Improvement of Antimicrobial Activity of Pediocin PA-1 by Site-directed Mutagenesis in C-terminal Domain

2015 ◽  
Vol 22 (11) ◽  
pp. 1007-1012 ◽  
Author(s):  
Lin Sun ◽  
Hui Song ◽  
Wen Zheng
Keyword(s):  
Antimicrobial Activity ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Terminal Domain

scholarly journals Catalytic roles of lysines (K9, K27, K31) in the N-terminal domain in human adenylate kinase by random site-directed mutagenesis

IUBMB Life ◽  
1996 ◽  
Vol 40 (5) ◽  
pp. 897-906
Author(s):  
Takanori Ayabe ◽  
Seung Kyu Park ◽  
Hitoshi Takenaka ◽  
Michihiro Sumida ◽  
Seiichi Uesugi ◽  
...  
Keyword(s):  
Adenylate Kinase ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Terminal Domain ◽  
Random Site

scholarly journals Site-directed mutagenesis of the leech-derived factor Xa inhibitor antistasin. Probing of the reactive site

1992 ◽  
Vol 287 (3) ◽  
pp. 943-949 ◽  
Author(s):  
K J Hofmann ◽  
E M Nutt ◽  
C T Dunwiddie
Keyword(s):  
Amino Acid ◽  
Proteinase Inhibitors ◽  
Expression System ◽  
Factor Xa ◽  
Site Directed Mutagenesis ◽  
Arginine Residue ◽  
Common Mechanism ◽  
Directed Mutagenesis ◽  
Reactive Site ◽  
Terminal Domain

Antistasin (ATS) is a leech-derived 119-amino-acid protein which exhibits potent and highly selective inhibition of coagulation Factor Xa. It inhibits Factor Xa according to a common mechanism of serine-proteinase inhibitors in which a conformationally rigid substrate-like reactive site is presented to the enzyme. In this study a recombinant version of ATS was expressed and purified utilizing a yeast expression system in order to probe the reactive site P1 (Arg-34) and P1′ (Val-35) residues by site-directed mutagenesis. The results demonstrate the requirement for a positively charged residue in the P1 position of ATS, with an arginine residue preferred over a lysine, yielding K1 values of 61 pM and 1.28 nM respectively. Mutation of the P1 arginine residue to the non-polar amino acid leucine abolished its inhibitory potency toward Factor Xa. The role of the C-terminal domain of ATS, which shares significant amino acid sequence identity with the N-terminal domain, was investigated by creating a second reactive site in the corresponding position of the C-terminal domain. The inhibitory activity of this mutant demonstrated that the C-terminal domain of ATS is not folded into the proper conformation necessary to create a functional inhibitory domain.

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