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Comparative enzymology of the adenosine triphosphate sulphurylases from leaf tissue of selenium-accumulator and non-accumulator plants.

ABSTRACT: 1. ATP sulphurylases were partially purified (20-40-fold) from leaf tissue of Astragalus bisulcatus, Astragalus racemosus (selenium-accumulator species) and Astragalus hamosus and Astragalus sinicus (non-accumulator species). Activity was measured by sulphate-dependent PP(i)-ATP exchange. The enzymes were separated from pyrophosphatase and adenosine triphosphatase activities. The properties of the Astragalus ATP sulphurylases were similar to the spinach enzyme. 2. The ATP sulphurylases from both selenium-accumulator and non-accumulator species catalysed selenate-dependent PP(i)-ATP exchange; selenate competed with sulphate. The ratio of V(selenate)/V(sulphate) and K(m)(selenate)/K(m)(sulphate) was approximately the same for the enzyme from each species. 3. Sulphate-dependent PP(i)-ATP exchange was inhibited by ADP, chlorate and nitrate. The kinetics of the inhibition for each enzyme were consistent with an ordered reaction mechanism, in which ATP is the first substrate to react with the enzyme and PP(i) is the first product released. 4. Synthesis of adenosine 5'-[(35)S]sulphatophosphate from [(35)S]sulphate was demonstrated by coupling the Astragalus ATP sulphurylases with Mg(2+)-dependent pyrophosphatase; the reaction was inhibited by selenate. An analogous reaction using [(75)Se]selenate as substrate could not be demonstrated.

SUBMITTER: Shaw WH

PROVIDER: S-EPMC1166248 | biostudies-other | 1974 Apr

REPOSITORIES: biostudies-other

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The enzymology of adenosine triphosphate sulphurylase from spinach leaf tissue.

Project description:1. Sulphate-dependent PP(i)-ATP exchange, catalysed by purified spinach leaf ATP sulphurylase, was correlated with the concentration of MgATP(2-) and MgP(2)O(7) (2-); ATP sulphurylase activity was not correlated with the concentration of free Mg(2+). 2. Sulphate-dependent PP(i)-ATP exchange was independent of PP(i) concentration, but dependent on the concentration of ATP and sulphate. The rate of sulphate-dependent PP(i)-ATP exchange was quantitatively defined by the rate equation applicable to the initial rate of a bireactant sequential mechanism under steady-state conditions. 3. Chlorate, nitrate and ADP inhibited the exchange reaction. The inhibition by chlorate and nitrate was uncompetitive with respect to ATP and competitive with respect to sulphate. The inhibition by ADP was competitive with respect to ATP and non-competitive with respect to sulphate. 4. ATP sulphurylase catalysed the synthesis of [(32)P]ATP from [(32)P]PP(i) and adenosine 5'-sulphatophosphate in the absence of sulphate; some properties of the reaction are described. Enzyme activity was dependent on the concentration of PP(i) and adenosine 5'-sulphatophosphate. 5. The synthesis of ATP from PP(i) and adenosine 5'-sulphatophosphate was inhibited by sulphate and ATP. The inhibition by sulphate was non-competitive with respect to PP(i) and adenosine 5'-sulphatophosphate; the inhibition by ATP was competitive with respect to adenosine 5'-sulphatophosphate and non-competitive with respect to PP(i). It was concluded that the reaction catalysed by spinach leaf ATP sulphurylase was ordered; expressing the order in the forward direction, MgATP(2-) was the first product to react with the enzyme and MgP(2)O(7) (2-) was the first product released. 6. The expected exchange reaction between sulphate and adenosine 5'-sulphatophosphate could not be demonstrated.

| S-EPMC1166247 | biostudies-other

Purification, properties and substrate specificity of adenosine triphosphate sulphurylase from spinach leaf tissue.

Project description:1. ATP sulphurylase was purified up to 1000-fold from spinach leaf tissue. Activity was measured by sulphate-dependent [(32)P]PP(i)-ATP exchange. The enzyme was separated from Mg(2+)-requiring alkaline pyrophosphatase (which interferes with the PP(i)-ATP-exchange assay) and from other PP(i)-ATP-exchange activities. No ADP sulphurylase activity was detected. 2. Sulphate was the only form of inorganic sulphur that catalysed PP(i)-ATP exchange; K(m) (sulphate) was 3.1mm, K(m) (ATP) was 0.35mm and the pH optimum was 7.5-9.0. The enzyme was insensitive to thiol-group reagents and required either Mg(2+) or Co(2+) for activity. 3. The enzyme catalysed [(32)P]PP(i)-dATP exchange; K(m) (dATP) was 0.84mm and V (dATP) was 30% of V (ATP). Competition between ATP and dATP was demonstrated. 4. Selenate catalysed [(32)P]PP(i)-ATP exchange and competed with sulphate; K(m) (selenate) was 1.0mm and V (selenate) was 30% of V (sulphate). No AMP was formed with selenate as substrate. Molybdate did not catalyse PP(i)-ATP exchange, but AMP was formed. 5. Synthesis of adenosine 5'-[(35)S]sulphatophosphate was demonstrated by coupling purified ATP sulphurylase and Mg(2+)-dependent alkaline pyrophosphatase (also prepared from spinach) with [(35)S]sulphate and ATP as substrates; adenosine 5'-sulphatophosphate was not synthesized in the absence of pyrophosphatase. Some parameters of the coupled system are reported.

| S-EPMC1178578 | biostudies-other

Adenosine diphosphate sulphurylase activity in leaf tissue.

Project description:1. A new method is described for the assay of ADP sulphurylase. The method involves sulphate-dependent [(32)P]P(i)-ADP exchange; the method is simpler, more sensitive and more direct than the method involving adenosine 5'-sulphatophosphate-dependent uptake of P(i). 2. ADP sulphurylase activity was demonstrated in crude extracts of leaf tissue from a range of plants. Crude spinach extract catalysed the sulphate-dependent synthesis of [(32)P]ADP from [(32)P]P(i); spinach extracts did not catalyse sulphate-dependent AMP-P(i), ADP-PP(i) or ATP-P(i) exchange under standard assay conditions. ADP sulphurylase activity in spinach leaf tissue was associated with chloroplasts and was liberated by sonication. 3. Some elementary kinetics of crude spinach leaf and purified yeast ADP sulphurylases in the standard assay are described; addition of Ba(2+) was necessary to minimize endogenous P(i)-ADP exchange of the yeast enzyme and crude extracts of winter-grown spinach. 4. Spinach leaf ADP sulphurylase was activated by Ba(2+) and Ca(2+); Mg(2+) was ineffective. The yeast enzyme was also activated by Ba(2+). The activity of both enzymes decreased with increasing ionic strength. 5. Purified yeast and spinach leaf ADP sulphurylases were sensitive to thiol-group reagents and fluoride. The pH optimum was 8. ATP inhibited sulphate-dependent P(i)-ADP exchange. Neither selenate nor molybdate inhibited sulphate-dependent P(i)-ADP exchange and crude spinach extracts did not catalyse selenate-dependent P(i)-ADP exchange. 6. The presence of ADP sulphurylase activity jeopardizes the enzymic synthesis of adenosine 5'-sulphatophosphate from ATP and sulphate with purified ATP sulphurylase and pyrophosphatase.

| S-EPMC1177721 | biostudies-other

Transition State Analysis of Adenosine Triphosphate Phosphoribosyltransferase.

Project description:Adenosine triphosphate phosphoribosyltransferase (ATP-PRT) catalyzes the first step in histidine biosynthesis, a pathway essential to microorganisms and a validated target for antimicrobial drug design. The ATP-PRT enzyme catalyzes the reversible substitution reaction between phosphoribosyl pyrophosphate and ATP. The enzyme exists in two structurally distinct forms, a short- and a long-form enzyme. These forms share a catalytic core dimer but bear completely different allosteric domains and thus distinct quaternary assemblies. Understanding enzymatic transition states can provide essential information on the reaction mechanisms and insight into how differences in domain structure influence the reaction chemistry, as well as providing a template for inhibitor design. In this study, the transition state structures for ATP-PRT enzymes from Campylobacter jejuni and Mycobacterium tuberculosis (long-form enzymes) and from Lactococcus lactis (short-form) were determined and compared. Intrinsic kinetic isotope effects (KIEs) were obtained at reaction sensitive positions for the reverse reaction using phosphonoacetic acid, an alternative substrate to the natural substrate pyrophosphate. The experimental KIEs demonstrated mechanistic similarities between the three enzymes and provided experimental boundaries for quantum chemical calculations to characterize the transition states. Predicted transition state structures support a dissociative reaction mechanism with a DN*AN‡ transition state. Weak interactions from the incoming nucleophile and a fully dissociated ATP adenine are predicted regardless of the difference in overall structure and quaternary assembly. These studies establish that despite significant differences in the quaternary assembly and regulatory machinery between ATP-PRT enzymes from different sources, the reaction chemistry and catalytic mechanism are conserved.

| S-EPMC6693643 | biostudies-literature

Adenosine triphosphate stimulates Aquifex aeolicus MutL endonuclease activity.

Project description:BACKGROUND:Human PMS2 (hPMS2) homologues act to nick 5' and 3' to misincorporated nucleotides during mismatch repair in organisms that lack MutH. Mn(++) was previously found to stimulate the endonuclease activity of these homologues. ATP was required for the nicking activity of hPMS2 and yPMS1, but was reported to inhibit bacterial MutL proteins from Thermus thermophilus and Aquifex aeolicus that displayed homology to hPMS2. Mutational analysis has identified the DQHA(X)(2)E(X)(4)E motif present in the C-terminus of PMS2 homologues as important for endonuclease activity. METHODOLOGIES/PRINCIPAL FINDINGS:We examined the effect ATP had on the Mn(++) induced nicking of supercoiled pBR322 by full-length and mutant A. aeolicus MutL (Aae MutL) proteins. Assays were single time point, enzyme titration experiments or reaction time courses. The maximum velocity for MutL nicking was determined to be 1.6+/-0.08x10(-5) s(-1) and 4.2+/-0.3x10(-5) s(-1) in the absence and presence of ATP, respectively. AMPPNP stimulated the nicking activity to a similar extent as ATP. A truncated Aae MutL protein composed of only the C-terminal 123 amino acid residues was found to nick supercoiled DNA. Furthermore, mutations in the conserved C-terminal DQHA(X)(2)E(X)(4)E and CPHGRP motifs were shown to abolish Aae MutL endonuclease activity. CONCLUSIONS:ATP stimulated the Mn(++) induced endonuclease activity of Aae MutL. Experiments utilizing AMPPNP implied that the stimulation did not require ATP hydrolysis. A mutation in the DQHA(X)(2)E(X)(4)E motif of Aae MutL further supported the role of this region in endonclease activity. For the first time, to our knowledge, we demonstrate that changing the histidine residue in the conserved CPHGRP motif abolishes endonucleolytic activity of a hPMS2 homologue. Finally, the C-terminal 123 amino acid residues of Aae MutL were sufficient to display Mn(++) induced nicking activity.

| S-EPMC2744016 | biostudies-literature

Adenosine 5'-sulphatophosphate kinase activity in spinach leaf tissue.

Project description:1. An F(-)-insensitive 3'-nucleotidase was purified from spinach leaf tissue; the enzyme hydrolysed 3'-AMP, 3'-CMP and adenosine 3'-phosphate 5'-sulphatophosphate but not adenosine 5'-nucleotides nor PP(i). The pH optimum of the enzyme was 7.5; K(m) (3'-AMP) was approx. 0.8mm and K(m) (3'-CMP) was approx. 3.3mm. 3'-Nucleotidase activity was not associated with chloroplasts. Purified Mg(2+)-dependent pyrophosphatase, free from F(-)-insensitive 3'-nucleotidase, catalysed some hydrolysis of 3'-AMP; this activity was F(-)-sensitive. 2. Adenosine 5'-sulphatophosphate kinase activity was demonstrated in crude spinach extracts supplied with 3'-AMP by the synthesis of the sulphate ester of 2-naphthol in the presence of purified phenol sulphotransferase; purified ATP sulphurylase and pyrophosphatase were also added to synthesize adenosine 5'-sulphatophosphate. Adenosine 5'-sulphatophosphate kinase activity was associated with chloroplasts and was released by sonication. 3. Isolated chloroplasts synthesized adenosine 3'-phosphate 5'-sulphatophosphate from sulphate and ATP in the presence of a 3'-nucleotide; the formation of adenosine 5'-sulphatophosphate was negligible. In the absence of a 3'-nucleotide the synthesis of adenosine 3'-phosphate 5'-sulphatophosphate was negligible, but the formation of adenosine 5'-sulphatophosphate was readily detected. Some properties of the synthesis of adenosine 3'-phosphate 5'-sulphatophosphate by isolated chloroplasts are described. 4. Adenosine 3'-phosphate 5'-sulphatophosphate, synthesized by isolated chloroplasts, was characterized by specific enzyme methods, electrophoresis and i.r. spectrophotometry. 5. Isolated chloroplasts catalysed the incorporation of sulphur from sulphate into cystine/cysteine; the incorporation was enhanced by 3'-AMP and l-serine. It was concluded that adenosine 3'-phosphate 5'-sulphatophosphate is an intermediate in the incorporation of sulphur from sulphate into cystine/cysteine.

| S-EPMC1177844 | biostudies-other

Supplementation of exogenous adenosine 5'-triphosphate enhances mechanical properties of 3D cell-agarose constructs for cartilage tissue engineering.

Project description:Formation of tissue-engineered cartilage is greatly enhanced by mechanical stimulation. However, direct mechanical stimulation is not always a suitable method, and the utilization of mechanisms underlying mechanotransduction might allow for a highly effective and less aggressive alternate means of stimulation. In particular, the purinergic, adenosine 5'-triphosphate (ATP)-mediated signaling pathway is strongly implicated in mechanotransduction within the articular cartilage. We investigated the effects of transient and continuous exogenous ATP supplementation on mechanical properties of cartilaginous constructs engineered using bovine chondrocytes and human mesenchymal stem cells (hMSCs) encapsulated in an agarose hydrogel. For both cell types, we have observed significant increases in equilibrium and dynamic compressive moduli after transient ATP treatment applied in the fourth week of cultivation. Continuous ATP treatment over 4 weeks of culture only slightly improved the mechanical properties of the constructs, without major changes in the total glycosaminoglycan (GAG) and collagen content. Structure-function analyses showed that transiently ATP-treated constructs, and in particular those based on hMSCs, had the highest level of correlation between compositional and mechanical properties. Transiently treated groups showed intense staining of the territorial matrix for GAGs and collagen type II. These results indicate that transient ATP treatment can improve functional mechanical properties of cartilaginous constructs based on chondrogenic cells and agarose hydrogels, possibly by improving the structural organization of the bulk phase and territorial extracellular matrix (ECM), that is, by increasing correlation slopes between the content of the ECM components (GAG, collagen) and mechanical properties of the construct.

| S-EPMC3761430 | biostudies-literature

Synthesis of a Biologically Important Adenosine Triphosphate Analogue, ApppD.

Project description:The chemical synthesis of a adenosine triphosphate analogue, 1-adenosin-5'-yl 3-(3-methylbut-2-enyl) triphosphoric acid diester (ApppD), is described. ApppD is known to be an active metabolite of the mevalonate pathway in the human body like its structural isomer isopentenyl ester of ATP (ApppI). Very recently, ApppI has been found to possess novel function(s); now it will also be possible to examine the effects of ApppD more precisely because it can be synthesized in reasonable amounts. 1-Adenosin-5'-yl 3-(3-methylbut-2-enyl) diphosphoric acid diester (AppD; a adenosine diphosphate analogue) was also isolated from the synthesis mixture. Both ApppD and AppD were characterized by 1H, 13C, 31P NMR and mass spectrometry methods.

| S-EPMC6044877 | biostudies-literature

The apparent stimulation of proteolysis by adenosine triphosphate in tissue homogenates.

Project description:Reports that ATP promotes proteolysis in tissue homogenates were reinvestigated. Although ATP increased production of material reacting with ninhydrin or Folin-phenol reagent, ATP did not stimulate protein degradation in such extracts. AMP and adenosine acted similarly to ATP. Deamination of the added nucleotides to produce ammonia caused the increase in ninhydrin-positive material.

| S-EPMC1177883 | biostudies-other

Adenosine Triphosphate-Sensitive Potassium Currents in Heart Disease and Cardioprotection.

Project description:The subunit makeup of the family of adenosine triphosphate-sensitive potassium channel (KATP) channels is more complex and labile than thought. The growing association of Kir6.1 and SUR2 variants with specific cardiovascular electrical and contractile derangements and the clear association with Cantu syndrome establish the importance of appropriate activity in normal function of the heart and vasculature. Further studies of such patients will reveal new mutations in KATP subunits and perhaps in proteins that regulate KATP synthesis, trafficking, or location, all of which may ultimately benefit therapeutically from the unique pharmacology of KATP channels.

| S-EPMC4894346 | biostudies-literature

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