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.     

Effect of culture conditions on the isocitrate dehydrogenase and isocitrate lyase activities in Chlamydomonas reinhardtii

In the green alga Chlamydomonas reinhardtii , nitrogen staravation induced a reversible increase (2-fold) in NAD-isocitrate dehydrogenase (NAD-IDH; EC 1.1.1.41) and NADP-isocitrate dehydrogenase (NADP-IDH; EC 1.1.1.42) activities. Both enzymes were not affected by the concentration of CO₂, the dark or the nature of the nitrogen source (nitrate, nitrite, or ammonium). When cells growing autotrophically were transferred to heterotrophic conditions, a 40% reduction of the NAD-IDH activity was detected, a 2-fold increase of NADP-IDH was observed and isocitrate lyase (ICL; EC 4.1.3.1) activity was induced. The replacement of autotrophic conditions led to the initial activity levels. NAD- and NADP-IDH activities showed markedly different patterns of increase in synchronous cultures of this alga obtained by 12 h light/12 h dark transitions. While NAD-IDH increased in the last 4 h of the dark period, NADP-IDH increased during the last 4 h of the light period, remaining constant for the rest of the cycle.     

Mitochondrial isocitrate dehydrogenase and isocitrate oxidation of rat ventral prostate.

Mitochondrial preparations isolated from rat ventral prostate were capable of oxidizing isocitrate by way of NADP isocitrate dehydrogenase (NADP-IDH) and NAD-IDH. NAD-IDH activity required ADP for activation. The pH responses for NAD-IDH and NADP-IDH were quite different. The results indicated that two different enzymes were involved in the NAD- and NADP-IDH activities. Indirect evidence indicated that NADPH-NAD transhydrogenase activity might also be involved in the mitochondrial pathway for isocitrate oxidation. NADP-IDH activity was significantly greater than NAD-IDH activity. The oxidation of isocitrate through IDH activity was coupled to the cytochrome system by NADPH- and NADH-cytochrome c reductase activities. Citrate, via isocitrate, oxidation proceeded at a much slower rate suggesting that aconitase activity could be limiting in the oxidation of citrate. In comparison to other tissues, the prostate oxidative enzyme activities are considerably lower. The results suggest that the accumulation of high prostate citrate levels is not due to a limitation imposed by a lack of IDH activity in prostate mitochondria.     

Studies on isocitrate lyase isolated from Lupinus cotyledons

Isocitrate lyase (threo-DS-isocitrate glyoxylate-lyase, EC 4.1.3.1) was isolated from cotyledons of Lupinus seedlings, purified 100-fold with respect to its initial specific activity and characterized (Km, pH optimum, Mg2+ requirement, sulfhydryl inhibitors, and synthase activity). The final purified preparation consisted of two homogeneous protein bands clearly separated by electrophoresis on polyacrylamide gel and chromatography on Sephadex G 200. Reducing agents are necessary for the maintenance of enzyme activity. The most effective reducing agent studied was 1,4-dithioerythreitol. The effect of several metabolites (oxalate, malonate, phosphoenolpyruvate, succinate, malate, tartrate, gluconate-6-phosphate, sorbose, sorbitol, and inositol) on the activity of purified preparations was tested. Oxalate proved to be the strongest inhibitor, seconded closely by phosphoenolpyruvate. The spectral characteristics of the purified enzyme are as follows: ultraviolet peak at 280 nm and fluorescence peak at 340 nm. The solid state infrared spectrum of the enzyme (lyophilized) showed that the enzyme was mostly in the alpha-helix conformation with very slight random orientation.     

Substrate activity of structural analogs of isocitrate for isocitrate dehydrogenases from bovine heart.

D-Garcinia acid (D-threo-1,2-dihydroxy-1,2,3-propanetricarboxylate), like D-isocitrate, has an alpha-DS-hydroxyl group and a beta-LS configuration of the second carboxyl group. The maximal velocity of pyridine nucleotide reduction with D-garcinia acid is 8 and 21% of D-threo-isocitrate with the DPN-linked and TPN-linked isocitrate dehydrogenase from bovine heart, respectively. The other stereoisomers of hydroxycitrate [L-garcinia acid, D- and L-hibiscus acid (D- and L-erythro-1,2-dihydroxy-1,2,3-propanetricarboxylate)] are inactive. DL-threo-Homoisocitrate (DL-threo-1-hydroxy-1,2,4-butanetricarboxylate) supports DPN+ reduction at 10-15% of the rate observed for isocitrate with the DPN-specific enzyme, but is not a substrate for TPN-linked isocitrate dehydrogenase. The values of apparent S0.5 for total isocitrate and total garcinia acid are similar with both enzymes; the apparent S0.5 of total homoisocitrate is two- to threefold higher than that of total isocitrate with the DPN-linked enzyme. Enzymatic oxidative decarboxylation of garcinia acid and homoisocitrate leads to formation of alpha-keto-beta-hydroxyglutarate and alpha-ketoadipate, respectively. DL-Methylmalate (DL-1-hydroxy-2-methylsuccinate) is inactive as a substrate for either dehydrogenase as are the newly synthesized compounds: DL-threo-gamma-isocitrate amide (DL-threo-1-hydroxy-3-carbamy01,2-propanedicarboxylate), beta-methyl-DL-isocitrate (DL-1-hydroxy-2-methyl-1,2,3-propanetricarboxylate), beta-methyl-DL-garcinia acid (DL-threo-1-hydroxyl-2-methoxy-1,2,3-propanetricarboxylate), DL-1-hydroxyl-1,2,2-ethanetricarboxylate, and DL-1,4-dihydroxy-1,2-butanedicarboxylate.     

The isocitrate dehydrogenase from cyanobacteria

The present communication describes the properties of isocitrate dehydrogenase in crude extracts from the unicellular Anacystis nidulans and from heterocysts and vegetative cells of Nostoc muscorum and Anabaena cylindrica. The activity levels of this enzyme are much higher in heterocysts than in vegetative cells of N. muscorum and A. cylindrica. Isocitrate dehydrogenase is virtually inactive in vegetative cells of A. cylindrica. The enzyme is negatively regulated by the reduction charge and scarcely affected by oxoglutarate in the three cyanobacteria. The inhibition by ATP and ADP is competitive with respect to isocitrate and NADP+ in A. cylindrica and N. muscorum and noncompetitive in A. nidulans. Isocitrate dehydrogenase from the three cyanobacteria seems to be a hysteretic enzyme. All the experimental data suggest that the major physiological role of isocitrate and the isocitrate dehydrogenase in heterocysts is not to generate reducing equivalents for N2-fixation. Oxoglutarate formed by the enzyme reaction is likely required for the biosynthesis of glutamate inside the heterocysts. Thioredoxin preparations from spinach chloroplasts or from A. cylindrica activate isocitrate dehydrogenase from either heterocysts or vegetative cells of A. cylindrica. Activation is completed within seconds and requires dithiothreitol besides thioredoxin. The thioredoxin preparation which activates isocitrate dehydrogenase also activates NADP+-dependent malate dehydrogenase from spinach chloroplasts or heterocysts of A. cylindrica. Isocitrate dehydrogenase from A. cylindrica is deactivated by oxidized glutathione. It is speculated that isocitrate dehydrogenase and thioredoxin play a role in the differentiation of vegetative cells to heterocysts.     

Purification of yeast isocitrate dehydrogenase

The NAD-linked isocitrate dehydrogenase from baker's yeast was purified to homogeneity (as judged by gel filtration and polyacrylamide-gel electrophoresis) with an overall yield of 50% by using dilute solutions of the allosteric effector (AMP) to elute the enzyme specifically from CM-cellulose. This method preserves the allosteric properties of the crude enzyme. Although the pure enzyme shows only a single band on electrophoresis in the presence of sodium dodecyl sulphate, two types of subunit are observed in 8m-urea. The isoelectric point of the enzyme rises during purification, and this may reflect the partial loss of an additional low-molecular-weight component. Values are included for the amino acid composition and extinction coefficients of the pure enzyme.     

Steady-state kinetic analysis of isocitrate lyase from Lupinus seeds: considerations on a possible catalytic mechanism of isocitrate lyase from plants

Isocitrate lyase catalyzes the reversible cleavage of isocitrate into glyoxylate and succinate. The kinetic mechanism of bacterial isocitrate lyase has been reported to be ordered uni-bi. Moreover, it has been proposed that isocitrate lyase in higher plants may be switched on and off by a succinylation/desuccinylation mechanism. Similarly to bacterial citrate lyase, in which an acetylation/deacetylation mechanism is operative, succinylation might also play a role in the catalytic mechanism of plant isocitrate lyase. We have investigated the kinetic mechanism of isocitrate lyase from Lupinus seeds. The results reported in this paper show that the system follows a preferentially ordered uni-bi pathway in which the succinate is released first. On the basis of our results and some other recently reported data, we conclude that it is unlikely that bacterial and plant isocitrate lyases have different catalytic mechanisms.     

Phosphorylation of Acinetobacter isocitrate lyase.

During growth on succinate, Acinetobacter calcoaceticus contains two forms of the enzyme isocitrate dehydrogenase. Addition of acetate to a lag-phase culture grown on succinate causes a dramatic increase in activity of form II of isocitrate dehydrogenase and in isocitrate lyase. Form II of isocitrate dehydrogenase may be responsible for the partition of isocitrate between the TCA cycle and the glyoxylate by-pass. This report describes the phosphorylation of the enzyme isocitrate lyase from A. calcoaceticus. This phosphorylation may be a regulatory mechanism for the glyoxylate by-pass.     

Effect of glucose on isocitrate lyase in Phycomyces blakesleeanus.

Repression of the synthesis of isocitrate lyase by glucose and/or induction of the synthesis of isocitrate lyase by acetate in Phycomyces blakesleeanus were demonstrated. Both glycerol and ethanol failed to induce isocitrate lyase activity. Furthermore, glucose appeared to cause an in vivo catabolite inactivation of the derepressed enzyme. Isocitrate lyase was inactivated both reversibly and irreversibly by glucose.