Locations of the regulatory sites for isocitrate dehydrogenase kinase/phosphatase.
Isocitrate dehydrogenase (IDH)(1) of Escherichia coli is regulated by a bifunctional protein, IDH kinase/phosphatase. In this paper, we demonstrate that the effectors controlling these activities belong to two distinct classes that differ in mechanism and in the locations of their binding sites. NADPH and isocitrate are representative members of one of these effector classes. NADPH inhibits both IDH kinase and IDH phosphatase, whereas isocitrate inhibits only IDH kinase. Isocitrate can "activate" IDH phosphatase by reversing product inhibition by dephospho-IDH. Mutations in icd, which encodes IDH, had parallel effects on the binding of these ligands to the IDH active site and on their effects on IDH kinase and phosphatase, indicating that these ligands regulate IDH kinase/phosphatase through the IDH active site. Kinetic analyses suggested that isocitrate and NADPH prevent formation of the complex between IDH kinase/phosphatase and its protein substrate. AMP, 3-phosphoglycerate, and pyruvate represent a class of regulatory ligands that is distinct from that which includes isocitrate and NADPH. These ligands bind directly to IDH kinase/phosphatase, a conclusion which is supported by the observation that they inhibit the IDH-independent ATPase activity of this enzyme. These effector classes can also be distinguished by the observation that mutant derivatives of IDH kinase/phosphatase expressed from aceK3 and aceK4 exhibited dramatic changes in their responses to AMP, 3-phosphoglycerate, and pyruvate but not to NADPH and isocitrate.
Miller
S P
SP
Department of Biochemistry, University of Minnesota, Minneapolis, Minnesota 55455, USA.
Chen
R
R
Karschnia
E J
EJ
Romfo
C
C
Dean
A
A
LaPorte
D C
DC
D000249:Adenosine Monophosphate / Q000378:metabolism; D019943:Amino Acid Substitution; D004926:Escherichia coli / Q000201:enzymology*; D005988:Glyceric Acids / Q000494:pharmacology; D007700:Kinetics; D008956:Models, Chemical; D009249:NADP / Q000378:metabolism / Q000494:pharmacology; D010749:Phosphoprotein Phosphatases / Q000737:chemistry / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity
D005988:Glyceric Acids; D011994:Recombinant Proteins; D000249:Adenosine Monophosphate; D009249:NADP; C005156:3-phosphoglycerate; D017346:Protein Serine-Threonine Kinases; C042301:isocitrate dehydrogenase kinase; C042300:isocitrate dehydrogenase phosphatase; D010749:Phosphoprotein Phosphatases