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1.
Although succinate thiokinase from mammalian sources has hitherto been described as showing substrate specificity for guanine nucleotide, a range of mammalian tissues has here been found to display succinate thiokinase activity with both guanine and adenine nucleotides as substrates. Evidence is presented for the existence of two distinct succinate thiokinases and this is confirmed by their separation by affinity chromatography. Each enzyme is specific for one nucleotide and is inhibited by the non-substrate nucleotide. The physiological roles of the two enzymes is yet to be established. 相似文献
2.
Two distinct succinate thiokinases in both bloodstream and procyclic forms of Trypanosoma brucei 总被引:1,自引:0,他引:1
T M Jenkins R Eisenthal P D Weitzman 《Biochemical and biophysical research communications》1988,151(1):257-261
Two succinate thiokinase activities specific for either adenine or guanine nucleotides have been found in Trypanosoma brucei. Key glycolytic and citric acid cycle enzymes were measured to show repression of glycolysis and derepression of the citric acid cycle in the procyclic form, relative to the bloodstream form. A marked rise in adenine-linked succinate thiokinase activity accompanied a rise in activity of citric acid cycle enzymes. However, guanine-linked succinate thiokinase was found to increase only slightly in activity. These results implicate the adenine-linked enzyme as an essential component of the citric acid cycle, whereas the guanine-linked enzyme appears to be under separate control. This communication also reports for the first time the occurrence of citrate synthase activity in the bloodstream (long slender) form of T. brucei. 相似文献
3.
The nucleotide specificity of succinate thiokinases, isolated from Escherichia coli, Aerobacter aerogenes, and Pseudomonas citronellolis, was determined and found to be nonspecific for adenine and 6-oxopurine nucleotides, guanine, and hypoxanthine. The enzyme from Herellae vaginicola was specific for the 6-oxopurine nucleotides. Succinate thiokinases from E. coli, A. aerogenes, and P. citronellolis also demonstrated purine nucleoside diphosphokinase activity (P-NDPK), which was 4, 9, and 40%, respectively, of the succinate thiokinase activity. P-NDPK activity was slightly stimulated by coenzyme A (CoA) and slightly inhibited by succinate; in the presence of both CoA and succinate, P-NDPK activity increased three-, three-, and sevenfold for the E. coli, A. aerogenes, and P. citronellolis enzymes, respectively. Isoelectric focusing demonstrated multiple forms of each enzyme, and the molecular weights of the A. aerogenes, P. citronellolis, and H. vaginicola enzymes were approximately 155,000. 相似文献
4.
The discovery of two distinct succinate thiokinases in mammalian tissues, one (G-STK) specific for GDP/GTP and the other (A-STK) for ADP/ATP, poses the question of their differential metabolic roles. Evidence has suggested that the A-STK functions in the citric acid cycle in the direction of succinyl-CoA breakdown (and ATP formation) whereas one role of the G-STK appears to be the re-cycling of succinate to succinyl-CoA (at the expense of GTP) for the purpose of ketone body activation. A third metabolic participation of succinyl-CoA is in haem biosynthesis. This communication shows that in chemically induced hepatic porphyria, when the demand for succinyl-CoA is increased, it is the level of G-STK only which is elevated, that of A-STK being unaffected. The results implicate G-STK in the provision of succinyl-CoA for haem biosynthesis, a conclusion which is further supported by the observation of a high G-STK/A-STK ratio in bone marrow. 相似文献
5.
Acetate:succinate CoA-transferase in the hydrogenosomes of Trichomonas vaginalis: identification and characterization 总被引:1,自引:0,他引:1
van Grinsven KW Rosnowsky S van Weelden SW Pütz S van der Giezen M Martin W van Hellemond JJ Tielens AG Henze K 《The Journal of biological chemistry》2008,283(3):1411-1418
Acetate:succinate CoA-transferases (ASCT) are acetate-producing enzymes in hydrogenosomes, anaerobically functioning mitochondria and in the aerobically functioning mitochondria of trypanosomatids. Although acetate is produced in the hydrogenosomes of a number of anaerobic microbial eukaryotes such as Trichomonas vaginalis, no acetate producing enzyme has ever been identified in these organelles. Acetate production is the last unidentified enzymatic reaction of hydrogenosomal carbohydrate metabolism. We identified a gene encoding an enzyme for acetate production in the genome of the hydrogenosome-containing protozoan parasite T. vaginalis. This gene shows high similarity to Saccharomyces cerevisiae acetyl-CoA hydrolase and Clostridium kluyveri succinyl-CoA:CoA-transferase. Here we demonstrate that this protein is expressed and is present in the hydrogenosomes where it functions as the T. vaginalis acetate:succinate CoA-transferase (TvASCT). Heterologous expression of TvASCT in CHO cells resulted in the expression of an active ASCT. Furthermore, homologous overexpression of the TvASCT gene in T. vaginalis resulted in an equivalent increase in ASCT activity. It was shown that the CoA transferase activity is succinate-dependent. These results demonstrate that this acetyl-CoA hydrolase/transferase homolog functions as the hydrogenosomal ASCT of T. vaginalis. This is the first hydrogenosomal acetate-producing enzyme to be identified. Interestingly, TvASCT does not share any similarity with the mitochondrial ASCT from Trypanosoma brucei, the only other eukaryotic succinate-dependent acetyl-CoA-transferase identified so far. The trichomonad enzyme clearly belongs to a distinct class of acetate:succinate CoA-transferases. Apparently, two completely different enzymes for succinate-dependent acetate production have evolved independently in ATP-generating organelles. 相似文献
6.
Hydrogenosomal succinate thiokinase in Tritrichomonas foetus and Trichomonas vaginalis 总被引:3,自引:0,他引:3
T M Jenkins T E Gorrell M Müller P D Weitzman 《Biochemical and biophysical research communications》1991,179(2):892-896
Succinate thiokinase displays a diversity of nucleotide specificity and molecular size throughout Nature. Eukaryotes and Gram-positive bacteria possess distinct 'small' (dimeric) thiokinase enzymes which are specific for adenine (ADP) or guanine (GDP) nucleotides, whereas Gram-negative bacteria contain a single 'large' (tetrameric) enzyme which utilizes both nucleotides. Succinate thiokinase activities, both ADP- and GDP-dependent, were shown to be hydrogenosomal in Tritrichomonas foetus and Trichomonas vaginalis. Surprisingly, the 'small' enzyme was found in T. foetus whereas T. vaginalis contained a 'large' enzyme. 相似文献
7.
The kinetic properties of acetate kinase from Veillonella alcalescens were investigated. In the presence of high concentrations of nucleotide both forward and reverse reactions were observed. In the presence of succinate the degree of cooperativity between subunits of the homodimer decreased, i.e. the Hill coefficient, n, decreased from 2.5 to 1.4 for acetyl phosphate in the presence of succinate. At low substrate concentrations hyperbolic kinetic data were observed with succinate. We have proposed a modified version of the concerted symmetry model to describe the kinetics observed with this enzyme. The primary differentiating feature of the proposed model is the requirement for activator ligand binding for catalysis. In the absence of succinate, the substrate (acetate or acetyl phosphate) also functions as an activating ligand. 相似文献
8.
R H Dunstan F R Whatley W Greenaway 《Proceedings of the Royal Society of London. Series B, Containing papers of a Biological character. Royal Society (Great Britain)》1988,233(1270):1-15
The biosynthesis in vivo of a number of amino acids, sugars, and purines in Paracoccus denitrificans grown on either [2,3-13C]succinate or [1,4-13C]succinate was investigated by using gas chromatography-mass spectrometry. The distribution of label in the TCA-cycle-related amino acids indicated that carbon intermediates of energy metabolism were utilized as precursors for the biosynthesis of these amino acids in vivo. The biosynthesis of glycine, serine, phenylalanine and glycerol from labelled succinate in vivo were consistent with phosphoenol pyruvate as an intermediate. A mechanism for the formation of C4, C5 and C6 sugars without the use of fructose-1,6-bisphosphate aldolase (which has not been detected in P. denitrificans) is proposed. The 13C-enrichments of ribose in the bacterium indicate that there are at least three routes of ribose biosynthesis operating during growth on labelled succinate. The probability distribution of labelled purine molecules was successfully predicted for adenine, guanine and adenosine, thus confirming their generally accepted route of biosynthesis in vivo. 相似文献
9.
Mitochondria from tomato fruit (Lycopersicon esculentum Mill.) exhibited a respiratory control ratio of 2.5 and an ADP:O ratio of 1.3 for succinate oxidation for 24 hours after isolation. They also showed a delay in response to the first addition of ADP. The addition of ATP and ADP before succinate eliminated the delayed response as did chelation of endogenous cations with ethylenediaminetetraacetic acid. The addition of ATP after succinate resulted in a longer delay in response than that obtained with ADP. Exogenous oxaloacetate in low concentration inhibited respiration in states 3 and 4 with succinate and resulted in delayed response to ADP. The function of adenine nucleotide during the delay in response may be to promote the metabolism of oxaloacetate or to decrease the affinity of oxaloacetate to its site of inhibition. 相似文献
10.
Purine metabolism in Toxoplasma gondii 总被引:11,自引:0,他引:11
We have studied the incorporation and interconversion of purines into nucleotides by freshly isolated Toxoplasma gondii. They did not synthesize nucleotides from formate, glycine, or serine. The purine bases hypoxanthine, xanthine, guanine, and adenine were incorporated at 9.2, 6.2, 5.1, and 4.3 pmol/10(7) cells/h, respectively. The purine nucleosides adenosine, inosine, guanosine, and xanthosine were incorporated at 110, 9.0, 2.7, and 0.3 pmol/10(7) cells/h, respectively. Guanine, xanthine, and their respective nucleosides labeled only guanine nucleotides. Inosine, hypoxanthine, and adenine labeled both adenine and guanine nucleotide pools at nearly equal ratios. Adenosine kinase was greater than 10-fold more active than the next most active enzyme in vitro. This is consistent with the metabolic data in vivo. No other nucleoside kinase or phosphotransferase activities were found. Phosphorylase activities were detected for guanosine and inosine; no other cleavage activities were detected. Deaminases were found for adenine and guanine. Phosphoribosyltransferase activities were detected for all four purine nucleobases. Interconversion occurs only in the direction of adenine to guanine nucleotides. 相似文献
11.
Mutations in sdh (succinate dehydrogenase genes) alter the thiamine requirement of Salmonella typhimurium.
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Mutants lacking the first enzyme in de novo purine synthesis (PurF) can synthesize thiamine if increased levels of pantothenate are present in the culture medium (J. L. Enos-Berlage and D. M. Downs, J. Bacteriol. 178:1476-1479, 1996). Derivatives of purF mutants that no longer required pantothenate for thiamine-independent growth were isolated. Analysis of these mutants demonstrated that they were defective in succinate dehydrogenase (Sdh), an enzyme of the tricarboxylic acid cycle. Results of phenotypic analyses suggested that a defect in Sdh decreased the thiamine requirement of Salmonella typhimurium. This reduced requirement correlated with levels of succinyl-coenzyme A (succinyl-CoA), which is synthesized in a thiamine pyrophosphate-dependent reaction. The effect of succinyl-CoA on thiamine metabolism was distinct from the role of pantothenate in thiamine synthesis. 相似文献
12.
David A. Morris 《Physiologia plantarum》1981,52(2):315-319
The pattern of incorporation of label into the nucleotides of axillary bud ribonucleic acid was investigated in Pisum sativum L. cv. Meteor following the application of N 6 [8-I4 C]furfuryladenine or of [8-14 C]adenine to the root system of decapitated plants and to cultured excised buds. When N 6 [8-14 C]furifaryladenine was applied to the root system label was confined to the guanine nucleotide moiety of the axillary bud ribonucleic acid; label from [8-14 C]adenine was incorporated preferentially into adenine nucleotide in the molar ratio adenine nucleotide/guanine nucleotide = 3.23. When isolated buds were incubated in media containing [8-14 C]adenine or N 6 [8-14 C]furfuryladenine, label was incorporated into both purine moieties of the ribonucleic acid. However, the relative incorporation into the guanine nucleotide fraction was considerably greater for N 6 [8-I4 C]furfuryladenine (adenine nucleotide/guanine nucleotide = 2.23) than for [8-14 C]adenine (ratio = 4.67).
It was concluded that the pattern of metabolism of adenine to guanine and its incorporation into the guanine nucleotide moiety of pea axillary bud ribonucleic acid, is influenced by the presence of a substitution in the N6 position of the adenine base. 相似文献
It was concluded that the pattern of metabolism of adenine to guanine and its incorporation into the guanine nucleotide moiety of pea axillary bud ribonucleic acid, is influenced by the presence of a substitution in the N
13.
Lee CS 《Molecules and cells》2000,10(6):723-727
The mechanisms of anticancer activity of 2,5-diaziridinyl-1,4-benzoquinone (DZQ) are believed to involve the alkylation of guanine and adenine bases. In this study, it has been investigated whether bacterial and mammalian 3-methyladenine-DNA glycosylases are able to excise DZQ-DNA adduct with a differential substrate specificity. DZQ-induced DNA adduct was first formed in the radiolabeled restriction enzyme DNA fragment, and excision of the DNA adduct was analyzed following treatment with homogeneous 3-methyladenine-DNA glycosylase from E. coli, rat, and human, respectively. Abasic sites generated by DNA glycosylases were cleaved by the associated lyase activity of the E. coli formamidopyrimidine-DNA glycosylase. Resolution of cleaved DNA on a sequencing gel with Maxam-Gilbert sequencing reactions showed that DZQ-induced adenine and guanine adducts were very good substrates for bacterial and mammalian enzymes. The E. coli enzyme excises DZQ-induced adenine and guanine adducts with similar efficiency. The rat and human enzymes, however, excise the adenine adduct more efficiently than the guanine adduct. These results suggest that the 3-methyladenine-DNA glycosylases from different origins have differential substrate specificity to release DZQ-DNA lesions. The use of 3-methyladenine-DNA glycosylase incision analysis could possibly be applied to quantify a variety of DNA adducts at the nucleotide level. 相似文献
14.
The relative abilities of ATP and GTP to support succinyl-CoA synthesis by mitochondrial matrix fractions prepared from rabbit heart and liver mitoplasts were investigated. The activity supported by ATP in rabbit heart preparations was less than 15% of that obtained with GTP, while no ATP-supported activity was observed in rabbit liver preparations. However, the addition of 30 micromolar GDP to matrix fractions from either heart or liver stimulated the ATP-supported activity to 40% of that observed with GTP, and the further addition of bovine liver nucleoside diphosphate kinase in the presence of 8 microM added GDP increased the activity to near that observed with GTP. The specific activity of nucleoside diphosphate kinase assayed directly in mitochondrial matrix from heart was about 15% of the specific activity of ATP-supported succinate thiokinase induced upon adding GDP. Evidence for a complex between nucleoside diphosphate kinase and succinate thiokinase in mitochondrial matrix from rabbit heart was obtained by glycerol density gradient centrifugation. It is proposed that binding of nucleoside diphosphate kinase to succinate thiokinase activates the former enzyme, accounts for the ATP-supported succinyl-CoA synthetase activity observed, and is involved in the channeling of high energy phosphate from GTP produced in the Krebs cycle to the ATP pool. 相似文献
15.
Succinyl-CoA synthetase catalyzes the reversible reaction succinyl-CoA + NDP + P(i) <--> succinate + CoA + NTP (N denoting adenosine or guanosine). The enzyme consists of two different subunits, designated alpha and beta. During the reaction, a histidine residue of the alpha-subunit is transiently phosphorylated. This histidine residue interacts with Glu 208 alpha at site I in the structures of phosphorylated and dephosphorylated Escherichia coli SCS. We postulated that Glu 197 beta, a residue in the nucleotide-binding domain, would provide similar stabilization of the histidine residue during the actual phosphorylation/dephosphorylation by nucleotide at site II. In this work, these two glutamate residues have been mutated individually to aspartate or glutamine. Glu 197 beta has been additionally mutated to alanine. The mutant proteins were tested for their ability to be phosphorylated in the forward or reverse direction. The aspartate mutant proteins can be phosphorylated in either direction, while the E208 alpha Q mutant protein can only be phosphorylated by NTP, and the E197 beta Q mutant protein can only be phosphorylated by succinyl-CoA and P(i). These results demonstrate that the length of the side chain at these positions is not critical, but that the charge is. Most significantly, the E197 beta A mutant protein could not be phosphorylated in either direction. Its crystal structure shows large differences from the wild-type enzyme in the conformation of two residues of the alpha-subunit, Cys 123 alpha-Pro 124 alpha. We postulate that in this conformation, the protein cannot productively bind succinyl-CoA for phosphorylation via succinyl-CoA and P(i). 相似文献
16.
A late-log-phase culture of an Escherichia coli nadB pncA double mutant took up 6-[7-14C]aminonicotinic acid and excreted 6-[14C]aminonicotinamide. This mutant also accumulated intracellularly several radioactive compounds which have been tentatively identified as 6-amino analogs of compounds in the pyridine nucleotide cycle. It is concluded that 6-aminonicotinamide and 6-aminonicotinic acid probably exert at least a portion of their bacteriostatic effects by being metabolized, by the enzymes of the pyridine nucleotide cycle, to 6-aminonicotinamide adenine dinucleotide and 6-aminonicotinamide adenine dinucleotide phosphate. These compounds are not electron acceptors and are known inhibitors of some pyridine nucleotide-linked dehydrogenases. 相似文献
17.
Chemical modification of Escherichia coli succinyl-CoA synthetase with the adenine nucleotide analogue 5''-p-fluorosulphonylbenzoyladenosine.
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Escherichia coli succinyl-CoA synthetase (EC 6.2.1.5) was irreversibly inactivated on incubation with the adenine nucleotide analogue 5'-p-fluorosulphonylbenzoyladenosine (5'-FSBA). Optimal inactivation by 5'-FSBA took place in 40% (v/v) dimethylformamide. ATP and ADP protected the enzyme against inactivation by 5'-FSBA, whereas desulpho-CoA, an analogue of CoA, did not. Inactivation of succinyl-CoA synthetase by 5'-FSBA resulted in total loss of almost four thiol groups per alpha beta-dimer, of which two groups appeared to be essential for catalytic activity. 5'-FSBA at the first instance appeared to interact non-specifically with non-essential thiol groups, followed by a more specific reaction with essential thiol groups in the ATP(ADP)-binding region. Plots of the data according to the method of Tsou [(1962) Sci. Sin. 11, 1535-1558] revealed that, of the two slower-reacting thiol groups, only one was essential for catalytic activity. When succinyl-CoA synthetase that had been totally inactivated by 5'-FSBA was unfolded in acidic urea and then refolded in the presence of 100 mM-dithiothreitol, 85% of the activity, in comparison with the appropriate control, was restored. These data are interpreted to indicate that inactivation of succinyl-CoA synthetase by 5'-FSBA involves the formation of a disulphide bond between two cysteine residues. Disulphide bond formation likely proceeds via a thiosulphonate intermediate between 5'-p-sulphonylbenzoyladenosine and one of the reactive thiol groups of the enzyme. 相似文献
18.
Chong-Soon Lee 《Molecules and cells》2001,10(6):723-727
The mechanisms of anticancer activity of 2,5-diaziridinyl-1,4-benzoquinone (DZQ) are believed to involve the alkylation of guanine and adenine bases. In this study, it has been investigated whether bacterial and mammalian 3-methyladenine-DNA glycosylases are able to excise DZQ-DNA adduct with a differential substrate specificity. DZQ-induced DNA adduct was first formed in the radiolabeled restriction enzyme DNA fragment, and excision of the DNA adduct was analyzed following treatment with homogeneous 3-methyladenine-DNA glycosylase from E. coli, rat, and human, respectively. Abasic sites generated by DNA glycosylases were cleaved by the associated lyase activity of the E. coli formami-dopyrimidine-DNA glycosylase. Resolution of cleaved DNA on a sequencing gel with Maxam-Gilbert sequencing reactions showed that DZQ-induced adenine and guanine adducts were very good substrates for bacterial and mammalian enzymes. The E. coli enzyme excises DZQ-induced adenine and guanine adducts with similar efficiency. The rat and human enzymes, however, excise the adenine adduct more efficiently than the guanine adduct. These results suggest that the 3-methyladenine-DNA glycosylases from different origins have differential substrate specificity to release DZQ-DNA lesions. The use of 3-methyladenine-DNA glycosylase incision analysis could possibly be applied to quantify a variety of DNA adducts at the nucleotide level. 相似文献
19.
Adenosine triphosphate-linked control of Pseudomonas aeruginosa glucose-6-phosphate dehydrogenase 总被引:31,自引:22,他引:9
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Extracts of Pseudomonas aeruginosa (ATCC 7700) cells grown on glucose, gluconate, or glycerol had enzyme activities related to the Entner-Doudoroff pathway. These activities were present in no more than trace amounts when the bacteria were grown on succinate. Fructose-1,6-diphosphate aldolase could not be detected in extracts of the bacteria grown on any of the above carbon sources. Therefore, it appears that P. aeruginosa degrades glucose via an inducible Entner-Doudoroff pathway. The apparent absence of fructose-1,6-diphosphate aldolase in cells growing on succinate suggests that the bacteria can form hexose and pentose phosphates from succinate by an alternate route. d-Glucose-6-phosphate dehydrogenase, a branch-point enzyme of the Entner-Doudoroff pathway, was purified 50-fold from glucose-grown cells. Its molecular weight, estimated by sucrose density gradient centrifugation, was found to be approximately 190,000. The enzyme was strongly inhibited by adenosine triphosphate, guanosine triphosphate, and deoxyguanosine triphosphate, which decreased the apparent binding of glucose-6-phosphate to the enzyme. It is suggested that adenine nucleotide-linked control of glucose-6-phosphate dehydrogenase may regulate the overall catabolism of hexose phosphates and prevent their wasteful degradation under certain conditions requiring gluconeogenesis. 相似文献
20.
Purification and Properties of Succinyl-CoA:3-Oxo-Acid CoA-Transferase from Rat Brain 总被引:1,自引:0,他引:1
Abstract: Rat brain succinyl-CoA:3-oxo-acid CoA-transferase (3-Oxo-acid CoA-transferase, EC 2.8.3.5), the first committed enzyme in the oxidation of ketone bodies in mitochondria, was purified to apparent homogeneity as judged by polyacrylamide gel electrophoresis. The enzyme has an apparent molecular weight of 90,000 as determined by (3-150 Sephadex chromatography, and an apparent subunit molecular weight of 53,000 as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The specific activity of the purified enzyme was approximately 161 μmol/min/mg of protein. Initial velocity studies of the forward reaction (acetoacetate → acetoacetyl-CoA) are consistent with a "ping pong" mechanism. Substrate inhibition appears above approximately 1 m M acetoacetate. Apparent Km , values were 70 μM for acetoacetate and 156 μ M for succinyl-CoA (the forward reaction), and 59 μ M for acetoacetyl-CoA and 25 m M for succinate (the reverse reaction). These values are markedly different from those reported for this enzyme from pig heart. 相似文献