The elucidation of the microheterogeneity of highly purified p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens by various biochemical techniques.
Highly purified p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens can be separated into at least five fractions by anion-exchange chromatography. All fractions exhibit the same specific activity and the enzyme exists mainly in the dimeric form in solution. Sodium dodecyl sulfate/polyacrylamide gel electrophoresis of a mixture of the different fractions reveals two apparent forms of enzyme molecules, while isoelectric focusing experiments, on the other hand, reveal six apparently different forms of enzyme molecules. It is shown that the different forms of enzyme molecules are due to the (partial) oxidation of Cys-116 in the sequence of the enzyme. This interpretation of the data is supported by kinetic measurements of the formation of hybrid dimeric molecules monitored by fast protein liquid chromatography, using purified enzyme containing Cys-116 either in the native and or the fully oxidized (sulfonic acid) state. By chemical modification studies using maleimide derivatives, 5,5'-dithiobis(2-nitrobenzoate) and H2O2, it is shown that sulfenic, sulfinic and sulfonic acid derivatives of Cys-116 are products of oxidation. The results are briefly discussed with respect to the possibility that this isolation artifact might also be partially responsible for the appearance of multiple forms of enzyme molecules in other biochemical preparations.
D015109:4-Hydroxybenzoate-3-Monooxygenase / Q000032:analysis* / Q000378:metabolism; D002852:Chromatography, Ion Exchange; D004228:Dithionitrobenzoic Acid / Q000494:pharmacology; D004578:Electron Spin Resonance Spectroscopy; D004591:Electrophoresis, Polyacrylamide Gel; D006861:Hydrogen Peroxide / Q000494:pharmacology; D007525:Isoelectric Focusing; D007527:Isoenzymes / Q000032:analysis* / Q000378:metabolism; D008301:Maleimides / Q000494:pharmacology; D008433:Mathematics; D006899:Mixed Function Oxygenases / Q000032:analysis*; D011551:Pseudomonas fluorescens / Q000201:enzymology*
D007527:Isoenzymes; D008301:Maleimides; D004228:Dithionitrobenzoic Acid; D006861:Hydrogen Peroxide; D006899:Mixed Function Oxygenases; D015109:4-Hydroxybenzoate-3-Monooxygenase