Modulation of the oxidation-reduction potential of the flavin in lipoamide dehydrogenase from Escherichia coli by alteration of a nearby charged residue, K53R.
The epsilon-amino group of a lysine residue occupies a position within bonding distance of the flavin N5 and the bound NADPH pyridinium C4' in glutathione reductase, and it has been suggested that this positive charge influences the redox potential of the FAD [Pai & Schulz (1983) J. Biol. Chem. 258, 1752]. A conserved lysine residue occupies a similar position in lipoamide dehydrogenase. This residue has been replaced by an arginine in lipoamide dehydrogenase from Escherichia coli to give K53R. The spectral and redox properties of the FAD in K53R as well as the interaction of the flavin with bound NAD+ are profoundly affected by the change. K53R does not catalyze either the dihydrolipoamide-NAD+ or the NADH-lipoamide reactions except at very low concentrations of the reducing substrate. The absorbance spectrum of K53R in the visible and near-ultraviolet is little changed from that of wild-type enzyme, but in contrast, the spectrum of K53R is sensitive to pH with an apparent pKa = 7.0. Unlike the wild-type enzyme, the binding of beta-NAD+ to K53R alters the spectrum and indicates an apparent Kd = 7.0 microM at pH 7.6. The flavin fluorescence is partially quenched, and the visible and near-ultraviolet circular dichroism spectrum is changed by beta-NAD+. K53R is extensively reduced (mostly EH4) by 2 equiv of dihydrolipoamide/FAD while the wild-type enzyme is only partially reduced (mostly EH2). The rate of this reduction is lowered by approximately 3-fold relative to the wild-type enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)
Maeda-Yorita
K
K
Department of Biological Chemistry, University of Michigan, Ann Arbor 48109.
Russell
G C
GC
Guest
J R
JR
Massey
V
V
Williams
C H
CH
D002942:Circular Dichroism; D008058:Dihydrolipoamide Dehydrogenase / Q000737:chemistry* / Q000378:metabolism; D004563:Electrochemistry; D004926:Escherichia coli / Q000201:enzymology*; D005182:Flavin-Adenine Dinucleotide / Q000737:chemistry* / Q000378:metabolism; D007700:Kinetics; D008239:Lysine / Q000737:chemistry; D009243:NAD / Q000378:metabolism; D010084:Oxidation-Reduction; D010777:Photochemistry; D013050:Spectrometry, Fluorescence; D013053:Spectrophotometry; D008063:Thioctic Acid / Q000031:analogs & derivatives / Q000378:metabolism
D009243:NAD; D005182:Flavin-Adenine Dinucleotide; C007409:dihydrolipoamide; D008063:Thioctic Acid; C013091:lipoamide; D008058:Dihydrolipoamide Dehydrogenase; D008239:Lysine