Characterization of the ATP2B gene family in blood pressure

The 12q21 locus, which lies near the plasma membrane calcium-transporting ATPase 1 gene (ATP2B1), has one of the strongest associations with blood pressure and hypertension in Europeans and Asians. We performed an association analysis of the ATP2B isomers ATP2B2, ATP2B3, and ATP2B4 with blood pressure and hypertension in 7,551 Korean individuals and observed a link with ATP2B2 and ATP2B4. To examine the regulation of blood pressure by ATP2B, ATP2B1 and ATP2B4 mRNA was reduced in vascular smooth muscle cells by siRNA. The expression pattern of 23 ATP2B-related genes was analyzed by quantitative real-time PCR, which differed between treatment with ATP2B1 and ATP2B4 siRNA. The reduction inATP2B1 mRNA induced a significant change in mRNA levels in the calcium pump RYR1 and the ATP2B-binding protein HOMER1. Conversely, the decrease in ATP2B4 mRNA significantly altered mRNA expression of the calcium pump SLC8A1 and the ATP2B-binding proteins CASK and DLG4. These results suggest that blood pressure is differentially regulated through calcium signaling between ATP2B1 and ATP2B4.     

Functional Characterization of TbMCP5, a Conserved and Essential ADP/ATP Carrier Present in the Mitochondrion of the Human Pathogen Trypanosoma brucei

Trypanosoma brucei is a kinetoplastid parasite of medical and veterinary importance. Its digenetic life cycle alternates between the bloodstream form in the mammalian host and the procyclic form (PCF) in the bloodsucking insect vector, the tsetse fly. PCF trypanosomes rely in the glucose-depleted environment of the insect vector primarily on the mitochondrial oxidative phosphorylation of proline for their cellular ATP provision. We previously identified two T. brucei mitochondrial carrier family proteins, TbMCP5 and TbMCP15, with significant sequence similarity to functionally characterized ADP/ATP carriers from other eukaryotes. Comprehensive sequence analysis confirmed that TbMCP5 contains canonical ADP/ATP carrier sequence features, whereas they are not conserved in TbMCP15. Heterologous expression in the ANC-deficient yeast strain JL1Δ2Δ3u⁻ revealed that only TbMCP5 was able to restore its growth on the non-fermentable carbon source lactate. Transport studies in yeast mitochondria showed that TbMCP5 has biochemical properties and ADP/ATP exchange kinetics similar to those of Anc2p, the prototypical ADP/ATP carrier of S. cerevisiae. Immunofluorescence microscopy and Western blot analysis confirmed that TbMCP5 is exclusively mitochondrial and is differentially expressed with 4.5-fold more TbMCP5 in the procyclic form of the parasite. Silencing of TbMCP5 expression in PCF T. brucei revealed that this ADP/ATP carrier is essential for parasite growth, particularly when depending on proline for energy generation. Moreover, ADP/ATP exchange in isolated T. brucei mitochondria was eliminated upon TbMCP5 depletion. These results confirmed that TbMCP5 functions as the main ADP/ATP carrier in the trypanosome mitochondrion. The important role of TbMCP5 in the T. brucei energy metabolism is further discussed.     

Regulation of photosynthetic cyclic electron flow pathways by adenylate status in higher plant chloroplasts

Cylic electron flow (CEF) around Photosystem I in photosynthetic eukaryotes is likely to be necessary to augment ATP production, rapidly- and precisely balancing the plastid ATP/NADPH energy budget to meet the demands of downstream metabolism. Many regulatory aspects of this process are unclear. Here we demonstrate that the higher plant plastid NADH/Fd:plastoquinone reductase (NDH) and proposed PGR5/PGRL1 ferredoxin:plastoquinone reductase (FQR) pathways of CEF are strongly, rapidly and reversibly inhibited in vitro by ATP with K_i values of 670 μM and 240 μM respectively, within the range of physiological changes in ATP concentrations. Control experiments ruled out effects on secondary reactions, e.g. FNR- and cytochrome b₆f activity, nonphotochemical quenching of chlorophyll fluorescence etc., supporting the view that ATP is an inhibitor of CEF and its associated pmf generation and subsequent ATP production. The effects are specific to ATP, with the ATP analog AMP-PNP showing little inhibitory effect, and ADP inhibiting only at higher concentrations. For the FQR pathway, inhibition was found to be classically competitive with Fd, and the NDH pathway showing partial competition with Fd. We propose a straightforward model for regulation of CEF in plants in which CEF is activated under conditions when stromal ATP low, but is downregulated as ATP levels build up, allowing for effective ATP homeostasis. The differences in K_i values suggest a two-tiered regulatory system, where the highly efficient proton pumping NDH is activated with moderate decreases in ATP, with the less energetically-efficient FQR pathway being activated under more severe ATP depletion.     

Energy metabolism of Bdellovibrio bacteriovorus

Bdellovibrio bacteriovorus, strain Bd. 109 Sa, generates ATP mainly by oxidative phosphorylation during electron transport. During exponential growth the ATP pool is constant (9 nmoles/100 μg N) indicating that energy-producing and energy-consuming reactions are well balanced. The ratio of substrate respiration/endogenous respiration is approx. 2.5/1. Energy charge is constant both in endogenous and substrate respiration at values of 0.62 to 0.64. During endogenous respiration (starvation) the ATP pool oscillates at regular intervals. ATP over-production is started after the ATP pool has decreased to a minimum level of 6 nmoles/100 μg N. The alternating over- and under-production of ATP is interpreted as a special regulation which enables the organism to make economic use of its own cellular materials. Addition of substrate (glutamate) to starving cells does not influence the type of ATP pool oscillation as observed in endogenous respiration. The parasitic strain Bd. 109 Pa exhibits the same periodicity of ATP overproduction as does its saprophytic derivative, Bd. 109 Sa. Decrease of viability during starvation is paralleled by a decrease of the ATP pool.     

Highly efficient production of ATP by a methanol yeast,Candida boidinii (Kloeckera sp.) No. 2201

Improvement of efficiency of ATP production from adenosine with sorbitol-treated cells of Candida boidinii (Kloeckera sp.) no. 2201 was investigatedOrthophosphate, pyrophosphate and divalent metal ions inhibited the deamination of AMP to IMP, a major by-product. No hypoxanthine nucleotidic compound was accumulated by addition of coformycin. By successive feeding of K₂HPO₄ to maintain the phosphorus concentration at over 100 mM, the conversion rate from adenosine to ATP was improved to 70%. Simultaneous feeding of K₂HPO₄ and adenosine resulted in the accumulation of 100 mM ATP (50.7 g/l) after 28 h of incubation and the increase of IMP without decrease of ATP for 48 h.ATP production was further prolonged until 60 h of incubation and 198 mM ATP (100 g/l) was accumulated at the conversion rate of 77.4% by control of pH in the range of 6.5 to 6.8 during the reaction.     

The dependence of intracellular ATP level on the nutrition mode of the acidophilic bacteria <Emphasis Type="Italic">Sulfobacillus thermotolerans</Emphasis> and <Emphasis Type="Italic">Alicyclobacillus tolerans</Emphasis>

The dynamics of the ATP pool in the aerobic spore-forming acidothermophilic mixotrophic bacteria Sulfobacillus thermotolerans Kr1^T and Alicyclobacillus tolerans K1^T were studied in the course of their chemolithoheterotrophic, chemoorganoheterotrophic, and chemolithoautotrophic growth. It was established that, during mixotrophic growth, the maximum ATP concentrations in the cells of S. thermotolerans Kr1 and A. tolerans K1 were 3.8 and 0.6 nmol/mg protein, respectively. The ATP concentrations in sulfobacilli and alicyclobacilli during organotrophic growth were 2.2 and 3.1 nmol/mg protein, respectively. In the cells of the obligately heterotrophic bacterium Alicyclobacillus cycloheptanicus 4006^T, the maximum ATP concentration was several times higher and reached 12.3 nmol/mg protein. During lithotrophic growth, the maximum values of the ATP concentration in the cells of S. thermotolerans Kr1 and A. tolerans K1 were 0.3 and <0.1 nmol/mg protein, respectively; in the cells of the autotrophic bacterium Acidithiobacillus ferrooxidans TFBk, the ATP content was about 60–300 times higher (17.0 nmol/mg protein). It is concluded that low ATP content is among the possible causes of growth cessation of S. thermotolerans Kr1 and A. tolerans K1 under auto-and heterotrophic conditions after several culture transfers.     

Influence of the redox state and the activation of the chloroplast ATP synthase on proton-transport-coupled ATP synthesis/hydrolysis

The membrane-bound ATP synthase from chloroplasts can occur in different redox and activation states. In the absence of reductants the enzyme usually is oxidized and inactive, E^ox_i. Illumination in the presence of dithiothreitol leads to an active, reduced enzyme, E^red_a. If this form is stored in the dark in the presence of dithiothreitol an inactive, reduced enzyme E^red_i is formed. The rates of ATP synthesis and ATP hydrolysis catalyzed by the different enzyme species are measured as a function of ΔpH (Δψ = 0 mV). The ΔpH was generated with an acid-base transition using a rapid-mixing quenched flow apparatus. The following results were obtained. (1) The oxidized ATP synthase catalyzes high rates of ATP synthesis, v^ox_max = 400 ATP per CF₀F₁ per s. The half-maximal rate is obtained at ΔpH = 3.4. (2) The active, reduced ATP synthase catalyzes high rates of ATP synthesis, v^red_max = 400 ATP per CF₀F₁ per s. The half-maximal rate is obtained at ΔpH = 2.7. It catalyzes also high rates of ATP hydrolysis v^red_max = −90 ATP per CF₀F per s at ΔpH = 0. (3) The inactive species (both oxidized and reduced) catalyze neither ATP synthesis nor ATP hydrolysis. The activation/inactivation of the reduced enzyme is completely reversible. (4) The activation of the reduced, inactive enzyme is measured as a function of ΔpH by measuring the rate of ATP hydrolysis catalyzed by the active species. Half-maximal activation is observed at ΔpH = 2.2. (5) On the basis of these results a reaction scheme is proposed relating the redox reaction, the activation and the catalytic reaction of the chloroplast ATP synthase.     

Modulation of intracellular chloride channels by ATP and Mg2+

We report the effects of ATP and Mg²⁺ on the activity of intracellular chloride channels. Mitochondrial and lysosomal membrane vesicles isolated from rat hearts were incorporated into bilayer lipid membranes, and single chloride channel currents were measured. The observed chloride channels (n = 112) possessed a wide variation in single channel parameters and sensitivities to ATP. ATP (0.5–2 mmol/l) modulated and/or inhibited the chloride channel activities (n = 38/112) in a concentration-dependent manner. The inhibition effect was irreversible (n = 5/93) or reversible (n = 15/93). The non-hydrolysable ATP analogue AMP-PNP had a similar inhibition effect as ATP, indicating that phosphorylation did not play a role in the ATP inhibition effect. ATP modulated the gating properties of the channels (n = 6/93), decreased the channels' open dwell times and increased the gating transition rates. ATP (0.5–2 mmol/l) without the presence of Mg²⁺ decreased the chloride channel current (n = 12/14), whereas Mg²⁺ significantly reversed the effect (n = 4/4). We suggest that ATP-intracellular chloride channel interactions and Mg²⁺ modulation of these interactions may regulate different physiological and pathological processes.     

The ASCT/SCS cycle fuels mitochondrial ATP and acetate production in Trypanosoma brucei

Acetate:succinate CoA transferase (ASCT) is a mitochondrial enzyme that catalyzes the production of acetate and succinyl-CoA, which is coupled to ATP production with succinyl-CoA synthetase (SCS) in a process called the ASCT/SCS cycle. This cycle has been studied in Trypanosoma brucei (T. brucei), a pathogen of African sleeping sickness, and is involved in (i) ATP and (ii) acetate production and proceeds independent of oxygen and an electrochemical gradient. Interestingly, knockout of ASCT in procyclic form (PCF) of T. brucei cause oligomycin A-hypersensitivity phenotype indicating that ASCT/SCS cycle complements the deficiency of ATP synthase activity. In bloodstream form (BSF) of T. brucei, ATP synthase works in reverse to maintain the electrochemical gradient by hydrolyzing ATP. However, no information has been available on the source of ATP, although ASCT/SCS cycle could be a potential candidate. Regarding mitochondrial acetate production, which is essential for fatty acid biosynthesis and growth of T. brucei, ASCT or acetyl-CoA hydrolase (ACH) are known to be its source. Despite the importance of this cycle, direct evidence of its function is lacking, and there are no comprehensive biochemical or structural biology studies reported so far. Here, we show that in vitro–reconstituted ASCT/SCS cycle is highly specific towards acetyl-CoA and has a higher k_cat than that of yeast and bacterial ATP synthases. Our results provide the first biochemical basis for (i) rescue of ATP synthase-deficient phenotype by ASCT/SCS cycle in PCF and (ii) a potential source of ATP for the reverse reaction of ATP synthase in BSF.     

Rapid Modulation of Spinach Leaf Nitrate Reductase by Photosynthesis : II. In Vitro Modulation by ATP and AMP

Assimilatory nitrate reductase activity (NRA) in crude spinach leaf (Spinacia oleracea) extracts undergoes rapid changes following fluctuations in photosynthesis brought about by changes in external CO₂ or by water stress (WM Kaiser, E Brendle-Behnisch [1991] Plant Physiol 96:363-367). A modulation of NRA sharing several characteristics (stability, response to Mg²⁺ or Ca²⁺, kinetic constants) with the in vivo modulation was obtained in vitro by preincubating desalted leaf extracts with physiological concentrations of Mg²⁺ and ATP (deactivating) or AMP (activating). When nitrate reductase (NR) was inactivated in vivo by illuminating leaves at the CO₂ compensation point, it could be reactivated in vitro by incubating leaf extracts with AMP. For the in vitro inactivation, ATP could be replaced by GTP or UTP. Nonhydrolyzable ATP analogs (β, γ-imido ATP, β, γ-methyl-ATP) had no effect on NR, whereas γ-S-ATP caused an irreversible inactivation. This suggests that NR modulation involves ATP hydrolysis. In contrast to NR in crude leaf extracts, partially purified NR did not respond to ATP or AMP. ATP and AMP levels in whole leaf extracts changed in the way predicted by the modulation of NRA when leaves were transferred from photosynthesizing (low ATP/AMP) to photorespiratory (high ATP/AMP) conditions. Adenine nucleotide levels in leaves could be effectively manipulated by feeding mannose through the leaf petiole. NRA followed these changes as expected from the in vitro results. This suggests that cytosolic ATP/AMP levels are indeed the central link between NRA in the cytosol and photosynthesis in the chloroplast. Phosphorylation/dephosphorylation of NR or of NR-regulating protein factors is discussed as a mechanism for a reversible modulation of NR by ATP and AMP.     

The reversal of the myosin and actomyosin ATPase reactions and the free energy of ATP binding to myosin

Evidence is presented that both myosin and actomyosin in presence of Mg²⁺ and KCl catalyze an incorporation of ³²P_i into ATP. The rate with actomyosin is about $\text{1}\text{500}$ the rate of ATP hydrolysis; the rate with myosin is less than $\text{1}\text{100}$ of that with actomyosin. With myosin, but not with actomyosin, an apparent initial “burst” of ³²P_i incorporation into ATP is observed. Actin binding thus promotes ATP dissociation. The data with myosin allow estimation of both the amount of enzyme-bound [³²P]-ATP present and the rate constant, k_?1, for dissociation of the myosin· ATP. From these results and other data a ?ΔG^o for ATP binding to myosin of 12–13 kcal/mole may be estimated, with a much lower ?ΔG^o for hydrolysis of enzyme-bound ATP. Protein conformational change accompanying ATP binding appears to be the principal means of capture of energy from the overall reaction of ATP cleavage.     

Essential Role of the ε Subunit for Reversible Chemo-Mechanical Coupling in F1-ATPase

F₁-ATPase is a rotary motor protein driven by ATP hydrolysis. Among molecular motors, F₁ exhibits unique high reversibility in chemo-mechanical coupling, synthesizing ATP from ADP and inorganic phosphate upon forcible rotor reversal. The ε subunit enhances ATP synthesis coupling efficiency to > 70% upon rotation reversal. However, the detailed mechanism has remained elusive. In this study, we performed stall-and-release experiments to elucidate how the ε subunit modulates ATP association/dissociation and hydrolysis/synthesis process kinetics and thermodynamics, key reaction steps for efficient ATP synthesis. The ε subunit significantly accelerated the rates of ATP dissociation and synthesis by two- to fivefold, whereas those of ATP binding and hydrolysis were not enhanced. Numerical analysis based on the determined kinetic parameters quantitatively reproduced previous findings of two- to fivefold coupling efficiency improvement by the ε subunit at the condition exhibiting the maximum ATP synthesis activity, a physiological role of F₁-ATPase. Furthermore, fundamentally similar results were obtained upon ε subunit C-terminal domain truncation, suggesting that the N-terminal domain is responsible for the rate enhancement.     

Quantitative Extraction of Adenosine Triphosphate from Cultivable and Host-Grown Microbes: Calculation of Adenosine Triphosphate Pools

Existing data on adenosine triphosphate (ATP) pools in microbes are deficient for two reasons: (i) incomplete extractions of ATP, and (ii) the failure to take into account that the adverse effects of extracting procedures on standard ATP exert analogous effects on the ATP released from bacterial cells. Methods for correcting observed yields and calculating ATP pools have been demonstrated. Three bacterial species were used in the studies on extraction of ATP: Escherichia coli, Mycobacterium phlei, and Mycobacterium lepraemurium. Perchloric acid and n-butanol were disqualified because of their failure to extract total bacterial ATP even from E. coli and because of inconvenient procedures. The new extraction procedure had minimal effects on standard ATP, liberated 100% of the ATP pools from the three representative species of microbes, and caused no ionic imbalance or quenching of bioluminescence. This method involves vortexing of cell suspensions for 10 s with 23% chloroform (vol/vol), heating at 98 C for the required time (E. coli, 3 min; M. phlei, 5 min; M. lepraemurium, 10 min) and then 1 min at 98 C with vacuum to dry the samples. Heat or chloroform alone may suffice for some microbes and release total ATP from plant and animal cells.     

Net uptake of adenine nucleotides by newborn rat liver mitochondria

In newborn rat liver, the adenine nucleotide content (ATP + ADP + AMP) of mitochondria increases severalfold within 2 to 3 h of birth. The net increase in mitochondrial adenines suggests a novel mechanism by which mitochondria are able to accumulate adenine nucleotides from the cytosol (J. R. Aprille and G. K. Asimakis, 1980, Arch. Biochem. Biophys.201, 564.). This was investigated further in vitro. Isolated newborn liver mitochondria incubated with 1 mM ATP for 10 min at 30 °C doubled their adenine nucleotide content with effects on respiratory functions similar to those observed in vivo: State 3 respiration and adenine translocase activity increased, but uncoupled respiration was unchanged. The mechanism for net uptake of adenine nucleotides was found to be specific for ATP or ADP, but not AMP. Uptake was concentration dependent and saturable. The apparent K_m′s for ATP and ADP were 0.85 ± 0.27 mM and 0.41 ± 0.20 mM, respectively, measured by net uptake of [¹⁴C]ATP or [¹⁴C]ADP. The specific activities of net ATP and ADP uptake averaged 0.332 ± 0.062 and 0.103 ± 0.002 nmol/min/mg protein, respectively. ADP was a competitive inhibitor of net ATP uptake. If P_i was omitted from the incubations, net uptake of ATP or ADP was reduced by 51%. Either mersalyl or N-ethylmaleimide severely inhibited the accumulation of adenine nucleotides. Net ATP uptake was stoichiometrically dependent on MgCl₂, suggesting that Mg²⁺ is accumulated along with ATP (or ADP). Uptake was energy dependent as indicated by the following results: Net AdN uptake (especially ADP uptake) was stimulated by the addition of an oxidizable substrate (glutamate) and inhibited by FCCP (an uncoupler). Antimycin A had no effect on net ATP uptake but inhibited net ADP uptake, suggesting that ATP was able to serve as an energy source for its own accumulation. If carboxyatractyloside was added to inhibit the exchange translocase, thereby preventing rapid access of exogenous ATP to the matrix, net ATP uptake was inhibited; carboxyatractyloside had no effect on ADP uptake. It was concluded that the net uptake of adenine nucleotides from the extramitochondrial space occurs by a specific transport process distinct from the classic adenine nucleotide exchange translocase. The accumulation of adenine nucleotides may regulate matrix reactions which are allosterically affected by adenines or which require adenines as a substrate.     

A new adenine nucleotide transporter located in the ER is essential for maintaining the growth of Toxoplasma gondii

The lumen of the endoplasmic reticulum (ER) is the subcellular site where secretory protein folding, glycosylation and sulfation of membrane-bound proteins, proteoglycans, and lipids occur. The protein folding and degradation in the lumen of the ER require high levels of energy in the form of ATP. Biochemical and genetic approaches show that ATP must first be translocated across ER membrane by particular transporters before serving as substrates and energy sources in the lumenal reactions. Here we describe an ATP/ADP transporter residing in the ER membranes of T.gondii. Immunofluorescence (IFA) assay in transgenic TgANT1-HA tag revealed that TgANT1 is a protein specifically expressed in the ER. In vitro assays, functional integration of TgANT in the cytoplasmic membrane of intact E. coli cells reveals high specificity for an ATP/ADP antiport. The depletion of TgANT leads to fatal growth defects in T.gondii, including a significant slowdown in replication, no visible plaque formation, and reduced ability to invade. We also found that the amino acid mutations in two domains of TgANT lead to the complete loss of its function. Since these two domains are conserved in multiple species, they may share the same transport mechanism. Our results indicate that TgANT is the only ATP/ADP transporter in the ER of T. gondii, and the lack of ATP in the ER is the cause of the death of T. gondii.     

The enigmatic mitochondrial ORF ymf39 codes for ATP synthase chain b

ymf39 is a conserved hypothetical protein-coding gene found in mitochondrial genomes of land plants and certain protists. We speculated earlier, based on a weak sequence similarity between Ymf39 from a green alga and the atpF gene product from Bradyrhizobium, that ymf39 might code for subunit b of mitochondrial F₀F₁-ATP synthase. To test this hypothesis, we have sequenced ymf39 from five protists with minimally derived mitochondrial genomes, the jakobids. In addition, we isolated the mitochondrial ATP synthase complex of the jakobid Seculamonas ecuadoriensis and determined the partial protein sequence of the 19-kDa subunit, the size expected for Ymf39. The obtained peptide sequence matches perfectly with a 3′-proximal region of the ymf39 gene of this organism, confirming that Ymf39 is indeed an ATP synthase subunit. Finally, we employed statistical tests to assess the significance of sequence similarity of Ymf39 proteins with each other, their nucleus-encoded functional counterparts, ATP4/ATP5F, from fungi and animals and α-proteobacterial ATP synthase b-subunits. This analysis provides clear evidence that ymf39 is an atpF homolog, while ATP4/ATP5F appears to be a highly diverged form of ymf39 that has migrated to the nucleus. We propose to designate ymf39 from now on atp4.     

Mutation of mitochondrial ATP8 gene improves hepatic energy status in a murine model of acute endotoxemic liver failure

AimsMitochondria not only generate and modulate bioenergy but also serve as biosensors for oxidative stress, and eventually become effector organelles for cell viability. Therefore, the implications of mitochondrial (dys)function in the development of multiple organ failure are profound. We investigated whether a mutation in the ATPase subunit-8 gene affects the course of endotoxemic acute liver failure.Main methodsC57BL/6J (ATP8 wild type) and C57BL/6J-mt^FVB/N (ATP8 mutant) mice were challenged with d-galactosamine (GalN) and Escherichia coli lipopolysaccharide (LPS) for induction of acute liver failure, and studied 6 h thereafter. Control mice received physiological saline only. Analysis included in vivo fluorescence microscopy of hepatic microcirculation and levels of hepatocellular apoptosis, hepatic adenosine nucleotides and oxidative stress. Additionally, survival rates were assessed.Key findingsInduction of endotoxemic liver failure provoked marked liver damage, which was coexistent with a drop of total adenosine nucleotide levels and increased oxidative stress. Of interest, oxidative stress was higher in the GalN/LPS challenged ATP8 mutants compared to wild types. Concomitantly, adenosine triphosphate (ATP) levels in livers of mice carrying the ATP8 mutation remained higher than those in wild type mice. As net result, ATP8 mutants showed lower transaminase release and a tendency to better survival rate upon GalN/LPS exposure compared to wild types.SignificanceOur findings demonstrate that mutation in the ATPase subunit-8 partially protects mice against endotoxemic stress, most probably due to better hepatic energy status despite elevated oxidative stress. Thus, modulating mitochondrial function to preserve bioenergetic status may be an effective strategy to protect against sepsis-induced multiorgan dysfunction.     

A comparison of the thermal stability and substrate binding constants of prolyl-tRNA synthetase from Phaseolus aureus and Delonix regia

Pro-tRNA synthetase from P. aureus and D. regia was protected against thermal denaturation by various substrates; the kinetics of this protection was investigated. The affinity of substrates for each synthetase was studied by a thermal inactivation technique. In the presence of ATP, Pro and several Pro-analogues were bound to each enzyme more efficiently than when ATP was absent. The efficiency of imino acid analogue binding, relative to that of Pro, was greater when ATP was absent. Pyrrolidine and 3-pyrroline were able to bind to the enzyme only in the presence of ATP. The ratio of the ATP/Pro binding constants for the Delonix enzyme was greater than that for the Phaseolus enzyme. Values for several thermodynamic parameters involved in substrate binding were determined for each synthetase. The results are discussed in relation to the order of substrate binding and the known differences in substrate specificity between the enzymes from P. aureus and D. regia.