Purification and characterization of isocitrate lyase fromEscherichia coli

Isocitrate lyase has been purified to homogeneity, as determined by SDS-polyacrylamide gel electrophoresis and subsequent silver staining, fromEscherichia coli D₅H₃G₇. The enzyme was found to have a subunit molecular weight of 48,000 and a native molecular weight of 188,000 as determined by gel filtration chromatography. Thus, the enzyme appears to have tetrameric structure. The isoelectric point was determined to be 4.6, and the enzyme displayed a pH optimum at 7.3. The K_m of isocitrate lyase forthreo-Ds-isocitrate was determined to be 8 M. The purification procedure is highly reproducible and results in a 39% net yield of purified protein.     

Properties of isocitrate lyase fromEscherichia coli K12 grown on acetate or glycolate

Properties of isocitrate lyase fromEscherichia coli, the first enzyme of the glyoxylate bypass, have been compared from cells grown on either acetate or glycolate as the sole carbon source. Michaelis constants for isocitrate, isoelectric points, native and subunit molecular weights, antigenic properties, peptide mapping with V-8 or trypsin, and several other properties were examined. Our data suggest that only one isocitrate lyase form exists inE. coli regardless of carbon source used for growth.     

A catalytically inactive form of isocitrate dehydrogenase fromEscherichia coli

A protein exhibiting immunological cross-reactivity with NADP-specific isocitrate dehydrogenase, but containing no catalytic activity, has been isolated from nalidixic acid-resistantEscherichia coli. The two proteins have, within the limits of experimentation, identical molecular weight, subunit structure, and amino acid homology. The absence of catalytic activity in the protein isolated from nalidixic acid-resistant mutants may result from a mutation in the isocitrate dehydrogenase structural gene.     

Multisite inhibition of Pinus pinea isocitrate lyase by phosphate

Our results show that the phosphate ion is a nonlinear competitive inhibitor of Pinus pinea isocitrate lyase. In addition, this compound induces a sigmoidal response of the enzyme, which usually exhibits standard Michaelis-Menten kinetics. This peculiar behavior of P. pinea isocitrate lyase could be explained by a dimer (two-site) model, in which phosphate binds cooperatively, but the affinity of the vacant site for substrate (the magnesium-isocitrate complex) remains the same. As a result, the interaction of phosphate with free enzyme produces an inhibitor-enzyme-inhibitor species that is of significant importance in determining reaction rate; a possible regulatory role of the glyoxylate cycle by inorganic phosphate is suggested. The mode of phosphate inhibition is consistent with both the mechanism for magnesium ion activation of P. pinea isocitrate lyase and its site heterogeneity. Our results explain the cooperative effects observed by some authors in kinetic studies of isocitrate lyase carried out in phosphate buffers and also account for the higher K(m) values determined by using such assay systems. Phosphate buffer should be avoided in performing isocitrate lyase kinetics.     

Phosphorylation of isocitrate lyase in Escherichia coli

Isocitrate lyase from Escherichia coli becomes phosphorylated in vitro by an endogenous kinase when partially purified extracts are incubated with [gamma-32P]ATP. Treatment of isocitrate lyase with histidine modifying reagents, and alkaline hydrolysis of in vitro phosphorylated enzyme indicated the presence of a phosphohistidine residue. Phosphorylation of isocitrate lyase can also occur in vivo, which indicates a possible regulatory significance of this modification. In addition to phosphorylation, isocitrate lyase is capable of incorporating label from both [alpha-32P]ATP and [14C]ATP suggesting that more than one type of covalent modification occurs on this enzyme. This report reviews the studies which have demonstrated the phosphorylation and modification of isocitrate lyase from Escherichia coli.     

Inhibiton of isocitrate dehydrogenase and isocitrate lyase from Acinetobacter calcoaceticus by acids of the citrate and glyoxylate cycle]

Acinetobacter calcoaceticus contains two forms of NADP+-dependent isocitrate dehydrogenases differing, among others, by their molecular weights and regulatory properties. The regulation of the high-molecular form of isocitrate dehydrogenase and of isocitrate lyase by organic acids, either belonging or related to the citrate and glyoxalate cycle, is investigated. While alpha-ketoglutarate and oxalacetate competitively inhibit the isocitrate dehydrogenase against Ds-isocitrate, glyoxylate and pyruvate were found to increase Vmax and to lower the KM value for Ds-isocitrate and NADP+. Simultaneous addition of oxalacetate and glyoxylate (not, however, addition of the nonenzymatically formed condensation product of both compound) nullified the activation of isocitrate dehydrogenase by glyoxylate, and potentiates the inhibitory effect of oxalacetate. Alpha-ketoglutarate, succinate, and phosphoenolpyruvate inhibit the isocitrate lyase in a noncompetitive fashion against DS-isocitrate; L-malate, oxalacetate and glyoxylate inhibit competitively. The intermediates of the citrate and glyoxylate cycle afford additive inhibition of the isocitrate lyase. The importance of organic acids of the citrate and glyoxylate cycle and of phosphoenolpyruvate for the regulation of the citrate and glyoxylate cycle at the level of isocitrate dehydrogenase and isocitrate lyase is discussed.     

Alkylation of isocitrate lyase from Escherichia coli by 3-bromopyruvate

The inactivation of tetrameric isocitrate lyase from Escherichia coli by 3-bromopyruvate, exhibiting saturation kinetics, is accompanied by the loss of one sulfhydryl per subunit. The substrates glyoxylate and isocitrate protect against inactivation whereas the substrate succinate does not. The modification by 3-bromopyruvate (equimolar to subunits) imparts striking resistance to digestion of isocitrate lyase by trypsin, chymotrypsin, and V8 protease as well as a major decrease in the intensity of tryptophan fluorescence. After alkylation, the sequence Gly-His-Met-Gly-Gly-Lys is found following the modified Cys residue in the tryptic peptide representing positions 196-201. Thus Cys195 is alkylated by 3-bromopyruvate.     

In vitro phosphorylation ofEscherichia coli isocitrate lyase

The in vitro phosphorylation of isocitrate lyase was demonstrated in partially purified sonic extracts ofEscherichia coli. Extracts were incubated with [gamma³²P]-ATP and subsequently analyzed by two-dimensional polyacrylamide gel electrophoresis. Isocitrate lyase was determined to be phosphorylated by autoradiography and Western blot analyses of the gels. Purified isocitrate lyase comigrates with the phosphorylated form of the enzyme; this suggests that the enzyme may become catalytically active concomitant with phosphorylation.     

The inhibition, by intermediary metabolites, of isocitrate lyase from Chlorella pyrenoidosa

1. Intermediary metabolites, arising from the glyoxylate by-pass, were tested for their effect on the activity of isocitrate lyase, the first enzyme of the by-pass. 2. Oxaloacetate and pyruvate inhibited the enzyme when present at concentrations similar to that of the substrate. 3. Inhibition was competitive and did not depend on a non-optimum pH. The affinities determined at the pH optimum were: K(i) (oxaloacetate), 3.7x10(-5)m; K(i) (pyruvate), 6.6x10(-5)m. 4. The significance of the inhibitions is discussed with particular reference to the known inhibition by phosphoenolpyruvate.     

Amino acid sequence of the phosphorylation site of isocitrate dehydrogenase fromEscherichia coli

InEscherichia coli, NADP⁺-specific isocitrate dehydrogenase (EC 1.1.1.42) may undergo a phosphorylation catalyzed by a cAMP-independent protein kinase, with a concomitant decrease in catalytic activity. In this report, we describe the purification and amino acid sequence of a³²P-labeled peptide obtained from in vivo³²P-labeled isocitrate dehydrogenase. The³²P-labeled peptide was isolated from a tryptic digest and found to contain seven amino acids, including a single serine residue. Following automated Edman degradation and reversephase high-pressure liquid chromatography of the phenylthiohydantoin-amino acids, the sequence of this peptide was established to be-Ser(P)-Leu-Asn-Val-Ala-Leu-Arg.     

Escherichia coli isocitrate lyase: properties and comparisons

The glyoxylate cycle was first discovered during studies on bacteria and fungi with the ability to grow on acetate or ethanol as the sole carbon source. Isocitrate lyase, the first enzyme unique to the glyoxylate cycle, has been studied in numerous prokaryotic and eukaryotic organisms. However, information on this enzyme from Escherichia coli is limited. We have recently reported the purification and in vitro phosphorylation of this enzyme. In the present study we have examined and characterized a variety of inhibitors, the divalent cation requirement and the amino acid composition of E. coli isocitrate lyase and compared these results to those obtained with other organisms.     

Caenorhabditis elegans and Ascaris suum: inhibition of isocitrate lyase by itaconate

The largest forms of isocitrate lyase from Caenorhabditis elegans and Ascaris suum of 543,000 and 549,000 daltons, respectively, can be purified from three- to five-fold in excellent yield by pelleting from extracts at 160,000g for 4 hr. Isocitrate lyase in the pellet is much more stable toward proteolysis. Itaconate which both inhibits isocitrate lyase and suppresses the level of this enzyme in bacteria inhibits the partially purified isocitrate lyase from both C. elegans and A. suum. The inhibition is noncompetitive with respect to d_s-isocitrate at one itaconate concentration. The K_i values at 30 C, pH 7.7, are 19 and 7.3 μM for the enzyme from C. elegans and A. suum, respectively. Itaconate inhibits the growth of C. elegans in random axenic as well as monoxenic cultures. At a concentration of 10 mM, itaconate is more effective in the inhibition of random axenic cultures than is oxalate, maleate, or succinate. At 60 mM itaconate, reproduction of C. elegans larvae is completely abolished.     

Dual role of isocitrate lyase 1 in the glyoxylate and methylcitrate cycles in Mycobacterium tuberculosis

The role of isocitrate lyase (ICL) in the glyoxylate cycle and its necessity for persistence and virulence of Mycobacterium tuberculosis has been well described. Recent reports have alluded to an additional role for this enzyme in M. tuberculosis metabolism, specifically for growth on propionate. A product of beta-oxidation of odd-chain fatty acids is propionyl-CoA. Clearance of propionyl-CoA and the by-products of its metabolism via the methylcitrate cycle is vital due to their potentially toxic effects. Although the genome of M. tuberculosis encodes orthologues of two of the three enzymes of the methylcitrate cycle, methylcitrate synthase and methylcitrate dehydratase, it does not appear to contain a distinct 2-methylisocitrate lyase (MCL). Detailed structural analysis of the MCL from Escherichia coli suggested that the differences in substrate specificity between MCLs and ICLs could be attributed to three conserved amino acid substitutions in the active site, suggesting an MCL signature. However, here we provide enzymatic evidence that shows that despite the absence of the MCL signature, ICL1 from M. tuberculosis can clearly function as a MCL. Furthermore, the crystal structure of ICL1 with pyruvate and succinate bound demonstrates that the active site can accommodate the additional methyl group without significant changes to the structure.     

In vivo phosphorylation of isocitrate lyase inEscherichia coli andAcinetobacter calcoaceticus

Isocitrate lyase inEscherichia coli and inAcinetobacter calcoaceticus is phosphorylated when the cells are grown with acetate as the sole carbon source in low-phosphate mineral salts medium containing³²P inorganic phosphate. The level of³²P incorporation into the enzyme in both microorganisms appears to be constant throughout the entire growth cycle. Further, theresults of immunoblots and rocket immunoelectrophoresis suggest that the amount of isocitrate lyase protein, although at different levels in each microorganism, also remains constant throughout the growth cycle.     

Evidence of histidine phosphorylation in isocitrate lyase from Escherichia coli

Escherichia coli isocitrate lyase (EC 4.1.3.1.) can be phosphorylated in vitro by an ATP-dependent reaction. The enzyme becomes phosphorylated by an endogenous kinase when partially purified sonic extracts are incubated with [gamma-32P]ATP. Treatment of isocitrate lyase with diethyl pyrocarbonate, a histidine-modifying reagent, blocked incorporation of [32P]phosphate from [gamma-32P]ATP. The isoelectric point of the enzyme was altered by treatment with phosphoramidate, a histidine phosphorylating agent, which suggests that isocitrate lyase can be phosphorylated at a histidine residue(s). Immunoprecipitated 32P-labeled isocitrate lyase was subjected to alkaline hydrolysis, mixed with chemically synthesized phosphohistidine standards, and analyzed by anion exchange chromatography. Characterization of the phosphoamino acid was based on the demonstration that the 32P-labeled product from alkali-hydrolyzed isocitrate lyase comigrated with synthetic 1-phosphohistidine. In addition, loss of catalytic activity after treatment with potato acid phosphatase indicates that catalytically active isocitrate lyase is the phosphorylated form of the enzyme.     

The importance of four histidine residues in isocitrate lyase from Escherichia coli.

By site-directed mutagenesis, substitutions were made for His-184 (H-184), H-197, H-266, and H-306 in Escherichia coli isocitrate lyase. Of these changes, only mutations of H-184 and H-197 appreciably reduced enzyme activity. Mutation of H-184 to Lys, Arg, or Leu resulted in an inactive isocitrate lyase, and mutation of H-184 to Gln resulted in an enzyme with 0.28% activity. Nondenaturing polyacrylamide gel electrophoresis demonstrated that isocitrate lyase containing the Lys, Arg, Gln, and Leu substitutions at H-184 was assembled poorly into the tetrameric subunit complex. Mutation of H-197 to Lys, Arg, Leu, and Gln resulted in an assembled enzyme with less than 0.25% wild-type activity. Five substitutions for H-266 (Asp, Glu, Val, Ser, and Lys), four substitutions for H-306 (Asp, Glu, Val, and Ser), and a variant in which both H-266 and H-306 were substituted for showed little or no effect on enzyme activity. All the H-197, H-266, and H-306 mutants supported the growth of isocitrate lyase-deficient E. coli JE10 on acetate as the sole carbon source; however, the H-184 mutants did not.