Site-Directed Mutagenesis of Human Dihydrolipoamide Dehydrogenase: Role of Lysine-54 and Glutamate-192 in Stabilizing the Thiolate–FAD Intermediate

The roles of lysine-54 (K54) and glutamate-192 (E192) of human dihydrolipoamide dehydrogenase (E3) in stabilizing the thiolate–FAD intermediate during electron transfer were investigated by site-directed mutagenesis. Recombinant human E3s, wild-type, K54E, S53K54–K53S54 (SK–KS), and E192Q, were over...

Full description

Saved in:
Bibliographic Details
Published in:Protein expression and purification 1999-06, Vol.16 (1), p.27-39
Main Authors: Liu, Te-Chung, Soo Hong, Young, Korotchkina, Lioubov G., Vettakkorumakankav, Nataraj N., Patel, Mulchand S.
Format: Article
Language:English
Subjects:
Base Sequence
Circular Dichroism
Dihydrolipoamide Dehydrogenase - chemistry
Dihydrolipoamide Dehydrogenase - genetics
Dihydrolipoamide Dehydrogenase - metabolism
DNA Primers - genetics
Electron Transport
Enzyme Stability
Escherichia coli - genetics
Flavin-Adenine Dinucleotide - metabolism
Genetic Vectors
Glutamic Acid - chemistry
Humans
In Vitro Techniques
Kinetics
Lysine - chemistry
Models, Molecular
Mutagenesis, Site-Directed
NAD - metabolism
Protein Conformation
Recombinant Proteins - chemistry
Recombinant Proteins - genetics
Recombinant Proteins - metabolism
Spectrometry, Fluorescence
Citations: Items that cite this one
Online Access:Get full text
Tags: Add Tag
No Tags, Be the first to tag this record!
Description
Summary:The roles of lysine-54 (K54) and glutamate-192 (E192) of human dihydrolipoamide dehydrogenase (E3) in stabilizing the thiolate–FAD intermediate during electron transfer were investigated by site-directed mutagenesis. Recombinant human E3s, wild-type, K54E, S53K54–K53S54 (SK–KS), and E192Q, were overexpressed, purified, and characterized. Only K54E and SK–KS E3s had about 25% less bound FAD compared to wild-type, implicating that K54 is crucial for the protein–FAD interaction. The specific activities of all mutant E3s were markedly decreased (
ISSN:1046-5928
1096-0279
DOI:10.1006/prep.1999.1047