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 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 (<5% wild-type). In the case of K54E E3, the Km for lipoamide in the reverse reaction was increased by about twofold. Surprisingly, for both SK-KS and E192Q E3s, the Kms for both dihydrolipoamide (forward reaction) and lipoamide (reverse reaction) were markedly reduced. The catalytic rate constants (kcat/Km) for both reactions for SK-KS E3 were significantly lower than wild-type, indicating that K54 is crucial for the catalytic efficiency of the enzyme. Fluorescence spectral analyses showed that the FAD in E3s were reduced by the addition of dihydrolipoamide, and that its reoxidation by NAD+ in the mutant E3s was slower than wild-type E3. Interestingly, in K54E E3 dihydrolipoamide reduced FAD efficiently only when NAD+ was present, indicating that K54 stabilizes the thiolate-FAD interaction. The lack of the formation of thiolate-FAD intermediate in the absence of NAD+ in K54E E3 was also confirmed by CD spectra. The SK-KS mutation demonstrates that the correct sequence of residues is as critical as the nature of the amino acid residues. These results suggest that K54 plays an important role in stabilizing the thiolate-FAD intermediate during the electron transfer in the reaction, and E192 is involved in maintaining correct orientation of K54 during catalysis.
Liu
T C
TC
Department of Biochemistry, School of Medicine and Biomedical Sciences, State University of New York at Buffalo, Buffalo, New York 14214, USA.
Hong
Y S
YS
Korotchkina
L G
LG
Vettakkorumakankav
N N
NN
Patel
M S
MS
D001483:Base Sequence; D002942:Circular Dichroism; D017931:DNA Primers / Q000235:genetics; D008058:Dihydrolipoamide Dehydrogenase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D004579:Electron Transport; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism; D005822:Genetic Vectors; D018698:Glutamic Acid / Q000737:chemistry; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D008239:Lysine / Q000737:chemistry; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009243:NAD / Q000378:metabolism; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013050:Spectrometry, Fluorescence
D017931:DNA Primers; D011994:Recombinant Proteins; D009243:NAD; D005182:Flavin-Adenine Dinucleotide; D018698:Glutamic Acid; D008058:Dihydrolipoamide Dehydrogenase; D008239:Lysine