Membrane-associated reactions in ubiquinone biosynthesis in Escherichia coli. 3-Octaprenyl-4-hydroxybenzoate carboxy-lyase.
A sensitive and quantitative assay for 3-octaprenyl-4-hydroxybenzoate carboxy-lyase has been developed. This enzyme, which catalyses the third reaction in ubiquinone biosynthesis in Escherichia coli, was partially purified and some of its properties determined. It was found that a considerable proportion of the carboxylyase activity could be separated from the membrane fraction in cell extracts prepared using a French press. Gel filtration showed the molecular weight of the enzyme to be about 340 000. For optimal activity the carboxy-lase was shown to require Mn2+, washed membranes or an extract of phospholipids, and an unidentified heat stable factor of molecular weight less than 10 000. The carboxy-lyase reaction was also shown to be strongly stimulated by dithiothreitol and methanol. The properties of the carboxy-lyase are compared with the three other enzymes concerned with ubiquinone biosynthesis in E. coli which have been studied in vitro. The fact that the substrate of the carboxy-lyase is membrane-bound and the enzyme is stimulated by phospholipid suggests that it normally functions in association with the cytoplasmic membrane in vivo.
D001426:Bacterial Proteins / Q000502:physiology; D002262:Carboxy-Lyases / Q000302:isolation & purification / Q000378:metabolism*; D002413:Cations, Divalent / Q000494:pharmacology; D002462:Cell Membrane / Q000201:enzymology*; D004229:Dithiothreitol / Q000494:pharmacology; D004789:Enzyme Activation; D004926:Escherichia coli / Q000201:enzymology*; D008722:Methods; D008970:Molecular Weight; D014451:Ubiquinone / Q000096:biosynthesis*
D001426:Bacterial Proteins; D002413:Cations, Divalent; D014451:Ubiquinone; D002262:Carboxy-Lyases; D004229:Dithiothreitol