Bacillus subtilis isocitrate dehydrogenase. A substrate analogue for Escherichia coli isocitrate dehydrogenase kinase/phosphatase.

In Escherichia coli, the homodimeric Krebs cycle enzyme isocitrate dehydrogenase (EcIDH) is regulated by reversible phosphorylation of a sequestered active site serine. The phosphorylation cycle is catalyzed by a bifunctional protein, IDH kinase/phosphatase (IDH-K/P). To better understand the nature of the interaction between EcIDH and IDH-K/P, we have examined the ability of an IDH homologue from Bacillus subtilis (BsIDH) to serve as a substrate for the kinase and phosphatase activities. BsIDH exhibits extensive sequence and structural similarities with EcIDH, particularly around the phosphorylated serine. Our previous crystallographic analysis revealed that the active site architecture of these two proteins is almost completely conserved. We now expand the comparison to include a number of biochemical properties. Both IDHs display nearly equivalent steady-state kinetic parameters for the dehydrogenase reaction. Both proteins are also phosphorylated by IDH-K/P in the same ratio (1 mole of phosphate per mole of monomer), and this stoichiometric phosphorylation correlates with an equivalent inhibition of IDH activity. Furthermore, tandem electrospray mass spectrometry demonstrates that BsIDH, like EcIDH, is phosphorylated on the corresponding active site serine residue (Ser-104). Despite the high degree of sequence, functional, and structural congruence between these two proteins, BsIDH is surprisingly a much poorer substrate of IDH-K/P than is EcIDH, with Michaelis constants for the kinase and phosphatase activities elevated by 60- and 3,450-fold, respectively. These drastically disparate values might result from restricted access to the active site cavity and/or from the lack of a potential docking site for IDH-K/P.     

Photoaffinity labelling of the ATP-binding site of the epidermal growth factor-dependent protein kinase.

Epidermal growth factor (EGF), after binding to its receptor, activates a tyrosine-specific protein kinase which phosphorylates several substrates, including the EGF receptor itself. The effects of a photoaffinity analogue of ATP, 3'-O-(3-[N-(4-azido-2-nitrophenyl)amino]propionyl)adenosine 5'-triphosphate (arylazido-beta-alanyl-ATP) on the EGF-dependent protein kinase in A431 human tumour cell plasma membrane vesicles was investigated. This analogue was capable of inactivating the EGF-receptor kinase in a photodependent manner. Partial inactivation occurred at an analogue concentration of 1 microM and complete inactivation occurred at 10 microM when a 2 min light exposure was used. Arylazido-beta-alanine at 100 microM and ATP at 100 microM were incapable of inactivating the enzyme with 2 min of light exposure. The photodependent inactivation of the enzyme by the analogue could be partially blocked by 20 mM-ATP and more effectively blocked by either 20 mM-adenosine 5'-[beta gamma-imido]triphosphate or 20 mM-guanosine 5'-[beta gamma-imido]triphosphate, indicating nucleotide-binding site specificity. Arylazido-beta-alanyl-[alpha-32P]ATP was capable of labelling membrane proteins in a photodependent manner. Numerous proteins were labelled, the most prominent of which ran with an apparent Mr of 53000 on polyacrylamide-gel electrophoresis. A band of minor intensity was seen of Mr corresponding to the EGF receptor (170000). Immunoprecipitation of affinity-labelled and solubilized membranes with an anti-(EGF receptor) monoclonal antibody demonstrated that the Mr 170000 receptor protein was photoaffinity labelled by the analogue. The Mr 53000 peptide was not specifically bound by the anti-receptor antibody. The affinity labelling of the receptor was not enhanced by EGF, suggesting that EGF stimulation of the kinase activity does not result from changes in the affinity of the kinase for ATP. These studies demonstrate that arylazido-beta-alanyl-ATP interacts with the ATP-binding site of the EGF-receptor kinase with apparent high affinity and that this analogue is an effective photoaffinity label for the kinase. Furthermore, these studies demonstrate that the EGF receptor, identified by using monoclonal antibodies, contains an ATP-binding site, providing further confirmation that the EGF receptor and EGF-dependent protein kinase are domains of the Mr 170000 protein.     

Crystal structure of Bacillus subtilis isocitrate dehydrogenase at 1.55 A. Insights into the nature of substrate specificity exhibited by Escherichia coli isocitrate dehydrogenase kinase/phosphatase

Isocitrate dehydrogenase from Bacillus subtilis (BsIDH) is a member of a family of metal-dependent decarboxylating dehydrogenases. Its crystal structure was solved to 1.55 A and detailed comparisons with the homologue from Escherichia coli (EcIDH), the founding member of this family, were made. Although the two IDHs are structurally similar, there are three notable differences between them. First, a mostly nonpolar beta-strand and two connecting loops in the small domain of EcIDH are replaced by two polar alpha-helices in BsIDH. Because of a 13-residue insert in this region of BsIDH, these helices protrude over the active site cleft of the opposing monomer. Second, a coil leading into this cleft, the so-called "phosphorylation" loop, is bent inward in the B. subtilis enzyme, narrowing the entrance to the active site from about 12 to 4 A. Third, although BsIDH is a homodimer, the two unique crystallographic subunits of BsIDH are not structurally identical. The two monomers appear to differ by a domain shift of the large domain relative to the small domain/clasp region, reminiscent of what has been observed in the open/closed conformations of EcIDH. In Escherichia coli, IDH is regulated by reversible phosphorylation by the bifunctional enzyme IDH kinase/phosphatase (IDH-K/P). The site of phosphorylation is Ser(113), which lies deep within the active site crevice. Structural differences between EcIDH and BsIDH may explain disparities in their abilities to act as substrates for IDH-K/P.     

Mutation of the predicted ATP binding site inactivates both activities of isocitrate dehydrogenase kinase/phosphatase

In Escherichia coli, the reversible phosphorylation of isocitrate dehydrogenase (IDH) is catalyzed by a bifunctional protein: IDH kinase/phosphatase. Although both IDH kinase and IDH phosphatase require ATP, the amino acid sequence of IDH kinase/phosphatase contains a single sequence that matches the consensus for ATP binding sites. A mutation that converted the "invariant" lysine (residue 336) of this consensus sequence to a methionine reduced the activities of both IDH kinase and IDH phosphatase by factors of greater than 500, to levels below the detection limits of the assays. The apparent elimination of both IDH kinase and IDH phosphatase by this mutation is consistent with the proposal that these activities share a common ATP binding site and that these reactions may occur at the same active site. Although conversion of Lys336 to a methionine eliminated detectable IDH kinase activity as measured in vitro, the mutant allele retained the ability to complement an aceK deletion mutation, restoring the ability of these cells to grow on minimal acetate medium. Complementation apparently resulted because the mutant protein retained sufficient activity to phosphorylate IDH in vivo. To determine whether the enzymatic assays performed in vitro had correctly reflected the activity of the mutant protein in vivo, we measured the rates at which mutant and wild-type cultures could incorporate [32P]inorganic phosphate into IDH. The wild-type culture achieved maximal incorporation in less than 3 min. In contrast, 32P incorporation was only barely detectable after 30 min in the mutant culture, indicating that the activity of the mutant protein is, indeed, greatly reduced in vivo. The ability of the mutant allele to complement an aceK null mutation thus suggests that IDH kinase/phosphatase levels in wild-type cells are in great excess over what is required for steady-state growth on acetate medium.     

A single gene codes for the kinase and phosphatase which regulate isocitrate dehydrogenase

The gene which codes for isocitrate dehydrogenase kinase/phosphatase of Escherichia coli, aceK, has been cloned. Physical and functional mapping of this clone indicated that both the isocitrate dehydrogenase kinase and isocitrate dehydrogenase phosphatase activities are encoded by an 1800-base pair sequence. This sequence produced a polypeptide with an apparent molecular weight of 66,000, which is identical to that of the purified protein. Since a protein of this size would require an 1800-base pair coding sequence, we conclude that isocitrate dehydrogenase kinase and isocitrate dehydrogenase phosphatase are expressed from a single gene. This strongly suggests that both activities reside on the same polypeptide chain. The cloning of aceK was made possible by the fortuitous addition of a second origin of replication to the expression vectors which were employed. These expression vectors were found to inhibit the growth of E. coli on the minimal acetate selective medium. The inclusion of a second origin of replication reduced the copy number and so reduced the inhibitory effects of these vectors. Control of the copy number through the addition of replication origins may have a general facility when manipulating plasmids which are potentially toxic to E. coli.     

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.     

Studies of the phosphorylation of Escherichia coli isocitrate dehydrogenase. Recognition of the enzyme by isocitrate dehydrogenase kinase/phosphatase and effects of phosphorylation on its structure and properties

Escherichia coli isocitrate dehydrogenase is completely inactivated by phosphorylation of a single serine residue per subunit. We have examined the conformations of the active and phosphorylated forms of the enzyme using circular dichroism spectroscopy. The results support the view that phosphorylation prevents the binding of NADP, probably by direct blocking of the coenzyme-binding site. Labelling studies suggest that an arginine residue at the coenzyme-binding site may be close to the phosphorylatable serine residue. The phosphorylation of isocitrate dehydrogenase is thus unusual in that it occurs at the active site of the enzyme. We therefore investigated the recognition of isocitrate dehydrogenase by isocitrate dehydrogenase kinase/phosphatase. The kinase activity of this enzyme can phosphorylate intact isocitrate dehydrogenase but not proteolytic fragments derived from it, nor a synthetic peptide corresponding to the sequence round the phosphorylation site.     

Phosphorylation inactivates Escherichia coli isocitrate dehydrogenase by preventing isocitrate binding

Equilibrium binding studies demonstrate that purified Escherichia coli isocitrate dehydrogenase binds isocitrate, alpha-ketoglutarate, NADP, and NADPH at 1:1 ratios of substrate to enzyme monomer. The phosphorylated enzyme, which is completely inactive, is unable to bind isocitrate but retains the ability to bind NADP and NADPH. Replacement of serine 113, which is the site of phosphorylation, by aspartate results in an inactive enzyme that is unable to bind isocitrate. Replacement of the same serine with other amino acids (lysine, threonine, cysteine, tyrosine, and alanine) produces active enzymes that bind both substrates. Hence, the negative charge of an aspartate or a phosphorylated serine at site 113 inactivates the enzyme by preventing the binding of isocitrate.     

Structural basis of the substrate specificity of bifunctional isocitrate dehydrogenase kinase/phosphatase

Isocitrate dehydrogenase kinase/phosphatase (AceK) regulates entry into the glyoxylate bypass by reversibly phosphorylating isocitrate dehydrogenase (ICDH). On the basis of the recently determined structure of the AceK-ICDH complex from Escherichia coli, we have classified the structures of homodimeric NADP(+)-ICDHs to rationalize and predict which organisms likely contain substrates for AceK. One example is Burkholderia pseudomallei (Bp). Here we report a crystal structure of Bp-ICDH that exhibits the necessary structural elements required for AceK recognition. Kinetic analyses provided further confirmation that Bp-ICDH is a substrate for AceK. We conclude that the highly stringent AceK binding sites on ICDH are maintained only in Gram-negative bacteria.     

Evidence for an arginine residue at the coenzyme-binding site of Escherichia coli isocitrate dehydrogenase.

Immobilization of biological systems in solid matrices is presently of great interest, in view of the many potential advantages associated with both the higher stability of the immobilized macromolecules and the potential utilization for biotechnology. In the present paper the electrochemical behaviour of the undecapeptide from cytochrome c (called microperoxidase) tightly entrapped in cellulose triacetate membrane is reported; its utilization as 'solid-state' promoter in the electrochemistry of soluble metalloproteins is presented. The results obtained indicate that: (i) membrane-entrapped microperoxidase undergoes rapid reversible electron transfer at a glassy carbon electrode; (ii) the electrochemical process is diffusion-controlled; (iii) entrapped microperoxidase acts as 'solid-state' promoter in the electrochemistry of soluble cytochrome c and of azurin.     

The "catalytic" triad of isocitrate dehydrogenase kinase/phosphatase from E. coli and its relationship with that found in eukaryotic protein kinases

The isocitrate dehydrogenase kinase/phosphatase (IDHK/P) of E. coli is a bifunctional enzyme responsible for the reversible phosphorylation of isocitrate dehydrogenase (IDH) on a seryl residue. As such, it belongs to the serine/threonine protein kinase family. However, only a very limited homology with the well-characterized eukaryotic members of that family was identified so far in its primary structure. In this report, a new region of amino acids including three putative residues involved in the kinase activity of IDHK/P was identified by sequence comparison with eukaryotic protein kinases. In IDHK/P, these residues are Asp-371, Asn-377, and Asp-403. Their counterpart eukaryotic residues have been shown to be involved in either catalysis (former residue) or magnesium binding (the two latter residues). Site-directed mutagenesis was performed on these three IDHK/P residues, and also on the Glu-439 residue equivalent to that of the Ala-Pro-Glu motif found in the eukaryotic protein kinases. Mutations of Asp-371 into either Ala, Glu, or Gln residues drastically lowered the yield and the quality of the purification. Nevertheless, the recovered mutant enzymes were barely able to phosphorylate IDH either in vitro or after expression in an aceK (-) mutant strain. In contrast, mutation of either Asn-377, Asp-403, or Glu-439 into an Ala residue altered neither the yield of purification nor the maximal phosphorylating capacity of the enzyme. However, when IDH was phosphorylated in the presence of increasing concentrations of magnesium ions, the two former mutants displayed a much lower affinity for this cation, with a K(m) value of 0.6 or 0.8 mM, respectively, as compared to 0.1 mM for the wild-type enzyme. On the other hand, the Glu439Ala mutant has an affinity for magnesium essentially unaffected. Therefore, and in contrast to the current opinion, our results suggest that the catalytic mechanism of IDHK/P exhibits some similarities with that found in the eukaryotic members of the protein kinase family.     

Structural and mechanistic insights into the bifunctional enzyme isocitrate dehydrogenase kinase/phosphatase AceK

The switch between the Krebs cycle and the glyoxylate bypass is controlled by isocitrate dehydrogenase kinase/phosphatase (AceK). AceK, a bifunctional enzyme, phosphorylates and dephosphorylates isocitrate dehydrogenase (IDH) with its unique active site that harbours both the kinase and ATP/ADP-dependent phosphatase activities. AceK was the first example of prokaryotic phosphorylation identified, and the recent characterization of the structures of AceK and its complex with its protein substrate, IDH, now offers a new understanding of both previous and future endeavours. AceK is structurally similar to the eukaryotic protein kinase superfamily, sharing many of the familiar catalytic and regulatory motifs, demonstrating a close evolutionary relationship. Although the active site is shared by both the kinase and phosphatase functions, the catalytic residues needed for phosphatase function are readily seen when compared with the DXDX(T/V) family of phosphatases, despite the fact that the phosphatase function of AceK is strictly ATP/ADP-dependent. Structural analysis has also allowed a detailed look at regulation and its stringent requirements for interacting with IDH.     

Nucleotide sequence of aceK, the gene encoding isocitrate dehydrogenase kinase/phosphatase.

In Escherichia coli, the phosphorylation and dephosphorylation of isocitrate dehydrogenase (IDH) are catalyzed by a bifunctional protein kinase/phosphatase. We have determined the nucleotide sequence of aceK, the gene encoding IDH kinase/phosphatase. This gene consists of a single open reading frame of 1,734 base pairs preceded by a Shine-Dalgarno ribosome-binding site. Examination of the deduced amino acid sequence of IDH kinase/phosphatase revealed sequences which are similar to the consensus sequence for ATP-binding sites. This protein did not, however, exhibit the extensive sequence homologies which are typical of other protein kinases. Multiple copies of the REP family of repetitive extragenic elements were found within the intergenic region between aceA (encoding isocitrate lyase) and aceK. These elements have the potential for combining to form an exceptionally stable stem-loop structure (delta G = -54 kcal/mol [ca. -226 kJ/mol]) in the mRNA. This structure, which masks the ribosome-binding site and start codon for aceK, may contribute to the downshift in expression observed between aceA and aceK. Another potential stem-loop structure (delta G = -29 kcal/mol [ca. 121 kJ/mol]), unrelated to the REP sequences, was found within aceK.     

Amino acid sequence round the site of phosphorylation in isocitrate dehydrogenase from Escherichia coli ML308

Isocitrate dehydrogenase from Escherichia coli is regulated by a reversible phosphorylation mechanism. We report here the amino acid sequence round the phosphorylation site; this is the first such sequence to be reported for a bacterial protein kinase. The sequence does not resemble sequences phosphorylated by cyclic AMP-dependent protein kinase.     

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.     

Isocitrate dehydrogenase kinase/phosphatase

In Escherichia coli, isocitrate dehydrogenase (IDH) is regulated by phosphorylation. This phosphorylation cycle is catalyzed by an unusual, bifunctional protein:IDH kinase/phosphatase. IDH kinase/phosphatase is expressed from a single gene, aceK, and both activities are catalyzed by the same polypeptide. The amino acid sequence of IDH kinase/phosphatase does not exhibit the characteristics which are typical of other protein kinases, although it does contain a consensus ATP binding site. The available evidence suggests that the IDH kinase and IDH phosphatase reactions occur at the same active site and that the IDH phosphatase reaction results from the back reaction of IDH kinase tightly coupled to ATP hydrolysis. The function of the IDH phosphorylation cycle is to control the flux of isocitrate through the glyoxylate bypass. This pathway is essential for growth on acetate because it prevents the quantitative loss of the acetate carbons as CO2 in the Krebs' cycle. IDH kinase/phosphatase monitors general metabolism by responding to the levels of a wide variety of metabolites, many of which activate IDH phosphatase and inhibit IDH kinase. The ability of IDH kinase/phosphatase to monitor general metabolism allows. the IDH phosphorylation cycle to compensate for substantial perturbations of the system, such as a 15-fold overproduction of IDH. The significance of the cellular level of IDH kinase/phosphatase has also been evaluated. The level of this protein is in great excess of that required for steady-state growth on acetate. In contrast, IDH kinase/phosphatase is, in some cases, rate-limiting for the dephosphorylation of IDH which results when preferred carbon sources are added to cultures growing on acetate.     

Purification and properties of phosphorylated isocitrate dehydrogenase of Escherichia coli.

Phosphorylated NADP+-isocitrate dehydrogenase (EC 1.1.1.42) has been purified to electrophoretic homogeneity from in vivo 32P-labeled Escherichia coli. The cells used as the source of phosphorylated enzyme were harvested 1 h after the addition of 5 mCi of [32P]orthophosphoric acid and 25 mM sodium acetate to cultures grown to early stationary phase on a low phosphate medium with limiting glucose. Double immunodiffusion and autoradiography demonstrated immunological identity between the 32P-labeled NADP+-isocitrate dehydrogenase and the enzyme isolated from glucose-grown E. coli. The phosphoenzyme had an apparent subunit molecular weight of 51,000 as determined by denaturing acrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, and the radioactivity co-electrophoresed with NADP+-isocitrate dehydrogenase activity when purified enzyme was subjected to nondenaturing gel electrophoresis. [32P]Phosphoserine was identified following partial acid hydrolysis of the purified phosphoenzyme.