Thiol groups of Escherichia coli citrate synthase and their influence on activity and regulation.

The modification of Escherichia coli citrate synthase (citrate oxaloacetatelyase(pro-3S-CH2.COO- leads to acetyl-CoA, EC 4.1.3.7) with 5,5'-dithiobis-(2-nitrobenzoic acid) has been investigated. (1) In low ionic strength (20 mM Tris.HCl, pH 8.0): (A) Eight thiol groups per tetramer of the native enzyme reacted with Nbs2. (b) Two of the eight accessible thiols were modified rapidly with the loss of 26% enzyme activity but with no change in the NADH inhibition. The remaining six were modified more slowly, resulting in a further 60% loss of activity and complete densensitization to NADH. (c) The 2nd-order rate constant for the modification of the rapidly reacting thiols is 2.5.10(4) M-1.min-1. At the reagent concentrations used (0.1 to 0.2 mM) the modification of the six thiols in the slow kinetic set appeared to be 1st-order; at 0.1 mM dithionitrobenzoic acid their rate of modification was approximately 30 times slower than the thiols in the fast kinetic set. (2) In high ionic strength (20 mM Tris.HCl, pH 8.0, 0.1 M KCl): (a) Four thiol groups were modified in a single kinetic set and it appeared that these thiols are four of the six slowly modified in the absence of KCl. (b) The modification resulted in 70% loss of enzyme activity and complete loss of NADH inhibition. (3) From the kinetic analysis it is proposed that the four thiol groups accessible to dithionitrobenzoic acid in the absence and presence of 0.1 M KCl are those involved in the response of NADH. Modification of any one of these four groups produced no reduction in the inhibition; instead, loss of NADH sensitivity was coincident with the appearance of tetrameric protein possessing three substituted thiols, whereas enzyme with one or two modified groups was still fully inhibited by NADH.

Physiological roles of animal succinate thiokinases. Specific association of the guanine nucleotide-linked enzyme with haem biosynthesis.

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.

Distinct physiological roles of animal succinate thiokinases. Association of guanine nucleotide-linked succinate thiokinase with ketone body utilization.

Two distinct succinate thiokinases have recently been shown to exist in animal tissues, one specific for guanine nucleotide and the other for adenine nucleotide. Their physiological roles have here been investigated by comparing the levels of the two enzymes in liver and brain of normal and diabetic rats. A marked rise in the level of brain guanine nucleotide-linked succinate thiokinase in the diabetic condition is consistent with an enhanced utilization of ketone bodies and hence with the associated elevated demand for succinyl-CoA for the activation of acetoacetate. Taken together with the reported mitochondrial values of the ATP/ADP and GTP/GDP ratios, the results are interpreted to indicate that the adenine nucleotide-linked enzyme functions as a component of the citric acid cycle whereas the guanine nucleotide-linked enzyme functions in the opposite metabolic direction to produce succinyl-CoA from succinate.

Organization of citric acid cycle enzymes into a multienzyme cluster.

The possibility that some of the enzymes of the citric acid cycle may be loosely associated into a multienzyme cluster has been investigated using extracts prepared by gentle disruption of cells. Gel filtration and sucrose density gradient centrifugation have shown that five sequential enzymes of the cycle specifically associate into a cluster: fumarase, malate dehydrogenase, citrate synthase, aconitase and isocitrate dehydrogenase. Ultrasonication destroys the abilities of the enzymes to associate. The cluster could catalyse the sequence of reactions leading from fumarate to oxoglutarate and has been found in extracts of several bacterial species as well as rat liver mitochondria.

Proteolysis of acinetobacter citrate synthase by subtilisin.

Citrate synthase from Acinetobacter calcoaceticus was subjected to proteolysis with subtilisin. Although the enzyme proved relatively resistant to inactivation by this treatment, SDS-polyacrylamide gel electrophoresis clearly revealed breakdown of the citrate synthase to smaller fragments. The regulatory responses of the native enzyme to inhibition by NADH and re-activation by AMP were retained on proteolysis, indicating that the fragments bind tightly to each other and preserve the overall cooperative molecular interactions.

Reversible effects of cross-linking on the regulatory cooperativity of Acinetobacter citrate synthase.

Citrate synthase was purified from Acinetobacter calcoaceticus and treated with the cleavable cross-linking reagent dithiobis(succinimidyl propionate). Cross-linking of the enzyme resulted in the abolition of the sigmoidal responses to inhibition by NADH and re-activation by AMP displayed by the native enzyme. Inhibition and re-activation were still observed but without any cooperativity. Cleavage of the disulphide bonds in the cross-links by treatment with dithiothreitol restored the sigmoidal characteristics of both inhibition and re-activation.

Occurrence of two distinct succinate thiokinases in animal tissues.

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.

Modulation of isocitrate dehydrogenase activity in Acinetobacter calcoaceticus by acetate.

The addition of acetate to a culture of Acinetobacter calcoaceticus grown in medium containing limiting succinate as the sole carbon and energy source leads to an increase in the specific activity of isocitrate dehydrogenase. This is in contrast to similar studies with several other microorganisms in which acetate induces an ATP-dependent phosphorylation and concomitant decrease in the specific activity of this enzyme.

Separation of isoenzymes of citrate synthase and isocitrate dehydrogenase by fast protein liquid chromatography.

Fast protein liquid chromatography (FPLC) has been shown to be a rapid and effective method of separating isoenzymes of citrate synthase and isocitrate dehydrogenase in extracts of Pseudomonas aeruginosa and Acinetobacter calcoaceticus. The advantages of FPLC over conventional methods of fractionation are discussed and it is suggested that this may be a valuable and more general technique for isoenzyme resolution.

Patterns of diversity of citric acid cycle enzymes.

The citric acid cycle performs a dual role in cell metabolism, acting as a source of both 'energy' and biosynthetic starting materials. The widespread occurrence of the cycle throughout Nature is an excellent example of the unity of biochemistry, but closer examination reveals that there is considerable diversity in the citric acid cycle of different organisms with respect to metabolic role, molecular enzymology and mode of regulation. Two enzymes of the cycle--citrate synthase and succinate thiokinase--have been found to exhibit particularly striking patterns of diversity in structure and catalytic and regulatory function. Some of these patterns show a correlation with the taxonomic groupings of the organisms and with their physiological characteristics. Comparative enzyme studies have a contribution to make to an ultimate understanding of the cycle and its cellular operation, and there are substantial benefits to be gained from interactive studies on both prokaryotic and eukaryotic systems.

GABA binding to receptor sites in locust supraoesophageal ganglia.

The binding of 4-amino-n-[2,3-3H]butyric acid (GABA) to receptor sites in the supraoesophageal ganglia of the locust Schistocerca gregaria is reported. Binding is saturable with a Kd of 30 nM and a Bmax of 150 fmol/mg protein. Binding is sodium-independent with a pH optimum of 6.8 and the pharmacological properties of the site suggest a receptor rather than an uptake or transport protein. The assay is being utilised in a comparative study of the binding sites of the GABA receptor and the enzyme 4-aminobutyrate: 2-oxoglutarate amino-transferase (EC 2.6.1.19, GABA-T). GABA binds to at least 4 proteins in the nervous system of vertebrates: the GABAA and GABAB receptors, GABA-T the enzyme involved in the GABA shunt, and the GABA transport system. In the invertebrates the status of these GABA-binding proteins is less well established. There are reports of a GABA receptor complex resembling the GABAA receptor; GABA-T activity has been reported and we have recently purified the enzyme from locust ganglia; it is assumed that GABA uptake systems are present in invertebrates. Proteins with different functions which specifically bind the same ligand are interesting from an evolutionary point of view. Are they distinct gene products or is the sub-unit of the receptor which binds GABA an enzyme which has lost the ability to bind pyroxidal phosphate? Do either receptor or enzyme differ significantly from their mammalian counterparts?