CHARACTERIZATION OF LIPOAMIDE DEHYDROGENASE FROM ESCHERICHIA-COLI LACKING THE REDOX-ACTIVE DISULFIDE - C44S AND C49S

Lipoamide dehydrogenase from Escherichia coli, a dimeric flavoprotein in the pyridine nucleotide-disulfide oxidoreductase family of enzymes, catalyzes the reduction of NAD(+) by dihydrolipoamide. The two electrons are transferred via a redox active disulfide and FAD. Cys(44) and Cys(49) comprise the redox active disulfide, Cys(44) interchanging with dihydrolipoamide and Cys(49) interacting with the flavin. Each of these residues has been mutated to serine (C44S, C49S). The altered enzymes showed minute amounts of activity, 0.003% for C44S and 0.012% for C49S using the physiological substrates dihydrolipoamide and NAD(+). These very low activities were expected, since the disulfide was no longer present in C44S and C49S, making dithiol-disulfide interchange impossible. However, the enzymes were capable of catalyzing reactions using NADH as the electron donor and alternate electron accepters: K3Fe(CN)(6), thio-NAD(+), DCIP, and O-2. These activities with NADH indicated that interaction of C44S and C49S with pyridine nucleotides was not affected greatly by the mutation. The pH dependence of the charge-transfer absorbance of C44S gives pK(a) values of 2.7, associated with titration of Cys(49), and 9.5, associated with titration of the acid-base catalyst, His(444'). A pK(a) of 5.1 was estimated for Cys(44) in C49S from the pH dependence of its reactivity with methyl methanethiosulfonate. The fluorescence of the FAD in oxidized wild type lipoamide dehydrogenase is markedly temperature dependent, while the remaining fluorescence of two-electron-reduced enzyme is independent of temperature. The fluorescence of the FAD in C44S and in C49S is likewise independent of temperature. The FAD of C44S and C49S is stoichiometrically titrated by 1 equiv of sodium dithionite. However, the FAD of C44S is markedly less completely reduced by 1 equiv of NADH than is the FAD of C49S, Ferricyanide stoichiometrically reoxidizes the FADH(2) of both altered forms of the enzyme.