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. 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 catalyst, His444'. A pKa of 5.1 was estimated for Cys44 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 FADH2 of both altered forms of the enzyme.

[1]  Wade H. Shafer,et al.  Chemistry and Biochemistry , 1997 .

[2]  C. Williams,et al.  Lipoamide dehydrogenase from Escherichia coli lacking the redox active disulfide: C44S and C49S. Redox properties of the FAD and interactions with pyridine nucleotides. , 1995, Biochemistry.

[3]  J. Guest,et al.  Properties of lipoamide dehydrogenase altered by site-directed mutagenesis at a key residue (I184Y) in the pyridine nucleotide binding domain. , 1991, Biochemistry.

[4]  N. Scrutton,et al.  Directed mutagenesis of the redox-active disulphide bridge in glutathione reductase from Escherichia coli , 1990, Proceedings of the Royal Society of London. Series B: Biological Sciences.

[5]  P. Karplus,et al.  Substrate binding and catalysis by glutathione reductase as derived from refined enzyme: substrate crystal structures at 2 A resolution. , 1994, Journal of molecular biology.

[6]  J. Guest,et al.  Oligonucleotide‐Directed Mutagenesis of the lpd Gene of Escherichia coli , 1989 .

[7]  L. Sahlman,et al.  Properties of Lipoamide Dehydrogenase and Thioredoxin Reductase from Escherichia coli Altered by Site‐Directed Mutagenesis a , 1989, Annals of the New York Academy of Sciences.

[8]  L. Sahlman,et al.  Lipoamide dehydrogenase from Escherichia coli. Steady-state kinetics of the physiological reaction. , 1989, The Journal of biological chemistry.

[9]  G. Vriend,et al.  X-ray structure of lipoamide dehydrogenase from Azotobacter vinelandii determined by a combination of molecular and isomorphous replacement techniques. , 1989, Journal of molecular biology.

[10]  C. Walsh,et al.  Mutagenesis of the redox-active disulfide in mercuric ion reductase: catalysis by mutant enzymes restricted to flavin redox chemistry. , 1989, Biochemistry.

[11]  J. Guest,et al.  Overexpression and mutagenesis of the lipoamide dehydrogenase of Escherichia coli. , 1988, The Biochemical journal.

[12]  P. Karplus,et al.  Refined structure of glutathione reductase at 1.54 A resolution. , 1987, Journal of molecular biology.

[13]  J. Shafer,et al.  Reactivity of small thiolate anions and cysteine-25 in papain toward methyl methanethiosulfonate. , 1986, Biochemistry.

[14]  P. Schultz,et al.  Directed mutagenesis of the redox-active disulfide in the flavoenzyme mercuric ion reductase. , 1985, Biochemistry.

[15]  C. Williams,et al.  Reconstitution of Escherichia coli thioredoxin reductase with 1-deazaFAD. Evidence for 1-deazaFAD C-4a adduct formation linked to the ionization of an active site base. , 1984, The Journal of biological chemistry.

[16]  L. Polgár,et al.  Current problems in mechanistic studies of serine and cysteine proteinases. , 1982, The Biochemical journal.

[17]  C. Thorpe,et al.  Lipoamide dehydrogenase from pig heart. Pyridine nucleotide induced changes in monoalkylated two-electron reduced enzyme. , 1981, Biochemistry.

[18]  K. Wilkinson,et al.  NADH inhibition and NAD activation of Escherichia coli lipoamide dehydrogenase catalyzing the NADH-lipoamide reaction. , 1981, The Journal of biological chemistry.

[19]  K. Wilkinson,et al.  Evidence for multiple electronic forms of two-electron-reduced lipoamide dehydrogenase from Escherichia coli. , 1979, The Journal of biological chemistry.

[20]  C. Thorpe,et al.  Spectral evidence for a flavin adduct in a monoalkylated derivative of pig heart lipoamide dehydrogenase. , 1976, The Journal of biological chemistry.

[21]  R. Matthews,et al.  Measurement of the oxidation-reduction potentials for two-electron and four-electron reduction of lipoamide dehydrogenase from pig heart. , 1976, The Journal of biological chemistry.

[22]  C. Thorpe,et al.  Differential reactivity of the two active site cysteine residues generated on reduction of pig heart lipoamide dehydrogenase. , 1976, The Journal of biological chemistry.

[23]  G. L. Kenyon,et al.  Simple alkanethiol groups for temporary blocking of sulfhydryl groups of enzymes. , 1975, Biochemistry.

[24]  D. Lambeth,et al.  The kinetics and mechanism of reduction of electron transfer proteins and other compounds of biological interest by dithionite. , 1973, The Journal of biological chemistry.

[25]  U. K. Laemmli,et al.  Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4 , 1970, Nature.

[26]  C. Williams,et al.  A method for titrating oxygen-sensitive organic redox systems with reducing agents in solution. , 1969, Analytical biochemistry.

[27]  G. Zanetti,et al.  Characterization of the active center of thioredoxin reductase. , 1967, The Journal of biological chemistry.

[28]  Williams Ch.  Studies on lipoyl dehydrogenase from Escherichia coli. , 1965 .

[29]  B. Anderson,et al.  THE THIONICOTINAMIDE ANALOGS OF DPN AND TPN. I. PREPARATION AND ANALYSIS. , 1963, Biochemistry.

[30]  V. Massey,et al.  The reaction mechanism of lipoamide dehydrogenase. II. Modification by trace metals. , 1962, Biochimica et biophysica acta.

[31]  V. Massey,et al.  Studies on the reaction mechanism of lipoyl dehydrogenase. , 1961, Biochimica et biophysica acta.

[32]  Q. Gibson,et al.  Intermediates in the catalytic action of lipoyl dehydrogenase (diaphorase). , 1960, The Biochemical journal.

[33]  L. Reed,et al.  alpha-Keto acid dehydrogenation complexes. III. Purification and properties of dihydrolipoic dehydrogenase of Escherichia coli. , 1960, The Journal of biological chemistry.

[34]  V MASSEY,et al.  The identity of diaphorase and lipoyl dehydrogenase. , 1958, Biochimica et biophysica acta.

[35]  H. Beinert,et al.  On the mechanism of dehydrogenation of fatty acyl derivatives of coenzyme A. VI. Isolation and properties of stable enzyme-substrate complexes. , 1958, The Journal of biological chemistry.

[36]  V. Massey.  The identity of diaphorase and lipoic dehydrogenase. , 1958, Biochimica et biophysica acta.

[37]  K. Foley,et al.  MOLECULAR INTERACTION OF ISOALLOXAZINE DERIVATIVES. , 1958, Proceedings of the National Academy of Sciences of the United States of America.

[38]  F. B. Straub,et al.  Isolation and properties of a flavoprotein from heart muscle tissue. , 1939, The Biochemical journal.