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Spectral evidence for a flavine adduct in a mono alkylated derivative of pig heart lipo amide dehydrogenase






Journal of Biological Chemistry 251(23): 7726-7728

Spectral evidence for a flavine adduct in a mono alkylated derivative of pig heart lipo amide dehydrogenase

A derivative of the flavoprotein pig heart lipoamide dehydrogenase has been described recently, in which 1 of the 2 cysteine residues generated on reduction of the intrachain active center disulfide bridge is selectively alkylated with iodoacetamide. This monolabeled enzyme exhibits a spectrum of oxidized bound flavin. The addition of 1 mM NAD+ to this derivative at pH 8.3 causes a decrease in absorbance of approximately 50% at 448 nm, with a concomitant increase at 380 nm. These spectral changes are complete within 3 ms and are reversible. NAD+ titrations generate isosbestic points at 408, 374 and 327 nm; allowing values for the apparent dissociation constant for NAD+ and the extent of bleaching at infinite ligand to be obtained from double reciprocal plots. Between pH 6.1 and 8.8, the apparent Kd decreases from 320-35 .mu.M, whereas the extrapolated .DELTA.epsilon.448 values remain approximately constant at 1/2 .epsilon.448. Direct measurement of NAD+ binding by gel filtration at pH 8.8 indicates that the spectral changes are associated with a stoichiometry of 1.2 mol of NAD+ bound/2 mol of FAD. The modified protein is a dimer containing 1 FAD and 1 alkylated cysteine residue/subunit; the native enzyme is also dimeric. The visible spectrum of the species absorbing at 380 nm, approximated by correction for the residual oxidized FAD, shows a single maximum at 384 nm, .epsilon.384 = 8.7 mM-1cm-1. Comparison of this spectrum with that of model compounds of known structure suggests that it may represent a reversible covalent flavin adduct induced on binding NAD+.


Accession: 006459676

PMID: 187594



Related references

Thorpe, C.; Williams, C.H., 1976: Spectral evidence for a flavin adduct in a monoalkylated derivative of pig heart lipoamide dehydrogenase. A derivative of the flavoprotein pig heart lipoamide dehydrogenase has been described recently (Thorpe, C., and Williams, C.H. (1976) J. Biol. Chem. 251, 3553-3557), in which 1 of the 2 cysteine residues generated on reduction of the intrachain ac...

O'donnell M.E.; Johnson F.A.; Williams C.H.Jr, 1983: Proton nmr investigation of the mechanism of flavin c 4a adduct formation induced by nad binding to mono alkylated pig heart lipoamide dehydrogenase. The active center thiol of monoalkylated pig heart lipoamide dehydrogenase, EHR, is induced to form an adduct to the enzyme-bound FAD at the C-4a position upon binding NAD. In light of hypotheses on covalent electron transfer between pyridine nucl...

Templeton D.M.; Hollebone B.R.; Tsai C.S., 1980: Magnetic circular dichroism studies on the active site flavine of lipo amide dehydrogenase. The magnetic circular dichroic (MCD) spectra of oxidized and reduced flavins are recorded in various solvents. They are sensitive to flavin environment. The MCD spectra of oxidized and reduced lipoamide dehydrogenase [porcine heart] are reported....

Anonymous, 1973: Purification of lipo amide dehydrogenase ec 1643 by affinity chromatography on propyl lipo amide glass columns

Texter F.L.; Lutton J.K.; Brandt K.G., 1977: Steady state kinetic studies of the lipo amide dehydrogenase component within the pig heart pyruvate dehydrogenase complex. Federation Proceedings 36(3): 875

Loechler, E.L.; Hollocher, T.C., 1980: Reduction of flavines by thiols 2. spectrophotometric evidence for a thiol c 4a flavine adduct and the kinetics of deprotonation of the sulfhydryl group of the di thio threitol adduct. In the reduction of 3-methylriboflavin [3] by dithiothreitol (DTT) a 2nd change in rate-determining step was observed at low [buffer] when [DTT] was low (5 mM range). The 3rd sequential step thus revealed was buffer catalyzed, followed the rate la...

Templeton, D.M.; Tsai, C.S., 1979: Histidine at the active center of pig heart lipo amide dehydrogenase ec 1.6.4.3. Dye-sensitized photooxidation of 1 or more histidine residues in lipoamide dehydrogenase adversely affects enzymic activity with lipoamide as acceptor. Other enzymic activities are relatively unaffected. Phosphorylation of a common histidine resid...

Thorpe, C.; Williams, C.H.Jr, 1981: Lipoamide dehydrogenase ec 1.6.4.3 from pig heart pyridine nucleotide induced changes in mono alkylated 2 electron reduced enzyme. Two-electron reduced lipoamide dehydrogenase from pig heart reacted with iodoacetamide was monoalkylated almost exclusively in the nascent thiol nearer the amino terminus of the protein. The charge-transfer absorbance, maximal at 530 nm, character...

Ogasahara, K.; Koike, K.; Hamada, M.; Hiraoka, T., 1976: Interaction of hydrophobic probes with the apo enzyme of pig heart lipo amide dehydrogenase ec 1.6.4.3. The interaction of hydrophobic probes, 8-anilinonaphthalene-1-sulfonate (ANS) and 4-benzoylamido-4'-aminostilbene-2,2'-disulfonate (MBAS), with pig heart lipoamide dehydrogenase [NADH: lipoamide oxidoreductase, EC 1.6.4.3] was investigat...

Nakamura, M.; Yamazaki, I., 1979: Salts induced oxidase activity of lipo amide dehydrogenase ec 1.6.4.3 from pig heart. A weak NADH oxidase activity of lipoamide dehydrogenase [EC 1.6.4.3] at neutral pH is increased as much as 15-fold by the addition of KI or (NH4)2SO4. The addition of NAD+ shifts the optimum pH for the KI-induced oxidase activity from 6.3-5.5 with...