13C NMR studies of carboxylate inhibitor binding to cobalt(II) carboxypeptidase A

Both 13C NMR and electronic absorption spectral studies on cobalt(II) carboxypeptidase A in the presence of acetate and phenylacetate provide evidence for two binding sites for each of these agents. The transverse relaxation rate T2-1 for the 13C-enriched carboxyl groups of the inhibitors is significantly increased when bound to the paramagnetic cobalt carboxypeptidase as compared to the diamagnetic zinc enzyme. The acetate concentration dependence of T2p-1 shows two inflections indicative of sequential binding of two inhibitor molecules. The cobalt-13C distances, calculated by means of the Solomon equation, indicate that the second acetate molecule binds directly to the metal ion while the first acetate molecule binds to a protein group at a distance 0.5-0.8 nm for the metal ion, consistent with it binding to one or more of the arginyl residues (Arg-145, Arg-127, or Arg-71). In the case of phenylacetate, perturbation of the cobalt electronic absorption spectrum shows that binding occurs stepwise. 13C NMR distance measurements indicate that one of the two phenylacetates is bound to the metal in the EI2 complex. These binding sites may correspond to those identified previously by kinetic means (one of which is competitive, the other noncompetitive) with peptide binding. The studies further indicate that it should be possible to map the protein interactions of the carbonyl groups of both substrate and noncompetitive inhibitors during catalysis by means of 13C NMR studies with suitably labeled substrates and inhibitors.     

Di-iron—carboxylate proteins

Di-iron centers bridged by carboxylate residues and oxide/hydroxide groups have so far been seen in four classes of proteins involved in dioxygen chemistry or phosphoryl transfer reactions. The dinuclear iron centers in these proteins are coordinated by histidines and additional carboxylate ligands. Recent structural data on some of these enzymes, combined with spectroscopic and kinetic data, can now serve as a base for detailed mechanistic suggestions. The di-iron sites in the major class of hydroxylase-oxidase enzymes, which contains ribonucleotide reductase and methane monooxygenase, show significant flexibility in the geometry of their coordination of three or more carboxylate groups. This flexibility, combined with a relatively low coordination number, and a buried environment suitable for reactive oxygen chemistry, explains their efficient harnessing of the oxidation power of molecular oxygen.     

Structural requirements for pyridine carboxylate effects on phosphoenolpyruvate carboxykinase

It has previously been established that quinolinic acid and 3-mercaptopicolinic acid cause hypoglycemia by inhibiting Phosphoenolpyruvate (PEP) carboxykinase and gluconeogenesis. In the rat, 3-aminopicolinic acid permits Fe²⁺ to activate this enzyme; it enhances gluconeogenesis and causes hyperglycemia. In the present study, other pyridine carboxylates were screened for effects on the activity of PEP carboxykinase. The structural requirement for an inhibitor or an activator of this enzyme has been defined: It must be a picolinic acid derivative with the α carboxyl unsubstituted and with another group on position 3. The group at position 3 determines the effect (inhibition or activation) and the potency of the compound. Compounds such as picolinic acid, all the isomers of quinolinic acid, 2-mercaptonicotinic acid, and 2-aminonicotinic acid were inactive. Fe²⁺ enhances the potency of quinolinate and 2-mercaptopicolinate 15- to 20-fold, and 3-aminopicolinate does not activate the carboxykinase in the absence of Fe²⁺. It is therefore assumed that Fe²⁺ binds to the ring nitrogen and the α-carboxyl group of one or more molecules of these compounds to form an effective coordination complex. Complexes involving picolinate derivatives with an acidic function at position 3 inhibit; the complex of Fe²⁺ with 3-aminopicolinate either delivers Fe²⁺ to the catalytic site and then dissociates or the Fe²⁺ in the complex is catalytically active. 3-Aminopicolinate causes hyperglycemia in the guinea pig. It activates guinea pig liver cytosolic PEP carboxykinase in vitro but does not activate the mitochondrial carboxykinase. If activation of PEP carboxykinase is the means by which 3-aminopicolinate causes hyperglycemia, our findings indicate that the cytosolic enzyme can play an important role in glucose synthesis in species having appreciable amounts of both carboxykinases.     

On the importance of orientation in general base catalysis by carboxylate

The carboxylate group serves as a general-base catalyst in numerous chemical and enzymatic reactions. The importance of the direction in which the proton is transferred to the carboxylate is discussed. syn (on the same side of the CO bond as the forming CO) protonation is estimated to be 10⁴-fold more favorable than anti protonation. To date, in the models studied where intramolecular reactions involve carboxylate, only anti protonation can occur. In these intramolecular models the catalytic efficiency of a carboxylate group may be underestimated due to this inability to achieve an optimal orientation for protonation; whereas for enzymatic reactions involving carboxylate side chains, structural studies support mechanisms involving syn protonation.     

Level and origin of carboxylate in buckwheat

Summary Buckwheat was grown in media in which the supply of nitrate and potassium was varied. The relation between nitrate assimilation and the carboxylate content of the plant was examined.In full supply of nitrate total carboxylate came from and was equivalent to the assimilated nitrate. Only when this nutrient was depleted, bicarbonate was absorbed and assimilated as the alternative source which caused carboxylate to accumulate in excess of the deficient level of organic nitrogen.Shortage of potassium had no effect on the level of carboxylate because increased uptake of calcium and magnesium replaced the missing potassium.     

Amine and carboxylate spin probe permeability in red cells

Summary Permeabilities for a homologous series of amine and carboxylate nitroxide spin probes were measured in human red blood cells by an electron paramagnetic resonance (EPR) method. Permeabilities determined in this study are much lower than would be predicted for a sheet of bulk hydrocarbon and the polarity of the rate-limiting region is shown to be greater than bulk hydrocarbon. This suggests that the rate-limiting region for permeation of these nonelectrolytes is somewhere in the membrane periphery rather than in the center of the membrane. The red cell membrane does not discriminate between these probes on the basis of molecular volume, as might be predicted by a simple free-volume theory of membrane permeation.     

Rhizosphere carboxylate concentrations of chickpea are affected by genotype and soil type

We investigated whether concentrations of carboxylates in the rhizosphere of chickpea (Cicer arietinum L.) roots were related to soil phosphorus levels. In a field experiment, cultivar Sona was grown at two P levels on eight soil types at three locations. There were large differences in extractable (0.2 mM CaCl₂) rhizosphere carboxylate concentrations amongst the locations. The effect of P fertiliser was variable and carboxylate concentrations depended on soil type. To examine the effect of soil P in more detail, a glasshouse experiment was carried out, in which three cultivars (Heera, Sona and Tyson) were grown at four P levels on one soil type. The biomass of chickpea plants increased with increasing P level of the soil, and the root mass ratio decreased at the highest soil P level. However, rhizosphere concentrations of the carboxylates malonate, malate and citrate did not differ significantly between P treatments. This implied that there was no simple relation between available P and root exudation rates, in contrast to earlier results in studies using hydroponics. Cultivars differed in carboxylate concentration pattern: Sona and Tyson showed a tendency towards increased rhizosphere carboxylate concentrations at the second harvest, whereas the carboxylate concentration of Heera tended to decrease. It is hypothesised that chickpea roots always exude a basal level of carboxylates into the rhizosphere. They only increase carboxylate exudation considerably when the P availability is extremely low, which may occur in soils that strongly bind P.     

Evolution of the carboxylate Jen transporters in fungi

Synteny analysis is combined with sequence similarity and motif identification to trace the evolution of the putative monocarboxylate (lactate/pyruvate) transporters Jen1p and the dicarboxylate (succinate/fumarate/malate) transporters Jen2p in Hemiascomycetes yeasts and Euascomycetes fungi. It is concluded that a precursor form of Jen1p, named here preJen1p, arose by the duplication of an ancestral Jen2p, during the speciation of Yarrowia lipolytica, which was transferred into a new syntenic context. The Jen1p transporters differentiated from preJen1p in Kluyveromyces lactis, before the Whole Genome Duplication (WGD), and are conserved as a single copy in the Saccharomyces species. In contrast, the ancestral Jen2p was definitively lost just prior to the WGD and is absent in Saccharomyces.     

The carboxylate type siderophore rhizoferrin and its analogs produced by directed fermentation

Summary Rhizoferrin is a novel carboxylate-type siderophore which has recently been isolated fromRhizopus microsporus and other fungi of the Mucorales (Zygomycetes). The present investigation shows that a variety of rhizoferrin analogs can be produced by directed fermentation. Thus both the diaminobutane backbone and the citric acid side chains of rhizoferrin have been substituted by diamine and citric acid analogs added to the culture medium. The new ligands as well as their iron complexes have been characterized by physicochemical methods. Conditions of precursor incorporation and implications for the biosynthesis of the new siderophores are discussed.     

Localization of the essential histidine and carboxylate group in D-xylose isomerases.

A light hepatic microsomal preparation was fractionated by sucrose-density centrifugation into one rough, one intermediate and two smooth fractions. The four fractions were characterized with respect to parameters relevant to Ca2+ sequestration. Ca2(+)-ATPase activity was similar in the rough, intermediate and smooth I fractions, but lower in the smooth II fraction. Ca2+ accumulation was the highest in the smooth I and intermediate fractions. On the other hand, Ca2+ efflux from the rough fraction was several-fold faster than from the smooth I fraction. All four subfractions exhibited specific binding sites for inositol 1,4,5-trisphosphate (IP3) and ryanodine; however, the receptors were especially enriched in the smooth I fraction. The total binding sites for ryanodine in that fraction exceeded the number of binding sites for IP3 by about 10-fold. The two receptors responded differently to pharmacological agents; caffeine and dantrolene strongly inhibited ryanodine binding but not IP3 binding, whereas heparin inhibited IP3 binding only. Thus the two receptors are distinct entities. The four fractions also showed distinct gel electrophoretic patterns. The use of two different SDS/polyacrylamide-gel gradients and two protein-staining methods revealed major differences in the distribution of the bands corresponding to Mr values of (x 10(-3) 380, 320, 260, 170, 90, 29 and 21. These proteins were enriched in the smooth fraction. The results indicate that the smooth I fraction might have special importance in stimulus-evoked Ca2(+)-release processes.     

The effects of cyclopropane carboxylate on hepatic pyruvate metabolism

The effects of cyclopropane carboxylate on gluconeogenesis and pyruvate decarboxylation from [1-14C]-labeled pyruvate and lactate were investigated in perfused livers from fasted rats. With high concentrations of pyruvate (greater than or equal to 0.5 mM) in the perfusion medium, infusion of cyclopropane carboxylate inhibited pyruvate decarboxylation and gluconeogenesis by 30 and 40%, respectively. With low, more physiological concentrations of pyruvate (50 microM) or with lactate (1 mM), cyclopropane carboxylate, at a concentration which elicits maximal inhibition of pyruvate decarboxylation from pyruvate (greater than or equal to 0.5 mM), did not affect either pyruvate decarboxylation or gluconeogenesis. Evidence is presented for the rapid formation of the coenzyme-A ester of cyclopropane carboxylate in perfused livers. Infusion of l-(-)carnitine (20 mM) prevented the inhibitory effects of cyclopropane carboxylate on pyruvate decarboxylation and gluconeogenesis from pyruvate (greater than or equal to 0.5 mM). Interestingly, no decrease in the tissue level of cyclopropanecarboxyl-CoA occurs under these conditions. The present study suggests that cyclopropane carboxylate, through a presently ill-defined mediator, inhibits pyruvate decarboxylation and gluconeogenesis by interfering with the pyruvate----oxalacetate----phosphoenolpyruvate----pyruvate cycle when pyruvate (greater than or equal to 0.5mM) supports gluconeogenesis.     

Polyelectrolytic effects in semi-flexible carboxylate polysaccharides. Part 2

A statistical model is presented in which an ionic polymer is taken as a linear arrangement of flexible segments (similar to the spring-bead model). This model is used to represent semi-flexible linear ionic polysaccharides in solution. The distribution of end-to-end distances is taken from Monte Carlo calculations of the amylosic chain conformation and combined with Manning's counterion condensation theory of linear polyelectrolytes. The excess thermodynamic properties are then calculated as a function of the degree of ionization and of a number of physical variables. The results show that the statistical average of thermodynamic functions taken over the conformational states is not equivalent to the thermodynamic function of the average conformation. This has important implications for the correct comparison of theoretical prediction with the experimental results.     

Retinylidene Schiff bases in alkylammonium carboxylate reversed micelles

All-trans-retinal, incorporated into dodecylammonium propionate, dodecylammonium 3-chloropropionate and dodecylammonium trifluoroacetate reversed micelles in cyclohexane containing different amounts of solubilized water, gave all-trans-N-retinylidene-n-dodecylamine. Formation of all-trans-N-retinylidene-n-dodecylamine was complete within a few minutes when all-trans-retinal was solubilized along with L-lysine in reversed-micellar dodecylammonium propionate in cyclohexane as compared to the several hours required for the comparable reaction to occur in the absence of lysine. In situ formed all-trans-N-retinylidene-n-dodecylamine was not protonated by the acidic moiety (i.e., propionate, 3-chloropropionate or trifluoroacetate anions) of dodecylammonium propionate, dodecylammonium 3-chloropropionate or dodecylammonium trifluoroacetate reversed micelles. Addition of propionic or 3-chloropropionic acid did not cause observable protonation of the reversed-micelle-incorporated all-trans-N-retinylidene-n-dedecylamine. Addition of strong trifluoroacetic acid to reversed micellar solutions of all-trans-N-retinylidene-n-dodecylamine caused protonation as well as hydrolysis to retinal. Retinylidene Schiff bases are protected from protonation by strongly held surfactant-ion pairs which may model proton channels whose function is controlled by protein conformational changes.     

Characterization of S1 nuclease. Involvement of carboxylate groups in metal binding.

Modification of the carboxylate groups of purified S1 nuclease resulted in a loss of its single-stranded DNAase, RNAase and phosphomonoesterase activities. The inactivation was due to the removal of zinc atoms from the enzyme and this in turn was dependent on the degree of modification. While the removal of one zinc atom resulted in the partial inactivation of the enzyme, removal of the remaining zinc atoms resulted in the complete inactivation of the enzyme. Similar results were obtained when the purified enzyme was incubated with various concentrations of the metal chelator, EDTA. The EDTA-(1 mM)-treated enzyme, depleted of one zinc atom, showing 40-45% residual activity, when incubated with 1 mM Zn2+ or 1 mM Co2+, regained a significant amount of its initial activity towards all the substrates. However, Woodward's-Reagent-K-modified enzyme depleted of one zinc atom and having the same level of activity (40-45%) could not regain its activity, indicating that the carboxylate groups are involved in the metal binding. Data obtained with carboxylate-group modification, EDTA-treatment, reconstitution with metal ions, zinc estimation and CD analysis of the enzyme suggests that, out of three zinc atoms present in S1 nuclease, zinc I is easily replaceable and is probably involved in the catalytic activity while zinc II and zinc III are involved in maintaining the enzyme structure.     

Alterations in carboxylate ligation at the active site of photosystem II.

Photosystem II (PSII) is the photosynthetic enzyme catalyzing the oxidation of water and reduction of plastoquinone (Q). This reaction occurs at a catalytic site containing four manganese atoms and cycling among five oxidation states, the Sn states, where n refers to the number of oxidizing equivalents stored. Biochemical and spectroscopic techniques have been used previously to conclude that aspartate 170 in the D1 subunit influences the structure and function of the PSII active site (Boerner, R. J., Nguyen, A. P., Barry, B. A., and Debus, R. J. (1992) Biochemistry 31, 6660-6672). Substitution of glutamate for aspartate 170 resulted in an assembled manganese cluster, which was capable of enzymatic turnover, but at lower steady-state oxygen evolution rates. Here, we obtained the difference (light-minus-dark) Fourier transform IR spectrum associated with the S2Q--minus-S1Q transition by illumination of oxygen-evolving wild-type and DE170D1 PSII preparations at 200 K. These spectra are known to be dominated by contributions from carboxylic acid and carboxylate residues that are close to or ligating the manganese cluster. Substitution of glutamate for aspartate 170 results in alterations in the S2Q--minus-S1Q spectrum; the alterations are consistent with a change in carboxylate coordination to manganese or calcium. In particular, the spectra are consistent with a shift from bridging/bidentate carboxylates in wild-type PSII to unidentate carboxylate ligation in DE170D1 PSII.     

Substrate specificity within a family of outer membrane carboxylate channels

Many Gram-negative bacteria, including human pathogens such as Pseudomonas aeruginosa, do not have large-channel porins. This results in an outer membrane (OM) that is highly impermeable to small polar molecules, making the bacteria intrinsically resistant towards many antibiotics. In such microorganisms, the majority of small molecules are taken up by members of the OprD outer membrane protein family. Here we show that OprD channels require a carboxyl group in the substrate for efficient transport, and based on this we have renamed the family Occ, for outer membrane carboxylate channels. We further show that Occ channels can be divided into two subfamilies, based on their very different substrate specificities. Our results rationalize how certain bacteria can efficiently take up a variety of substrates under nutrient-poor conditions without compromising membrane permeability. In addition, they explain how channel inactivation in response to antibiotics can cause resistance but does not lead to decreased fitness.     

A free carboxylate oxygen in the side chain of position 674 in transmembrane domain 7 is necessary for TSH receptor activation.

A specific H-bonding network formed between the central regions of transmembrane domain 6 and transmembrane domain 7 has been proposed to be critical for stabilizing the inactive state of glycoprotein hormone receptors. Many different constitutively activating TSH receptor point mutations have been identified in hyperfunctioning thyroid adenomas in the lower portion of transmembrane domain 6. Position D633 in transmembrane domain 6 of the human TSH receptor is the only one in which four different constitutively activating amino acid exchanges have been identified. Further in vitro substitutions led to constitutive activation of the TSH receptor (D633Y, F, C) as well as to the first inactivating TSH receptor mutation in transmembrane domain 6 without changes of membrane expression or TSH binding (D633R). Molecular modeling of this inactivating TSH receptor mutation revealed potential interaction partners of R633 in transmembrane domain 3 and/or transmembrane domain 7, presumably via hydrogen bonds that could be responsible for locking the TSH receptor in a completely inactive state. To further elucidate the H-bond network that most likely maintains the inactive state of the TSH receptor, we investigated these potential interactions by generating TSH receptor double mutants designed to break up possible H bonds. We excluded S508 in transmembrane domain 3 as a possible interaction partner of R633. In contrast, a partial response to TSH stimulation was rescued in a receptor construct with the double-substitution D633R/N674D. Our results therefore confirm the H bond between position 633 in transmembrane domain 6 and 674 in transmembrane domain 7 suggested by molecular modeling of the inactivating mutation D633R. Moreover, the mutagenesis results, together with a three-dimensional structure model, indicate that for TSH receptor activation and G protein-coupled signaling, at least one free available carboxylate oxygen is required as a hydrogen acceptor atom at position 674 in transmembrane domain 7.     

CO2 adducts as reactive analogues of carboxylate substrates for aconitase and other enzymes of carbohydrate metabolism

The CO2 adducts resulting from N-, O-, and S-carboxylation of suitable precursors are close analogues of carboxylate substrates in which -NH-CO2-, -O-CO2-, or -S-CO2- replaces -CH2-CO2- in the physiological substrate, -NOH-CO-2 replaces -CHOH-CO-2 and O-CO2- replaces -O-PO3H- R-XH + CO2 in equilibrium with R-X-CO2- + H+ X(-XH = -NH2, -NHOH, -OH or -SH). We find that aconitase catalyzes the CO2-dependent dehydration of N-hydroxy-DL-aspartate and erythro-beta-hydroxyl-L-aspartate with respective kcat values 62 and 90% of kcat for citrate and Km values of 3.6 and 3.2 mM, respectively. The CO2 adducts (carbamates) of the precursors would be structural and stereo analogues of the physiological substrate isocitrate. Detailed kinetic analyses of the behavior of intermediates and products show that aconitase catalyzes the formation of the enzyme-bound CO2 adducts from enzyme-bound precursors and CO2 and directs them, as well as the preformed CO2 adducts, into alpha,beta water elimination reactions formally identical to the isocitrate/cis-aconitate reaction. Six other enzymes of carbohydrate metabolism (succinate thiokinase and isocitrate, glucose-6-phosphate, succinate semialdehyde, glutamate, and malate dehydrogenase) utilize CO2 adducts as reactive substrate analogues. At least one of these (glucose-6-phosphate dehydrogenase) catalyzes the formation of the enzyme-bound CO2 adduct (presumed to be D-glucose 6-carbonate in this case) from enzyme-bound precursor (D-glucose) and CO2 in the manner of aconitase. The case of malate dehydrogenase is unique because the reactive malate analogue, -O2C-O-CHOH-CO-2, arises from nucleophilic attack of HCO-3 on the carbonyl of glyoxylate, rather than electrophilic attack of CO2 on the hydrated carbonyl of glyoxylate.     

Temporal changes in carboxylate content of ryegrass with stepwise change in nutrition

Summary A detailed scheme of carboxylate formation and retention by plant tissues as a result of ion uptake and utilization is given.By means of discontinuities in the supply with nutrient ions, carboxylate retention by the tissues of perennial ryegrass was followed as a function of growth. It was found that translocation of potassium nitrate to the shoot and subsequent nitrate metabolism was the only process capable of supplying the shoot with sufficient carboxylates and of removing the excess from the foliage to the root system with maintenance of the normal carboxylate content. Absorbed bicarbonate was a good source of carboxylates in the roots, but the rate of translocation to the plant tops was too slow relative to growth. Therefore, the carboxylate concentration in the foliage fell progressively to one half the normal value.Constancy of carboxylate concentration in the dry matter was related to the early establishment of the proportion of carboxylates to dry material in the new growth, making it independent of subsequent changes in water content of the tissues.Changes in carboxylate concentrations due to changes in the supply were continuous with time. Nitrate caused a depression in the roots during nitrate accumulation, but the nitrate metabolism in the follage made sufficient carboxylates available for replenishment and maintenance of their normal level in the whole plant.Agronomy Department, Paper No. 787