FAD analogues as prosthetic groups of human glutathione reductase

Abstract

1. Human glutathione reductase (NADPH + GSSG + H + + NADP' + 2 GSH) is a suitable enzyme for correlating spectroscopic properties and chemical reactivities of protein-bound FAD analogues with structural data. FAD, the prosthetic group of the enzyme, was replaced by FAD analogues, which were modified at the positions 8, 1, 2, 4, 5 and 6, respectively, of the isoalloxazine ring.2. When compared with a value of 100% for native glutathione reductase, the specific activities of most enzyme species ranged from 40% to 17%, in the order of the prosthetic groups 8-mercapto-FAD > 8-azido-The enzymic activities indicate a correct orientation of the bound analogues. The enzyme species containing 5-deaza-FAD and 6-OH-FAD, respectively, had no more glutathione reductase activity than the FAD-free apoenzyme. 5-Deaza-FAD . glutathione reductase was crystallized for X-ray diffraction analysis. 3. Detailed studies were focussed on position 8 of the flavin. 8-C1-FAD.glutathione reductase and 8-F-FAD. glutathione reductase reacted only poorly with HS-to give 8-mercapto-FAD . glutathione reductase, which suggests that the region around Val61 hinders the halogen anion from leaving the tetrahedral intermediate. Other experiments showed that position 8 is accessible to certain solvent-borne reagents. 8-Mercapto-FAD . glutathione reductase, for instance, reacted readily and stoichiometrically with the thiol reagent methylmethanethiosulfonate.4. 8-Mercapto-FAD . glutathione reductase does not exhibit a long wavelength charge transfer absorption band upon reduction, as it is the case for the 2-electron-reduced FAD-containing enzyme. This behaviour indicates that the charge transfer interaction between flavin and the tholate of Cys63 in the native enzyme is not per se essential for catalysis.The absorption spectrum of the blue anionic 8-mercapto-FAD bound to glutathione reductase suggests that the protein concurs to the stabilization of a negative charge in the pyrimidine subnucleus. In light of the protein structure this effect is attributed to the dipole moment of a-helix 338 -354 which starts out close to the N(I)/ C(2)/0(2cr) region of the flavin.5. 1-Deaza-FAD binds as tightly as FAD to the apoenzyme. The resulting holoenzyme was found to be enzymically active but structurally unstable. In this respect 1-deaza-FAD . glutathione reductase mimics the properties of the enzyme species found in inborn glutathione reductase deficiency. The high specific activity of 1-deaza-FAD.glutathione reductase shows that the hydrogen bond between N(l) and Thr339 in the native enzyme is not essential for catalysis. 6. 2-Thio-FAD and 4-thio-FAD were used as probes in order to confirm the crystallographic result that the region around N( 1)/0(2a)/N(3) and around O(

Absolute stereochemistry of flavins in enzyme-catalyzed reactions

“…II. The reconstituted enzyme had no detectable reductase activity, which is consistent with the results of Chan and Bruice (1977), who reported that 5-deazaflavins cannot transfer electrons to thiols, and also with the finding of Krauth-Siegel et al (1985) that glutathione reductase when reconstituted with 5-deaza-FAD has no reductase activity anymore. However, the addition of a 4-fold excess of NAD+ to a solution containing reduced 8-OH-5-deaza enzyme led to the complete reoxidation of the flavin chromophore within the time of mixing.…”

Role of the charged groups of glutathione disulfide in the catalysis of glutathione reductase: crystallographic and kinetic studies with synthetic analogs

“…The experiments showed that AH and ACP of GSSG analogues can be determined and that meaningful differences between the analogues may become available with careful measurements. It should be kept in mind, however, that the occupancies of the analogues have to be established accurately because they do not bind as well as "All enzyme concentrations were determined spectroscopically at 460 nm by using t = 11 mM"1 cm"1 per active site (Williams, 1976;Krauth-Siegel et al, 1985). The <2so/e460 ratio was always 6, indicating 100% holoenzyme (Krohne-Ehrich et al, 1977).…”

New flavins for old: artificial flavins as active site probes of flavoproteins

“…The attributions shown were confirmed by binding to flavoenzymes where the topography of the active centre was known from X-ray crystallography. Thus, with p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens, and with glutathione reductase from human erythrocytes, in which dipoles formed from a protein a-helix exert a partial positive charge directed toward the flavin N(1)-C(2) = 0 position [29][30][31], the spectrum predicted for (d) was obtained [28,32], and with flavodoxin, in which the flavin 8-position is exposed to solvent [33], the spectrum predicted for (c) was obtained [28].…”