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Title 

Single amino acid substitutions alpha1-antitrypsin that confer enhancement in thermal stability

Authors 

Ki Sun KwonJeong Ho KimHwa Soo ShinMyeong Hee Yu

Publisher 

American Society for Biochemistry and Molecular Biology

Issue Date 

1994

Citation 

Journal of Biological Chemistry, vol. 269, no. 13, pp. 9627-9631

Keywords 

alpha 1 antitrypsinenzyme stabilitythermostabilityalpha 1-Antitrypsin

Abstract 

A recombinant α1-antitrypsin variant which increased thermal stability was obtained from random mutagenesis followed by screening. The clone was identified as having a single mutation of Phe51 → Cys. Heat deactivation of purified recombinant α1-antitrypsin produced in Escherichia coli revealed that the mutation slowed down the deactivation rate 10-fold at 57 °C, increasing thermal stability of recombinant protein to almost that of natural glycosylated plasma form. The mutant protein also exhibited increased stability against denaturant. The urea-induced unfolding monitored by the changes in fluorescence intensity at 360 nm showed that the mutation shifted midpoint of the transition from 1.9 M to 2.8 M. The mutation site is particularly interesting in that some genetic variants mapped at adjacent positions were shown previously to cause aggregation of the polypeptides, while the Phe51 → Cys mutation decreased aggregation rate significantly during heat deactivation. The association rate constant with porcine pancreatic elastase revealed that the mutation did not affect inhibitory activity significantly. The site identified may be critical for regulating stability of α1-antitrypsin. Characterization of various single amino acid substitutions at position 51 suggests that volume and flexibility of hydrophobic side chain at the site are critical factors for enhancing the stability of α1-antitrypsin.

ISSN 

0021-9258

Appears in Collections

1. Journal Articles > Journal Articles

Registered Date

2017-04-19


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