Fluorescent labelling of 6-phosphogluconate dehydrogenase from Bacillus stearothermophilus.
The thermophilic 6-phosphogluconate dehydrogenase from Bacillus stearothermophilus was inhibited upon specific modification of the -SH group of cysteine residues by 7-chloro-4-nitrobenzo-2-oxa-1, 3-diazole (NBD-Cl) at pH 7.0. By using 20-100-fold molar excess of NBD-CL the reaction occurs slowly at pH 7.0 as a first order process. Partial protection from inactivation was observed when the substrate 6-phosphogluconate or the coenzyme NADP was added to the reaction mixture. Complete inactivation was achieved upon modification of 1.9 of the six cysteine residues per mole of enzyme, which corresponds to nearly one residue per enzyme subunit. Circular dichroism measurements suggest that the gross structure of the protein molecule is practically unchanged upon reaction of the enzyme with NBD-Cl. Melting profile experiments revealed a single transition occurring at about 65 degrees C. Analogously, the profile of intensity of the fluorescence emission at 520 nm of the enzyme-bound S-NBD groups versus temperature indicated a midpoint of transition near 65 degrees C. Since this melting temperature corresponds closely to that observed with the native enzyme, these results would indicate that the molecular organizations of the native and modified enzyme are similar and stabilized by similar interactions within the polypeptide chain.
D009327:4-Chloro-7-nitrobenzofurazan / Q000494:pharmacology; D002942:Circular Dichroism; D001411:Geobacillus stearothermophilus / Q000201:enzymology*; D005978:Glutathione / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D010734:Phosphogluconate Dehydrogenase / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013057:Spectrum Analysis; D013696:Temperature
D010734:Phosphogluconate Dehydrogenase; D009327:4-Chloro-7-nitrobenzofurazan; D005978:Glutathione