Isocitrate dehydrogenase kinase/phosphatase: aceK alleles that express kinase but not phosphatase activity.
For Escherichia coli, growth on acetate requires the induction of the enzymes of the glyoxylate bypass, isocitrate lyase and malate synthase. The branch point between the glyoxylate bypass and the Krebs cycle is controlled by phosphorylation of isocitrate dehydrogenase (IDH), inhibiting that enzyme's activity and thus forcing isocitrate through the bypass. This phosphorylation cycle is catalyzed by a bifunctional enzyme, IDH kinase/phosphatase, which is encoded by aceK. We have employed random mutagenesis to isolate novel alleles of aceK. These alleles were detected by the loss of ability to complement an aceK null mutation. The products of one class of these alleles retain IDH kinase activity but have suffered reductions in IDH phosphatase activity by factors of 200 to 400. Selective loss of the phosphatase activity also appears to have occurred in vivo, since cells expressing these alleles exhibit phenotypes which are reminiscent of strains lacking IDH; these strains are auxotrophic for glutamate. Assays of cell-free extracts confirmed that this phenotype resulted from nearly quantitative phosphorylation of IDH. The availability of these novel alleles of aceK allowed us to assess the significance of the precise control which is a characteristic of the IDH phosphorylation cycle in vivo. The fractional phosphorylation of IDH was varied by controlled expression of one of the mutant alleles, aceK3, in a wild-type strain. Reduction of IDH activity to 50% of the wild-type level did not adversely affect growth on acetate. However, further reductions inhibited growth, and growth arrest occurred when the IDH activity fell to 15% of the wild-type level. Thus, although wild-type cells maintain a precise effective IDH activity during growth on acetate, this precision is not critical.
Ikeda
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Department of Biochemistry, University of Minnesota, Minneapolis 55455.
LaPorte
D C
DC
D000085:Acetates / Q000378:metabolism; D000483:Alleles; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics* / Q000254:growth & development; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial; D007700:Kinetics; D016296:Mutagenesis; D010749:Phosphoprotein Phosphatases / Q000235:genetics*; D010957:Plasmids; D011494:Protein Kinases / Q000235:genetics*; D017346:Protein Serine-Threonine Kinases
D000085:Acetates; D011494:Protein Kinases; D017346:Protein Serine-Threonine Kinases; C042301:isocitrate dehydrogenase kinase; C042300:isocitrate dehydrogenase phosphatase; D010749:Phosphoprotein Phosphatases