Regulation of NAD+- and NADP+-linked isocitrate dehydrogenase in the obligate methylotrophic bacterium Pseudomonas W6.
Cell-free extracts of the obligate methanol-utilizing bacterium Pseudomonas W6 catalyze the oxydation of isocitrate to alpha-ketoglutarate in the presence of NAD+ and NADP+. After electro-focusing of the crude extract of Pseudomonas W6 actually two distinct bands each of NAD+-linked isocitrate dehydrogenase (NAD+-IDH) and of NADP+-linked isocitrate dehydrogenase (NADP+-IDH) could be observed. The NAD+-IDH was completely separated from the NADP+-IDH by employing DEAE ion exchange chromatography and further purified by affinity chromatography using Cibacron blue F 3G-A. The NAD+-IDH was inhibited by a high energy charge, whereas the NADP+-IDH was found to be independent of energy charge. Consequently the NAD+-IDH showed the control behaviour of an enzyme of an energy-generating sequence which, however, equally fulfils a catabolic and an anabolic function. With respect to the inhibition by reduced pyridine nucleotides and alpha-ketoglutarate differences between NAD+-IDH and NADP+-IDH were also found. Only the NADP+-linked enzyme exhibited a feedback inhibition by its reaction products alpha-ketoglutarate and NADPH. This control behaviour gives evidence for the biosynthetic function of the NADP+-IDH. These results confer an amphibolic character to the sequence from citrate to alpha-ketoglutarate in the incomplete citric-acid cycle of Pseudomonas W6.
D002474:Cell-Free System; D007521:Isocitrate Dehydrogenase / Q000302:isolation & purification / Q000378:metabolism*; D007525:Isoelectric Focusing; D007527:Isoenzymes / Q000302:isolation & purification / Q000378:metabolism*; D000432:Methanol / Q000378:metabolism; D009243:NAD / Q000378:metabolism; D009249:NADP / Q000378:metabolism; D011549:Pseudomonas / Q000201:enzymology* / Q000378:metabolism
D007527:Isoenzymes; D009243:NAD; D009249:NADP; D007521:Isocitrate Dehydrogenase; D000432:Methanol