Properties of lipoamide dehydrogenase altered by site-directed mutagenesis at a key residue (Il84Y) in the pyridine nucleotide binding domain

Abstract

The binding of pyridine nucleotide to human erythrocyte glutathione reductase, an enzyme of known three-dimensional structure, requires some movement of the side chain of Tyr197. Moreover, this side chain lies very close to the isoalloxazine ring of the FAD cofactor. The analogous residue, Ile184, in the homologous enzyme Escherichia coli lipoamide dehydrogenase has been altered by site-directed mutagenesis to a tyrosine residue (I184Y) [Russell, G. C., Allison, N., Williams, C. H., Jr., & Guest, J.R. (1989) Ann. N.Y. Acad. Sci. 573, 429-431]. Characterization of the altered enzyme shows that the rate of the pyridine nucleotide half-reaction has been markedly reduced and that the spectral properties have been changed to mimic those of glutathione reductase. Therefore, Ile184 is shown to be an important residue in modulating the properties of the flavin in lipoamide dehydrogenase. Turnover in the dihydrolipoamide/NAD+ reaction is decreased by 10-fold and in the NADH/lipoamide reaction by 2-fold in I184Y lipoamide dehydrogenase. The oxidized form of I184Y shows remarkable changes in the fine structure of the visible absorption and circular dichroism spectra and also shows nearly complete quenching of FAD fluorescence. The spectral properties of the altered enzyme are thus similar to those of glutathione reductase and very different from those of wild-type lipoamide dehydrogenase. On the other hand, spectral evidence does not reveal any change in the amount of charge-transfer stabilization at the EH2 level. Stopped-flow data indicate that, in the reduction of I184Y by NADH, the first step, reduction of the flavin, is only slightly slowed but the subsequent two-electron transfer to the disulfide is markedly inhibited.(ABSTRACT TRUNCATED AT 250 WORDS)

Protein Thiol Modifications Visualized In Vivo

“…To regenerate this complex, the disulfide bond in dihydrolipoamide is re-oxidized by the active-site disulfide bond of Lpd, which itself donates its electrons to the prosthetic flavin adenine dinucleotide and ultimately to nicotinamide adenine dinucleotide. Because the standard redox potentials of Lpd and the dihydrolipoic acid/lipoic acid redox pair of AceF are known (Table 1) (Maeda-Yorita et al 1991; Nelson et al 2000), we determined the 14 C activity/protein ratio of Lpd under regular and reverse-trapping conditions and determined its redox state in vivo.…”

“… a   Maeda-Yorita et al (1991) b  Determined from the ratios obtained in the trapping experiment and the standard redox potential c   Nelson et al (2000) d  Determined from the ratios obtained in the trapping experiment, assuming that AhpC is in equilibrium with the cellular redox potential as represented by the GSH/GSSG redox pair e  Calculated from the concentrations of GSH and GSSG in E. coli DHB4 (Aslund et al 1999), assuming that AhpC is in equilibrium with the cellular redox potential as represented by the GSH/GSSG redox pair…”

Zinc Is a Potent Inhibitor of Thiol Oxidoreductase Activity and Stimulates Reactive Oxygen Species Production by Lipoamide Dehydrogenase

“…More recently it has been proposed that NAD ϩ release directly yields the charge-transfer complex. The enzyme form with fully reduced FAD is not thought to be significantly populated (18).…”

Electrochemical Comparative Study of Riboflavin, FMN and FAD Immobilized on the Silica Gel Modified with Zirconium Oxide

“…However a major obstacle found in that kind of study is the relative low difference between the E o´ values of NADH and of free flavin which results in a slow kinetics of this redox reaction. Attempts to increase the reaction rate between flavins and NADH include derivating flavins, [13][14][15] immobilization of flavins on matrices to cause a shift of its E o´ toward more positive values. [16][17][18] Flavins have been adsorbed on graphite, glassy carbon, platinum and gold 19 electrodes surfaces and have also been attached through mercaptan 20 and thiourea 21 linkages to noble metal substrates or using of the Langmuir-Blodgett technique.…”