not account for the tight binding of serine proteases to .alpha.1-PI. The kcat values, a measure of the ease with which certain types of bond formation between proteases and .alpha.1-PI would occur, were higher for the P1' Ser octapeptide than for the Thr analogue, indicating that relatively minor amino acid substitutions in the .alpha.1-PI reactive site was profoundly influence its reactivity toward various proteases. Inactivation of .alpha.1-PI in the lung by oxidation and the resulting protease imbalance is the currently accepted model for the development of emphysema. In the majority of cases studied, oxidation of the P1 Met residue to simple peptides to the sulfoxide resulted in decreased binding to the enzymes studied, and a decreased kcat/KM. Reduction of substrate effectiveness was greatest with HL elastase for the P1' Ser peptides compared to the P1' Thr peptides. Reactive site substitutions could effect the degree to which oxidation is damaging to the inhibitor and may be one possible explanation for the greater susceptibility to emphysema of some individuals with normal .alpha.1-PI.