Nondecarboxylating and Decarboxylating Isocitrate Dehydrogenases: Oxalosuccinate Reductase as an Ancestral Form of Isocitrate Dehydrogenase

Isocitrate dehydrogenase (ICDH) from Hydrogenobacter thermophilus catalyzes the reduction of oxalosuccinate, which corresponds to the second step of the reductive carboxylation of 2-oxoglutarate in the reductive tricarboxylic acid cycle. In this study, the oxidation reaction catalyzed by H. thermophilus ICDH was kinetically analyzed. As a result, a rapid equilibrium random-order mechanism was suggested. The affinities of both substrates (isocitrate and NAD⁺) toward the enzyme were extremely low compared to other known ICDHs. The binding activities of isocitrate and NAD⁺ were not independent; rather, the binding of one substrate considerably promoted the binding of the other. A product inhibition assay demonstrated that NADH is a potent inhibitor, although 2-oxoglutarate did not exhibit an inhibitory effect. Further chromatographic analysis demonstrated that oxalosuccinate, rather than 2-oxoglutarate, is the reaction product. Thus, it was shown that H. thermophilus ICDH is a nondecarboxylating ICDH that catalyzes the conversion between isocitrate and oxalosuccinate by oxidation and reduction. This nondecarboxylating ICDH is distinct from well-known decarboxylating ICDHs and should be categorized as a new enzyme. Oxalosuccinate-reducing enzyme may be the ancestral form of ICDH, which evolved to the extant isocitrate oxidative decarboxylating enzyme by acquiring higher substrate affinities.     

Stereochemistry of the decarboxylation of l-ornithine with ornithine decarboxylase from mouse kidney

Using highly purified ornithine decarboxylase isolated from androgen-treated mice, [1R-²H]putrescine was generated by the decarboxylation of l-ornithine in D₂O, and [1S-²H]putrescine was generated from [2-²H]ornithine by carrying out the decarboxylation in H₂O. Chirality of the putrescines was then determined from the 200-MHz ¹H NMR spectra of their bis-camphanamides in the presence of Eu(fod)₃. These results demonstrated that decarboxylation had taken place with retention of configuration.     

Influence of substrates and coenzymes on the role of manganous ion in reactions catalyzed by pig heart triphosphopyridine nucleotide-dependent isocitrate dehydrogenase.

The interaction of manganous ions with pig heart triphosphopyridine nucleotide (TPN) specific isocitrate dehydrogenase has been studied by kinetic experiments and by direct ultrafiltration measurements of manganous ion binding. At low metal ion concentrations, a lag is observed in the time-dependent production of reduced triphosphopyridine nucleotide (TPNH) that can be eliminated by adding 20 muM TPNH to the initial reaction mixture. A plot of 1/upsilon vs. 1/ (Mn2+) obtained at relatively high TPNH concentrations (20 muM) is linear and yields of Km value of 2 muM for metal ion, which is comparable to the direct binding constant measured in the presence of isocitrate. A similar plot at low TPNH concentrations (2 muM) reveals a biphasic relationship: at high metal concentrations the points are collinear with those obtained at high levels of TPNH, but at low metal concentrations that line is characterized by a Km of 19 muM for Mn2+. A difference in the deuterium oxide solvent isotope effect on Vmax observed with 20 muM TPNH as compared with 2 muM TPNH suggests that at high TPNH concentrations or high manganous ion concentrations the rate-limiting step is the dehydrogenation of isocitrate, while at low manganous ion concentrations and low TPNH concentrations, the slow step is the decarboxylation of enzyme-bound oxalosuccinate. Evidence to support this hypothesis is provided by the sensitivity to isocitrate concentration of the Km for total manganese measured in the presence of 20 muM TPNH that contrasts with the relative insensitivity to isocitrate of the Km measured at 2 muM TPNH and low manganous ion concentration. Direct measurements of oxalosuccinate decarboxylation reveal that the Vmax and the Km for manganous ion are influenced by the presence of oxidized or reduced TPN with the Km being lowest (5-7 muM) in the presence of TPNH. The dependence of the Km for manganous ion on the presence of substrate, TPN, and TPNH, is responsible for the variation with conditions in the rate-determining step. The enzyme binds only 1 mol of metal ion and 1 mol of isocitrate/mol of protein under all conditions. The pH dependence of the binding of free manganous ion, free isocitrate, and manganous-isocitrate complex indicates differences in the interaction of these species with isocitrate dehydrogenase. These results can be described in terms of two functions for manganous ion in the reactions catalyzed by isocitrate dehydrogenase, each of which requires a distinct binding site for metal ion: in the dehydrogenation step, Mn2+ facilitates the binding of the substrate isocitrate, and in the decarboxylation step it may stabilize the enolate of alpha-ketoglutarate which is generated.     

Laser Raman spectroscopy of acid phosphatase from rat liver

The Raman spectra of isoenzyme I and II in aqueous solution and in solid state are reported. The results reveal differences between these isoenzymes. These tertiary structures vary depending on whether the isoenzymes are solid or dissolved in H₂O or D₂O.     

Crystal structure studies of NADP dependent isocitrate dehydrogenase from Thermus thermophilus exhibiting a novel terminal domain

NADP⁺ dependent isocitrate dehydrogenase (IDH) is an enzyme catalyzing oxidative decarboxylation of isocitrate into oxalosuccinate (intermediate) and finally the product α-ketoglutarate. The crystal structure of Thermus thermophilus isocitrate dehydrogenase (TtIDH) ternary complex with citrate and cofactor NADP⁺ was determined using X-ray diffraction method to a resolution of 1.80 Å. The overall fold of this protein was resolved into large domain, small domain and a clasp domain. The monomeric structure reveals a novel terminal domain involved in dimerization, very unique and novel domain when compared to other IDH’s. And, small domain and clasp domain showing significant differences when compared to other IDH’s of the same sub-family. The structure of TtIDH reveals the absence of helix at the clasp domain, which is mainly involved in oligomerization in other IDH’s. Also, helices/beta sheets are absent in the small domain, when compared to other IDH’s of the same sub family. The overall TtIDH structure exhibits closed conformation with catalytic triad residues, Tyr144-Asp248-Lys191 are conserved. Oligomerization of the protein is quantized using interface area and subunit–subunit interactions between protomers. Overall, the TtIDH structure with novel terminal domain may be categorized as a first structure of subfamily of type IV.     

Increased conformational stability of mitochondrial soluble ATPase (F1) by substitution of H2O for D2O.

Between 20 and 40 °C D₂O inhibits the hydrolytic activity of soluble mitochondrial ATPase F₁. The effect of D₂O is proportional to its concentration in the incubation mixture and at nearly 100% D₂O in the incubation mixture the ATPase activity is inhibited by 50–60%. The effect of D₂O is mainly on the V of the reaction. At temperatures above 45 °C, D₂O does not inhibit the activity. D₂O protects against the denaturation of the enzyme that is observed at relatively high temperatures and against the cold-induced inactivation of F₁. The intensity of fluorescence of 8-anilino-1-naphthalene sulfonate incubated with F₁ increases as the enzyme becomes inactivated by low temperatures; in D₂O the changes of fluorescence are almost nil. These observations indicate that H (or D) bonding between the solvent and the protein as well as the strength of the hydrophobic interactions within the enzyme as determined by the solvent are of central importance in determining the overall activity of F₁ and the stability of the enzyme to denaturing conditions. Moreover, the data indicate that the enzyme may exist in two different conformations, each with a characteristic activation energy. It is also proposed that D₂O may be employed with success in the isolation and purification of labile enzymes.     

A novel oxalosuccinate-forming enzyme involved in the reductive carboxylation of 2-oxoglutarate in Hydrogenobacter thermophilus TK-6

We have previously demonstrated that the reductive carboxylation of 2-oxoglutarate in Hydrogenobacter thermophilus TK-6 is not simply a reversal of the oxidative decarboxylation catalysed by isocitrate dehydrogenase (ICDH). The reaction involves a novel biotin protein (carboxylating factor for ICDH-CFI) and ATP. In this study, we have analysed the ICDH/CFI system responsible for the carboxylation reaction. Sequence analysis revealed a close relationship between CFI and pyruvate carboxylase. Rather unexpectedly, the rate of ATP hydrolysis was greater than that of isocitrate formation or NADH oxidation. Furthermore, ATP hydrolysis catalysed by CFI was dependent on 2-oxoglutarate but not on ICDH, suggesting that a carboxylated product is formed in the absence of ICDH. The product, which was detectable only at low temperatures, was identified as oxalosuccinate. Thus, CFI was confirmed to be a novel enzyme that catalyses the carboxylation of 2-oxoglutarate to form oxalosuccinate, which corresponds to the first step of the reductive carboxylation from 2-oxoglutarate to isocitrate. The CFI-ICDH system may also be present in mammals, where it could play a significant role in modulating central metabolism.     

Studies on the reactivity of malondialdehyde. I. Effects of acid concentration and solvent composition on deuterium exchange reactions

The rates of deuterium exchange reactions of malondialdehyde (MDA) and deuterated malondialdehyde (MDAd) have been studied as a function of acidity and the content of dimethyl sulfoxide (DMSO) in binary mixtures with D₂O . MDA incorporates deuterium from D₂O solutions in a first-order reaction with a rate constant (k_obs) that depends on the acid concentration. From this dependence, a catalytic constant, k_cat, can be derived (k_cat^MDA = 2.25 × 10⁵M^?s^?1). Similar kinetic behavior was found for MDAd in H₂O solutions, and in this case, k_cat^MDA = 1.56 × 10⁵M^?1s^?1. Results from reactions of MDA and MDAd in identical H₂OD₂O mixtures show that primary and secondary isotope effects are small (k_H/k_D = 1.13) and that solvent isotope effects cause most of the differences found between reactions in D₂O and H₂O. Reactions in binary DMSOd₆D₂O mixtures show a six-fold rate increase as the proportion of DMSOd₆ increases from 50% to 90%. These results also illustrate the relatively high reactivity of MDA at pH values well above its pK_a and the importance of medium composition on its reaction rate.     

Synthesis of Isocitrate Lyase in Sunflower Cotyledons during the Transition in Cotyledonary Microbody Function

Density-labeling with 10 millimolar K¹⁵NO₃/70% ²H₂O has been used to investigate isocitrate lyase synthesis during greening of sunflower (Helianthus annuus L.) cotyledons when the glyoxysomal enzyme activities sharply decline and the transition in cotyledonary microbody function occurs. A density shift of 0.0054 (kilograms per liter) was obtained for the profile of isocitrate lyase activity in the CsCl gradient with respect to the ¹H₂O control. Quantitative evaluation of the density-labeling data indicates that about 50% of the isocitrate lyase activity present towards the end of the transition stage in microbody function is due to enzyme molecules newly synthesized during this stage.     

Die zeitliche Dauer der Isocitrat-Lyase-Synthese in Kotyledonen von Wassermelonenkeimlingen

Summary Previously, it was deduced from inhibitor experiments that isocitrate lyase (EC 4.1.3.1.) is synthesized de novo in watermelon cotyledons during the first 3 days of germination, which explains the sharp increase of activity during this period. The following decrease of activity was interpreted as the result of a limited half life of the enzyme molecule (Hock and Beevers, 1966).This hypothesis has been confirmed now by density labeling experiments of isocitrate lyase with deuterium. Seedlings grown from day 0 on D₂O (80 vol. %) contained a heavier enzyme at the time of maximum activity than control seedlings grown on H₂O (Fig. 6). No incorporation of deuterium into isocitrate lyase, however, was detectable when the cotyledons were labeled only from day 3 1/2 on, i.e. after the stage of maximum activity had been passed (Fig. 10), in spite of the fact that D₂O was taken up from the cotyledons in considerable quantities. —These results prove at the same time that density labeling of the isocitrate lyase during early stages of germination was a result of de novo synthesis rather than a mere artifact produced by isotopic exchange.An improved method for the purification of isocitrate lyase from higher plants is introduced.     

Activation of O(2) Uptake and NAD-Specific Isocitrate Dehydrogenase in Mitochondria Isolated from Cotyledons of Castor Bean by cis, trans-Abscisic Acid

The rate of O₂ uptake and the activity of NAD-specific isocitrate dehydrogenase (NAD-ICDH) of mitochondria isolated from castor bean cotyledons were increased by added cis, trans-abscisic acid (ABA) in an in vitro system, while the NADP-specific isocitrate dehydrogenase (NADP-ICDH) was not affected by cis, trans-ABA. Trans, trans-ABA showed only a slightly inhibitory effect on O₂ uptake. The V_max value for the isotherm of isocitrate by the enzyme was also increased by cis, trans-ABA. The isocitrate K_m value for the enzyme with cis, trans-ABA was calculated to be approximately 249.8 micromolar, while the S_0.5 for the enzyme without the ABA was 151.6 micromolar. The n value calculated from the slopes of Hill plots of the reaction velocity of NAD-ICDH against isocitrate concentration was 1.5 in the mitochondrial fraction in the absence of ABA, and cis, trans-ABA treatment decreased the value to 1.0. Cis, trans-ABA also partly overcame the inhibition of NAD-ICDH activity by ATP.     

Solvent isotope effects on microtubule polymerization and depolymerization

The initial velocity of polymerization of purified beef brain tubulin has been determined at various values of pH or pD in water and in H₂O-D₂O mixtures. D₂O was shown to inhibit both polymerization at 37 °C and depolymerization measured at 5 °C and 37 °C. The microtubules formed in D₂O were indistinguishable from those formed in H₂O, by electron microscope examination. In 93% D₂O the pL²versus rate of polymerization curve was displaced about one unit towards higher pL values. In certain regions of the pL versus rate curve, a stimulation in the rate of polymerization by D₂O is observed. The extent of polymerization at the optimum pL value was not affected by D₂O.     

Local Control Model of Excitation–Contraction Coupling in Skeletal Muscle

The voltage-activated H⁺ selective conductance of rat alveolar epithelial cells was studied using whole-cell and excised-patch voltage-clamp techniques. The effects of substituting deuterium oxide, D₂O, for water, H₂O, on both the conductance and the pH dependence of gating were explored. D⁺ was able to permeate proton channels, but with a conductance only about 50% that of H⁺. The conductance in D₂O was reduced more than could be accounted for by bulk solvent isotope effects (i.e., the lower mobility of D⁺ than H⁺), suggesting that D⁺ interacts specifically with the channel during permeation. Evidently the H⁺ or D⁺ current is not diffusion limited, and the H⁺ channel does not behave like a water-filled pore. This result indirectly strengthens the hypothesis that H⁺ (or D⁺) and not OH⁻ is the ionic species carrying current. The voltage dependence of H⁺ channel gating characteristically is sensitive to pH_o and pH_i and was regulated by pD_o and pD_i in an analogous manner, shifting 40 mV/U change in the pD gradient. The time constant of H⁺ current activation was about three times slower (τ_act was larger) in D₂O than in H₂O. The size of the isotope effect is consistent with deuterium isotope effects for proton abstraction reactions, suggesting that H⁺ channel activation requires deprotonation of the channel. In contrast, deactivation (τ_tail) was slowed only by a factor ≤1.5 in D₂O. The results are interpreted within the context of a model for the regulation of H⁺ channel gating by mutually exclusive protonation at internal and external sites (Cherny, V.V., V.S. Markin, and T.E. DeCoursey. 1995. J. Gen. Physiol. 105:861–896). Most of the kinetic effects of D₂O can be explained if the pK _a of the external regulatory site is ∼0.5 pH U higher in D₂O.     

Hydrogen-Deuterium Exchange Effects on β-Endorphin Release from AtT20 Murine Pituitary Tumor Cells

Abundant evidences demonstrate that deuterium oxide (D₂O) modulates various secretory activities, but specific mechanisms remain unclear. Using AtT20 cells, we examined effects of D₂O on physiological processes underlying β-endorphin release. Immunofluorescent confocal microscopy demonstrated that 90% D₂O buffer increased the amount of actin filament in cell somas and decreased it in cell processes, whereas β-tubulin was not affected. Ca²⁺ imaging demonstrated that high-K⁺-induced Ca²⁺ influx was not affected during D₂O treatment, but was completely inhibited upon D₂O washout. The H₂O/D₂O replacement in internal solutions of patch electrodes reduced Ca²⁺ currents evoked by depolarizing voltage steps, whereas additional extracellular H₂O/D₂O replacement recovered the currents, suggesting that D₂O gradient across plasma membrane is critical for Ca²⁺ channel kinetics. Radioimmunoassay of high-K⁺-induced β-endorphin release demonstrated an increase during D₂O treatment and a decrease upon D₂O washout. These results demonstrate that the H₂O-to-D₂O-induced increase in β-endorphin release corresponded with the redistribution of actin, and the D₂O-to-H₂O-induced decrease in β-endorphin release corresponded with the inhibition of voltage-sensitive Ca²⁺ channels. The computer modeling suggests that the differences in the zero-point vibrational energy between protonated and deuterated amino acids produce an asymmetric distribution of these amino acids upon D₂O washout and this causes the dysfunction of Ca²⁺ channels.     

Hydrolysis of p-nitrotrifluoroacetanilide catalyzed by water and imidazole

The hydrolyses of p-nitrotrifluoroacetanilide catalyzed by water and imidazole were examined at 70°C. The pH-rate constant profile of the hydrolysis in H₂O was examined in the pH range 0.0–11.4. The hydrolysis was independent of pH in the region from pH 1.0 to 4.5, presumably a water-catalyzed reaction. The rate constant and the D₂O solvent isotope effect for this reaction were 1.0 × 10^?4 sec^?1 and 3.7, respectively. Both natural imidazole and imidazolium cation catalyzed hydrolysis. The rate constant of the hydrolysis catalyzed by neutral imidazole was determined to be 5.4 × 10^?3M^?1 sec^?1 and the D₂O solvent isotope effect was 1.8.     

Stereochemistry of Decarboxylation Reactions Catalyzed by a Constitutive Aromatic l-Amino Acid Decarboxylase

The stereochemistry of the decarboxylation reaction catalyzed by an aromatic l-amino acid decarboxylase, purified from Micrococcus percitreus, was studied using stereospecifically deuterium labelled phenylalanine (Phe). The ¹H NMR spectrum of [1,2-²H₂]-β-phenethylamine enzymatically derived from (2S, 3R)-[3-²H]-Phe in ²H₂O was compared with that of [1-²H]-β-phenethylamine from unlabelled Phe in ²H₂O. The results clearly indicate that the decarboxylation reaction of this enzyme proceeds exclusively through a course in which the configuration at C-2 of Phe is retained.     

The lateral diffusion coefficient of lecithin molecules in single bilayer vesicles studied by 14N NMR

The ¹⁴N nuclear relaxation times T₁ and T₂ in egg yolk phosphatidylcholine have been observed in single bilayer vesicles dispersed in the media of different viscosities, ¹H₂O and ²H₂O. The lateral diffusion coefficient of lipid molecule D has been calculated according to the method reported earlier: D = 2.2 × 10^?8cm²s^?1 in ¹H₂O and 2.1 × 10^?8cm²s^?1 in ²H₂O at 20°C. They are in excellent agreement. This result gives a strong basis of usefulness of ¹⁴N NMR method in the evaluation of D without introducing any system perturbation.     

A vibrational spectroscopic study of the ice-melting-induced transition of 1,2-dibehenoyl-sn-glycero-3-phosphocholine

The phase transition of bilayers of 1,2-dibehenoyl-sn-glycero-3-phosphocholine (DBPC) induced by ice (H₂O and D₂O) melting has been investigated by infrared and Raman spectroscopy. Spectral changes observed at this transition are smaller at lower water content. These spectral changes are interpreted in terms of increased molecular mobility. Slightly different temperature dependencies are observed for various spectral parameters between samples dispersed in H₂O and D₂O.     

Isotope effect studies of the chemical mechanism of pig heart NADP isocitrate dehydrogenase

The catalytic mechanism of porcine heart NADP isocitrate dehydrogenase has been investigated by use of the variation of deuterium and 13C kinetic isotope effects with pH. The observed 13C isotope effect on V/K for isocitrate increases from 1.0028 at neutral pH to a limiting value of 1.040 at low pH. The limiting 13C isotope effect with deuteriated isocitrate at low pH is 1.016. This decrease in 13(V/KIc) upon deuteriation indicates a stepwise mechanism for the oxidation and decarboxylation of isocitrate. This predicts a deuterium isotope effect on V/K of 2.9, but D(V/K) at low pH only increases to a maximum of 1.08. It is not known why 13(V/KIc) with deuteriated isocitrate decreases more than predicted. The pK seen in the 13(V/KIc) pH profile for isocitrate is 4.5. This pK is displaced 1.2 pH units from the true pK of the acid/base functionality of 5.7 seen in the pKi profile for oxalylglycine, a competitive inhibitor for isocitrate. From this displacement, catalysis is estimated to be 16 times faster than substrate dissociation. By use of the pH-dependent partitioning ratio of the reaction intermediate oxalosuccinate between decarboxylation to 2-ketoglutarate and reduction to isocitrate, the forward commitment to catalysis for decarboxylation was determined to be 7.3 at pH 5.4 and 3.2 at pH 5.0. This gives an intrinsic 13C isotope effect for decarboxylation of 1.050. 3-Fluoroisocitrate is a new substrate oxidatively decarboxylated by NADP isocitrate dehydrogenase. At neutral pH, D(V/K3-F-Ic) = 1.45 and 13(V/K3-F-Ic) = 1.0129. At pH 5.2, 13(V/K3-F-Ic) increases to 1.0186, indicating that a finite, but diminished, external commitment remains at neutral pH. The product of oxidative decarboxylation of 3-hydroxyisocitrate by NADP isocitrate dehydrogenase is 2-hydroxy-3-ketoglutarate. This results from enzymatic protonation of the cis-enediol intermediate at C2 rather than C3 (as seen with isocitrate and 3-fluoroisocitrate). 2-Hydroxy-3-ketoglutarate further decarboxylates in solution to 2-hydroxy-3-ketobutyrate, which further decarboxylates to acetol. This makes 3-hydroxyisocitrate unsuitable for 13C isotope effect studies.     

Thermal perturbation difference spectra and D2O vs H2O isothermal difference spectra of protein-model aromatic chromophores.

The thermal perturbation difference spectra of phenolic and indolic chromophores in water resemble the isothermal D₂O and H₂O spectra of these chromophores. For phenols approximately equal Δ? values are obtained in both types of spectra, but for their methyl ethers Δ? values of D₂O vs H₂O spectra are about half of those of the thermal perturbation spectra. Phenols and their methyl ethers were studied in deuterated ethylene glycol and glycerol vs the corresponding protiated solvent, and in nonprotic solvents containing 0.25–4% D₂O or H₂O. For phenols in D₂O vs H₂O, about one-third to one-half of the difference spectrum is attributed to solvent structure difference, and the remainder to the effects of replacing OH by OD and to differences in accepting hydrogen bonds from D₂O and H₂O. The refractive index difference between D₂O and H₂O was shown to be a minor contribution by means of experiments in which D₂O was at 5 dgC and H₂O at 47 dgC, conditions of equal refractive index (Na_D). D₂O vs H₂O and glycerol-d vs glycerol-h difference spectra of ribonuclease are about twice as large as expected from the known number of exposed tyrosyl side chains. Possible sources of error in D₂O vs H₂O spectra of proteins are discussed.