Regulation of an Enzyme by Phosphorylation at the Active Site

Abstract

The isocitrate dehydrogenase of Escherichia coli is an example of a ubiquitous class of enzymes that are regulated by covalent modification. In the three-dimensional structure of the enzyme-substrate complex, isocitrate forms a hydrogen bond with Ser113, the site of regulatory phosphorylation. The structures of Asp113 and Glu113 mutants, which mimic the inactivation of the enzyme by phosphorylation, show minimal conformational changes from wild type, as in the phosphorylated enzyme. Calculations based on observed structures suggest that the change in electrostatic potential when a negative charge is introduced either by phosporylation or site-directed mutagenesis is sufficient to inactivate the enzyme. Thus, direct interaction at a ligand binding site is an alternative mechanism to induced conformational changes from an allosteric site in the regulation of protein activity by phosphorylation.

Structures of Human Cytosolic NADP-dependent Isocitrate Dehydrogenase Reveal a Novel Self-regulatory Mechanism of Activity

“…The HcIDH monomer has a secondary structure fold and topology similar to other dimeric NADP-IDHs whose structures are known, specifically EcIDH (13,14), BsIDH (20), and PmIDH (22), even though these enzymes share varying sequence identity (ϳ16 -17% between mammalian and bacterial NADP-IDHs and ϳ69% between HcIDH and PmIDH) (Figs. 2 and 3).…”

“…In bacterial NADP-IDHs, the equivalent positions are occupied by two different, but conserved, residues corresponding to Lys 344 and Tyr 345 in EcIDH and Lys 350 and Tyr 351 in BsIDH, respectively. In the NADP-bound EcIDH structures, the 2Ј-phosphomonoester group of NADP has interactions with the side chains of Tyr 345 , Tyr 391 , and Arg 395 , which are suggested to be the specificity determinants of NADP in EcIDH; the side chain of Lys 344 is either disordered or has no contact with NADP (14,16).…”

“…In contrast, Escherichia coli as well as other bacterial cells contain only a single NADP-IDH (EcIDH) whose structures and functions have been studied extensively (13)(14)(15)(16)(17). EcIDH functions at a critical juncture between the Krebs cycle and the glyoxylate cycle, and regulation of its activity controls the substrate flux between the two reactions (18).…”

“…EcIDH functions at a critical juncture between the Krebs cycle and the glyoxylate cycle, and regulation of its activity controls the substrate flux between the two reactions (18). The activity of EcIDH is regulated by the phosphorylation of Ser 113 , which is located at the isocitrate-metal ion-binding site (13,14,16,19). The phosphorylation reaction is reversibly catalyzed by a bifunctional enzyme, IDH kinase/phosphatase (IDH-K/P) (18).…”

Phosphoproteome Analysis of E. coli Reveals Evolutionary Conservation of Bacterial Ser/Thr/Tyr Phosphorylation

“…glycolysis, sugar transport) and essential proteins, which further supports the emerging view of Ser/Thr/ Tyr phosphorylation as an important regulatory mechanism in the bacterial cell. One potential mechanism for such a regulation has already been described: phosphorylation of isocitrate dehydrogenase (icd) in E. coli occurs at Ser113, the residue involved in binding of the substrate (isocitrate), and therefore abolishes the enzyme action (37). Another such example may come directly from our study: the Thr93 residue of the essential protein nucleoside diphosphate kinase (ndk), found to be phosphorylated in both E. coli and B. subtilis and conserved from Archaea to humans, serves as an ATP-binding site.…”