Characterization of lipoamide dehydrogenase from Escherichia coli lacking the redox active disulfide: C44S and C49S

Biochemistry
N Hopkins, C H Williams

Abstract

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. Cys44 and Cys49 comprise the redox active disulfide, Cys44 interchanging with dihydrolipoamide and Cys49 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 acceptors: K3Fe(CN)6, thio-NAD+, DCIP, and O2. 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 pKa values of 2.7, associated with titration of Cys49, and 9.5, associated with titration of the acid-base catalys...Continue Reading

Citations

Apr 15, 1997·Archives of Biochemistry and Biophysics·J Marcinkeviciene, J S Blanchard
Aug 12, 2005·The Journal of Biological Chemistry·Kanagalaghatta R RajashankarChristopher D Lima
Oct 1, 2004·The Journal of Biological Chemistry·Argyrides ArgyrouJohn S Blanchard
Sep 10, 2020·ACS Catalysis·Derren J HeyesNigel S Scrutton
Feb 22, 2018·ACS Infectious Diseases·Ana NegriSelin Somersan-Karakaya

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