Lipoamide dehydrogenase from Escherichia coli lacking the redox active disulfide: C44S and C49S. Redox properties of the FAD and interactions with pyridine nucleotides.
The cysteines that comprise the active site disulfide in lipoamide dehydrogenase have been individually mutated to a serine residue to give the altered enzymes, C44S and C49S, making it possible to study the redox behavior of the FAD in the absence of the disulfide. The redox potential of the FAD in C44S and C49S was -379 and -345 mV, respectively, at pH 7.0, 25 degrees C. A plot of the redox potential as a function of pH for C49S gave slopes of 57 mV/pH from pH 5.0 to 7.9 and 10 mV/pH from pH 7.9 to 8.8. The plot of the redox potential as a function of pH for C44S gave slopes of 70 mV/pH from pH 5.0 to 7.9 and 4 mV/pH from pH 7.9 to 8.38. The change in the slope at pH 7.9 is associated with the ionization (pKa) of the FADH2 to FADH- in the reduced form of both enzymes. These determinations show that the redox potential of the FAD in C49S, in C44S, and in wild type enzyme is modulated by the electronegativity of its nearest neighbor, hydroxyl, thiolate, or disulfide, and that the flavin is bound more tightly to the oxidized forms of these enzymes than to the reduced forms. The redox potentials of these enzymes determined using NADH and NADPH at pH 7.6, 25 degrees C are as follows: C44S, -350 mV, -369 mV; C49S, -328 mV, -353 mV, respectively. Thus, pyridine nucleotide binding raises the redox potential of the flavin, showing that both substrates bind more tightly to the reduced form of the enzymes, as well as tighter binding of NADH to the enzymes than that of NADPH. Kd values for the binding of NADH and NADPH to oxidized C44S and C49S were determined in pre-steady-state kinetics at pH 7.6 and 25 degrees C, which were monophasic when NADPH was the reductant and biphasic with NADH. The binding constants for NADPH were 660 microM for C44S and 500 microM for C49S; using NADH, the binding constants were 137 microM for C44S and 23 microM for C49S. Fluorescence and absorbance spectrophotometry were used to determine the binding of NAD+ to the oxidized forms of the enzymes as 275 microM and 270 microM for C44S and C49S, respectively.
Hopkins
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Department of Biological Chemistry, University of Michigan, Ann Arbor, USA.
Williams
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D001665:Binding Sites; D004396:Coloring Agents / Q000737:chemistry; D003545:Cysteine / Q000737:chemistry; D008058:Dihydrolipoamide Dehydrogenase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004220:Disulfides / Q000737:chemistry; D004579:Electron Transport; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D005182:Flavin-Adenine Dinucleotide / Q000737:chemistry / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D009243:NAD / Q000378:metabolism; D009249:NADP / Q000378:metabolism; D010084:Oxidation-Reduction; D010619:Phenazines / Q000737:chemistry; D012694:Serine / Q000737:chemistry; D014755:Viologens / Q000737:chemistry
D004396:Coloring Agents; D004220:Disulfides; D010619:Phenazines; D014755:Viologens; D009243:NAD; D005182:Flavin-Adenine Dinucleotide; D012694:Serine; D009249:NADP; D008058:Dihydrolipoamide Dehydrogenase; D003545:Cysteine; C009195:safranine T