Protein Engineering, Vol 11, 1243-1247, Copyright © 1998 by Oxford University Press
SP Bottomley and SR Stone
The exposed Serpin reactive centre loop controls the specificity of the
serpin proteinase interaction. Mutations within this region have been used
to generate novel potentially therapeutic inhibitors. In this study we
examine the effect of the serpin scaffold and reactive centre loop length
upon the generation of such inhibitors. The reactive centre loop regions,
P7-P3', of alpha1-antitrypsin and alpha1-antichymotrypsin were replaced by
the corresponding residues of the viral serpin, Serp1, to form AT/Serp1 and
ACT/Serp1, respectively. AT/Serp1 formed SDS stable complexes with a range
of proteinases with association rate constants for plasmin, tissue
plasminogen activator, urokinase, thrombin and factor Xa of approximately
10(4) M(-1)s(-1) and a stoichiometry of inhibition of approximately 1 for
all of them. ACT/Serp1, however, formed SDS-stable complexes with only
plasmin and thrombin with association rate constant 100-fold slower than
AT/Serp1 and an increased stoichiometry of inhibition. The reactive centre
loop of ACT/Serp1 is four amino acid residues longer than AT/Serp1. These
four additional residues (VETR) were inserted into AT/Serp1 to form
AT/Serp1(VETR). AT/Serp1(VETR) formed SDS stable complexes with plasmin,
thrombin and tissue plasminogen activator similar to AT/Serp1, however, the
association rate constants were 10-fold slower than those observed with
AT/Serp1, while the stoichiometry of inhibition remained around 1. These
results suggest that the additional reactive centre loop residues effect
the rate of initial complex formation by placing the reactive centre loop
in a non-ideal conformation. This study demonstrates that both reactive
centre loop length and serpin scaffold are important in defining the
inhibitory characteristics of a serpin.
ARTICLES
Protein engineering of chimeric Serpins: an investigation into effects of the serpin scaffold and reactive centre loop length
Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria, Australia.
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