Non-equivalency of active centers of alpha-ketoglutarate dehydrogenase detected by modification of histidine residues

Using selective chemical modification of histidine residues of the alpha-ketoglutarate dehydrogenase component within the alpha-ketoglutarate dehydrogenase complex, the existence of interconvertible forms of the enzyme was demonstrated. These forms are distinguished by kinetics of inactivation caused by diethylpyrocarbonate. The interconversion of the enzyme forms involves alpha-ketoglutarate. Studies on substrate effects on the inactivation kinetics of individual enzyme forms revealed the non-equivalency of the enzyme active centers within the dimeric molecule of the alpha-ketoglutarate dehydrogenase component. The accessibility of an essential histidine residue in the active center of a neighbouring substrate-free monomer to the modifier increases as a result of interaction of the enzyme active centers during alpha-ketoglutarate binding by one of the subunits. The non-equivalency of the active centers manifests itself in different rates of interaction and in the unequal stability of binding of alpha-ketoglutarate to the alternate sites of the dimer. It is assumed that the biphasic kinetics of inactivation of pigeon breast muscle alpha-ketoglutarate dehydrogenase is due to tight binding of alpha-ketoglutarate in one of active centers of the enzyme dimeric molecule.     

Genetics of the alpha-ketoglutarate dehydrogenase complex of Bacillus subtilis.

The enzymatic defects in a number of Bacillus subtilis mutants of the alpha-ketoglutarate dehydrogenase complex lacking activity have been investigated. Mutants in the citK locus, as well as a series of deletions of unknown length covering the citK locus, are deficient in E1 of the complex, alpha-ketoglutarate dehydrogenase, but have normal activities of E2, dehydrolipoyl transsuccinylase, and E3, lipoamide dehydrogenase. The citK mutants and the citL22 mutant show in vitro complementation of alpha-ketoglutarate dehydrogenase complex activity. The citL22 mutant is severely deficient in lipoamide dehydrogenase activity, and, as a result, lacks activity for both the alpha-ketoglutarate and the pyruvate dehydrogenase complexes. Thus, the E3 components of both complexes are identical. The citL22 mutation maps between ura and metC on the chromosome.     

Inhibition of glycine oxidation by pyruvate, alpha-ketoglutarate, and branched-chain alpha-keto acids in rat liver mitochondria: presence of interaction between the glycine cleavage system and alpha-keto acid dehydrogenase complexes

Pyruvate, alpha-ketoglutarate, and branched-chain alpha-keto acids which were transaminated products of valine, leucine, and isoleucine inhibited glycine decarboxylation by rat liver mitochondria. However, glycine synthesis (the reverse reaction of glycine decarboxylation) was stimulated by those alpha-keto acids with the concomitant decarboxylation of alpha-keto acid added in the absence of NADH. Both the decarboxylation and the synthesis of glycine by mitochondrial extract were affected similarly by alpha-ketoglutarate and branched-chain alpha-keto acids in the absence of pyridine nucleotide, but not by pyruvate. This failure of pyruvate to have an effect was due to the lack of pyruvate oxidation activity in the mitochondrial extract employed. It indicated that those alpha-keto acids exerted their effects by providing reducing equivalents to the glycine cleavage system, possibly through lipoamide dehydrogenase, a component shared by the glycine cleavage system and alpha-keto acid dehydrogenase complexes. On the decarboxylation of pyruvate, alpha-ketoglutarate, and branched-chain alpha-keto acids in intact mitochondria, those alpha-keto acids inhibited one another. In similar experiments with mitochondrial extract, decarboxylations of alpha-ketoglutarate and branched-chain alpha-keto acid were inhibited by branched-chain alpha-keto acid and alpha-ketoglutarate, respectively, but not by pyruvate. NADH was unlikely to account for the inhibition. We suggest that the lipoamide dehydrogenase component is an indistinguishable constituent among alpha-keto acid dehydrogenase complexes and the glycine cleavage system in mitochondria in nature, and that lipoamide dehydrogenase-mediated transfer of reducing equivalents might regulate alpha-keto acid oxidation as well as glycine oxidation.     

alpha-Ketoglutarate dehydrogenase complex of Escherichia coli. A hybrid complex containing pyruvate dehydrogenase subunits from pyruvate dehydrogenase complex

The alpha-ketoglutarate dehydrogenase complex of Escherichia coli utilizes pyruvate as a poor substrate, with an activity of 0.082 units/mg of protein compared with 22 units/mg of protein for alpha-ketoglutarate. Pyruvate fully reduces the FAD in the complex and both alpha-keto[5-14C]glutarate and [2-14C]pyruvate fully [14C] acylate the lipoyl groups with approximately 10 nmol of 14C/mg of protein, corresponding to 24 lipoyl groups. NADH-dependent succinylation by [4-14C]succinyl-CoA also labels the enzyme with approximately 10 nmol of 14C/mg of protein. Therefore, pyruvate is a true substrate. However, the pyruvate and alpha-ketoglutarate activities exhibit different thiamin pyrophosphate dependencies. Moreover, 3-fluoropyruvate inhibits the pyruvate activity of the complex without affecting the alpha-ketoglutarate activity, and 2-oxo-3-fluoroglutarate inhibits the alpha-ketoglutarate activity without affecting the pyruvate activity. 3-Fluoro[1,2-14C]pyruvate labels about 10% of the E1 components (alpha-ketoacid dehydrogenases). The dihydrolipoyl transsuccinylase-dihydrolipoyl dehydrogenase subcomplex (E2E3) is activated as a pyruvate dehydrogenase complex by addition of E. coli pyruvate dehydrogenase, the E1 component of the pyruvate dehydrogenase complex. All evidence indicates that the alpha-ketoglutarate dehydrogenase complex purified from E. coli is a hybrid complex containing pyruvate dehydrogenase (approximately 10%) and alpha-ketoglutarate dehydrogenase (approximately 90%) as its E1 components.     

Regulation of citric acid cycle by calcium

The relationship of extramitochondrial Ca2+ to intramitochondrial Ca2+ and the influence of intramitochondrial free Ca2+ concentrations on various steps of the citric acid cycle were evaluated. Ca2+ was measured using the Ca2+ sensitive fluorescent dye fura-2 trapped inside the rat heart mitochondria. The rate of utilization of specific substrates and the rate of accumulation of citric acid cycle intermediates were measured at matrix free Ca2+ ranging from 0 to 1.2 microM. A change in matrix free Ca2+ from 0 to 0.3 microM caused a 135% increase in ADP stimulated oxidation of 0.6 mM alpha-ketoglutarate (K0.5 = 0.15 microM). In the absence of ADP and the presence of 0.6 mM alpha-ketoglutarate, Ca2+ (0.3 microM) increased NAD(H) reduction from 0 to 40%. On the other hand, when pyruvate (10 microM to 5 mM) was substrate, pyruvate dehydrogenase flux was insensitive to Ca2+ and isocitrate dehydrogenase was sensitive to Ca2+ only in the presence of added ADP. In separate experiments pyruvate dehydrogenase activation (dephosphorylation) was measured. Under the conditions of the present study, pyruvate dehydrogenase was found to be almost 100% activated at all levels of Ca2+, thus explaining the Ca2+ insensitivity of the flux measurements. However, if the mitochondria were incubated in the absence of pyruvate, with excess alpha-ketoglutarate and excess ATP, the pyruvate dehydrogenase complex was only 20% active in the absence of added Ca2+ and activity increased to 100% at 2 microM Ca2+. Activation by Ca2+ required more Ca2+ (K0.5 = 1 microM) than for alpha-ketoglutarate dehydrogenase. The data suggest that in heart mitochondria alpha-ketoglutarate dehydrogenase may be a more physiologically relevant target of Ca2+ action than pyruvate dehydrogenase.     

The form of alpha-ketoglutarate dehydrogenase showing no cooperative properties during substrate binding

A form of alpha-ketoglutarate dehydrogenase was detected, which is characterized by the non-equivalency of active centers for substrate binding normally revealed by chemical modification techniques and typical for other enzyme forms. The properties of various forms of alpha-ketoglutarate dehydrogenase (both soluble and immobilized on Sepharose) were compared. It was shown that despite its dimeric structure the newly detected enzyme form binds alpha-ketoglutarate in a way similar to the monomer; in this case no substrate-induced non-equivalency of the subunits due to intersubunit interactions is observed. It was found that the independent functioning of the active centers of the enzyme is due to the loosening of intersubunit contacts.     

Citrate Cycle and Related Metabolism of Listeria monocytogenes

The growth response of Listeria monocytogenes strains A4413 and 9037-7 to carbohydrates was determined in a defined medium. Neither pyruvate, acetate, citrate, isocitrate, alpha-ketoglutarate, succinate, fumarate, nor malate supported growth. Furthermore, inclusion of any of these carbohydrates in the growth medium with glucose did not increase the growth of Listeria over that observed on glucose alone. Resting cell suspensions of strain A4413 oxidized pyruvate but not acetate, citrate, isocitrate, alpha-ketoglutarate, succinate, fumarate, or malate. Cell-free extracts of strain A4413 contained active citrate synthase, aconitate hydratase, isocitrate dehydrogenase, malate dehydrogenase, fumarate hydratase, fumarate reductase, pyruvate dehydrogenase system, and oxidases for reduced nicotinamide adenine dinucleotide and reduced nicotinamide adenine dinucleotide phosphate. The alpha-ketoglutarate oxidation system, succinate dehydrogenase, isocitrate lyase, and malate synthase were not detected. Cytochromes were not detected. The data suggest that strain A4413, under these conditions, utilizes a split noncyclic citrate pathway which has an oxidative portion (citrate synthase, aconitate hydratase, and isocitrate dehydrogenase) and a reductive portion (malate dehydrogenase, fumarate hydratase, and fumarate reductase). This pathway is probably important in biosynthesis but not for a net gain in energy.     

Dihydrolipoamide dehydrogenase: functional similarities and divergent evolution of the pyridine nucleotide-disulfide oxidoreductases

Dihydrolipoamide dehydrogenase (E3) is the common component of the three alpha-ketoacid dehydrogenase complexes oxidizing pyruvate, alpha-ketoglutarate, and the branched-chain alpha-ketoacids. E3 also participates in the glycine cleavage system. E3 belongs to the enzyme family called pyridine nucleotide-disulfide oxidoreductases, catalyzing the electron transfer between pyridine nucleotides and disulfide compounds. This review summarizes the information available for E3 from a variety of species, from a halophilic archaebacterium which has E3 but no alpha-ketoacid dehydrogenase complexes, to mammalian species. Evidence is reviewed for the existence of two E3 isozymes (one for pyruvate dehydrogenase complex and alpha-ketoglutarate dehydrogenase complex and the other for branched-chain alpha-ketoacid dehydrogenase complex) in Pseudomonas species and for possible mammalian isozymes of E3, one associated with the three alpha-ketoacid dehydrogenase complexes and one for the glycine cleavage system. The comparison of the complete amino acid sequences of E3 from Escherichia coli, yeast, pig, and human shows considerable homologies of certain amino acid residues or short stretches of sequences, especially in the specific catalytic and structural domains. Similar homology is found with the limited available amino acid sequence information on E3 from several other species. Sequence comparison is also presented for other member flavoproteins [e.g., glutathione reductase and mercury(II) reductase] of the pyridine nucleotide-disulfide oxidoreductase family. Based on the known tertiary structure of human glutathione reductase it may be possible to predict the domain structures of E3. Additionally, the sequence information may help to better understand a divergent evolutionary relationship among these flavoproteins in different species.     

Use of trypsin and lipoamidase to study the role of lipoic acid moieties in the pyruvate and alpha-ketoglutarate dehydrogenase complexes of Escherichia coli

The relationships between release of (3)H-labeled lipoyl moieties by trypsin and lipoamidase and accompanying loss of overall enzymatic activity of the Escherichia coli pyruvate and alpha-ketoglutarate dehydrogenase complexes were studied. Trypsin releases lipoyl domains together with their covalently attached lipoyl moieties from the "inner" core of the dihydrolipoyl transacetylase and the dihydrolipoyl transsuccinylase whereas lipoamidase releases only the lipoyl moieties. The results show that release of lipoyl domains by trypsin and release of lipoyl moieties by lipoamidase proceeded at faster rates than the accompanying loss of overall activity of the two complexes. Trypsin released about half of the lipoyl domains in the pyruvate dehydrogenase complex without significant effect on the overall activity. A model is presented to explain these and other observations on active-site coupling via lipoyl moieties.     

Keto acid metabolism in Desulfovibrio.

Four strains of Desulfovibrio each excreted pyruvate to a constant level during growth; it was re-absorbed when the substrate (lactate) was exhausted. Malate, succinate, fumarate and malonate also accumulated during growth. One of the strains (Hildenborough) excreted alpha-ketoglutarate as well as pyruvate when incubated in nitrogen-free medium; the former was re-absorbed on addition of NH4Cl. In a low-lactate nitrogen-free medium, strain Hildenborough rapidly re-absorbed the pyruvate initially excreted, but did not re-absorb the alpha-ketoglutarate. Arsenite (I mM) prevented the accumulation of alpha-ketoglutarate; I mM-malonate did not affect the accumulation of keto acids. Isocitrate dehydrogenase activity (NAD-specific) in all strains was lower than NADP-specific glutamate dehydrogenase activity. Alpha-Ketoglutarate dehydrogenase could not be detected in any strain. NADPH oxidase activity was demonstrated. This and previous work indicate that a tricarboxylic acid pathway from citrate to alpha-ketoglutarate exists in Desulfovibrio spp., and that succinate can be synthesized via malate and fumarate; however, an intact tricarboxylic acid cycle is evidently not present. The findings are compared with observations on biosynthetic pathways in clostridia, obligate lithotrophs, phototrophs, and methylotrophs, and various facultative bacteria.     

β-Amyloid inhibits integrated mitochondrial respiration and key enzyme activities

Disrupted energy metabolism, in particular reduced activity of cytochrome oxidase (EC 1.9.3.1), alpha-ketoglutarate dehydrogenase (EC 1.2.4.2) and pyruvate dehydrogenase (EC 1.2.4.1) have been reported in post-mortem Alzheimer's disease brain. beta-Amyloid is strongly implicated in Alzheimer's pathology and can be formed intracellularly in neurones. We have investigated the possibility that beta-amyloid itself disrupts mitochondrial function. Isolated rat brain mitochondria have been incubated with the beta-amyloid alone or together with nitric oxide, which is known to be elevated in Alzheimer's brain. Mitochondrial respiration, electron transport chain complex activities, alpha-ketoglutarate dehydrogenase activity and pyruvate dehydrogenase activity have been measured. Beta-amyloid caused a significant reduction in state 3 and state 4 mitochondrial respiration that was further diminished by the addition of nitric oxide. Cytochrome oxidase, alpha-ketoglutarate dehydrogenase and pyruvate dehydrogenase activities were inhibited by beta-amyloid. The K(m) of cytochrome oxidase for reduced cytochrome c was raised by beta-amyloid. We conclude that beta-amyloid can directly disrupt mitochondrial function, inhibits key enzymes and may contribute to the deficiency of energy metabolism seen in Alzheimer's disease.     

Impact of short- and medium-chain organic acids, acylcarnitines, and acyl-CoAs on mitochondrial energy metabolism

Accumulation of organic acids as well as their CoA and carnitine esters in tissues and body fluids is a common finding in organic acidurias, beta-oxidation defects, Reye syndrome, and Jamaican vomiting sickness. Pathomechanistic approaches for these disorders have been often focused on the effect of accumulating organic acids on mitochondrial energy metabolism, whereas little is known about the pathophysiologic role of short- and medium-chain acyl-CoAs and acylcarnitines. Therefore, we investigated the impact of short- and medium-chain organic acids, acylcarnitines, and acyl-CoAs on central components of mitochondrial energy metabolism, namely alpha-ketoglutarate dehydrogenase complex, pyruvate dehydrogenase complex, and single enzyme complexes I-V of respiratory chain. Although at varying degree, all acyl-CoAs had an inhibitory effect on pyruvate dehydrogenase complex and alpha-ketoglutarate dehydrogenase complex activity. Effect sizes were critically dependent on chain length and number of functional groups. Unexpectedly, octanoyl-CoA was shown to inhibit complex III. The inhibition was noncompetitive regarding reduced ubiquinone and uncompetitive regarding cytochrome c. In addition, octanoyl-CoA caused a blue shift in the gamma band of the absorption spectrum of reduced complex III. This effect may play a role in the pathogenesis of medium-chain and multiple acyl-CoA dehydrogenase deficiency, Reye syndrome, and Jamaican vomiting sickness which are inherited and acquired conditions of intracellular accumulation of octanoyl-CoA.     

The effect of corticotropin and hydrocortisone on the dehydration of Krebs cycle substrates in E. coli cells

Corticotropin and hydrocortisone were studied for their effect on dehydrogenase activity of microbial E. coli cells in the medium with the tricarboxylic acid cycle substrates, glucose and beta-oxybutyric acid. Corticotropin, as distinct from hydrocortisone, is shown to increase the dehydrogenase activity of microbial cells when pyruvate, isocitrate, oxaloacetate, alpha-ketoglutarate, succinate, furmarate, glucose and beta-oxybutyrate are used as substrates. Hydrocortisone induced a rise of the dehydrogenase activity of microbial cells only in the medium with isocitrate, alpha-ketoglutarate and fumarate, however to a less extent than corticotropin; it lowered this activity in the medium with pyruvate and glucose and did not change it with oxaloacetate, succinate and beta-oxybutyrate. The corticotropin effect is supposed to be extra-adrenal because microbial cells are also subjected to its action.     

Complex I binds several mitochondrial NAD-coupled dehydrogenases

NADH:ubiquinone reductase (complex I) of the mitochondrial inner membrane respiratory chain binds a number of mitochondrial matrix NAD-linked dehydrogenases. These include pyruvate dehydrogenase complex, alpha-ketoglutarate dehydrogenase complex, mitochondrial malate dehydrogenase, and beta-hydroxyacyl-CoA dehydrogenase. No binding was detected between complex I and cytosolic malate dehydrogenase, glutamate dehydrogenase, NAD-isocitrate dehydrogenase, lipoamide dehydrogenase, citrate synthase, or fumarase. The dehydrogenases that bound to complex I did not bind to a preparation of complex II and III, nor did they bind to liposomes. The binding of pyruvate dehydrogenase complex, alpha-ketoglutarate dehydrogenase complex, and mitochondrial malate dehydrogenase to complex I is a saturable process. Based upon the amount of binding observed in these in vitro studies, there is enough inner membrane present in the mitochondria to bind the dehydrogenases in the matrix space. The possible metabolic significance of these interactions is discussed.     

Functional role of histidine residues of alpha-ketoglutarate dehydrogenase

The protective effect of alpha-ketoglutarate dehydrogenase substrate and its analogs on the enzyme inactivation by diethylpyrocarbonate was studied. The values of true rate constants for diethylpyrocarbonate-induced inactivation and the Kd values for the enzyme complexes with ligands were determined. A comparison of Kd values for a number of ligands suggests that the histidine residue of the enzyme active center interacts with the alpha-keto group of the substrate. A mechanism of this histidine residue involvement in the catalytic act is proposed. According to this mechanism, the imidazole ring of histidine which is responsible for the substrate activation causes a simultaneous formation of a catalytically active form of the coenzyme--thiamine pyrophosphate ilide. It is assumed that the lower (as compared with the enzyme-substrate complexes) values of rate constants of inactivation by diethylpyrocarbonate for alpha-ketoglutarate dehydrogenase complexes with succinate, glutarate, and oxaloacetate are due to additional protonation of the histidine residue, eventually resulting in the blocking of the analogs interaction with the coenzyme.     

Methanol metabolism by Pseudomonas oleovorans

A typical facultative methylotroph Pseudomonas oleovorans oxidizes methanol to formaldehyde by a specific dehydrogenase which is active towards phenazine metosulphate. Direct oxidation of formalydehyde to CO2 via formiate is a minor pathway because the activities of dehydrogenases of formaldehyde and formiate are lwo. Most formaldehyde molecules are involved in the hexulose phosphate cycle, which is confirmed by a high activity of hexulose phosphate synthase. Formaldehyde is oxidized to CO2 in the dissimilation branch of the cycle providing energy for biosynthesis; this confirmed by higher levels of dehydrogenases of glucose-6-phosphate and 6-phosphogluconate during the methylotrophous growth of the cells. The acceptor of formaldehyde (ribulose-5-phosphate) is regenerated and pyruvate is synthesized in the assimilation branch of the hexulose phosphate cycle. Aldolase of 2-keto-3-deoxy-6-phosphogluconate plays an important role in this process. Further metabolism of trioses involves reactions of the tricarboxylic acid cycle which performs mainly an anabolic function due to complete repression of alpha-ketoglutarate dehydrogenase during the methylotrophous growth. The carbon of methanol is partially assimilated as CO2 by the carboxylation of pyruvate or phosphoenolpyruvate. NH+4 is assimilated by the reductive amination of alpha-ketoglutarate.     

Mitochondrial protein lipoylation does not exclusively depend on the mtKAS pathway of de novo fatty acid synthesis in Arabidopsis

The photorespiratory Arabidopsis (Arabidopsis thaliana) mutant gld1 (now designated mtkas-1) is deficient in glycine decarboxylase (GDC) activity, but the exact nature of the genetic defect was not known. We have identified the mtkas-1 locus as gene At2g04540, which encodes beta-ketoacyl-[acyl carrier protein (ACP)] synthase (mtKAS), a key enzyme of the mitochondrial fatty acid synthetic system. One of its major products, octanoyl-ACP, is regarded as essential for the intramitochondrial lipoylation of several proteins including the H-protein subunit of GDC and the dihydrolipoamide acyltransferase (E2) subunits of two other essential multienzyme complexes, pyruvate dehydrogenase and alpha-ketoglutarate dehydrogenase. This view is in conflict with the fact that the mtkas-1 mutant and two allelic T-DNA knockout mutants grow well under nonphotorespiratory conditions. Although on a very low level, the mutants show residual lipoylation of H protein, indicating that the mutation does not lead to a full functional knockout of GDC. Lipoylation of the pyruvate dehydrogenase and alpha-ketoglutarate dehydrogenase E2 subunits is distinctly less reduced than that of H protein in leaves and remains unaffected from the mtKAS knockout in roots. These data suggest that mitochondrial protein lipoylation does not exclusively depend on the mtKAS pathway of lipoate biosynthesis in leaves and may occur independently of this pathway in roots.     

Catalysis of diaphorase reactions by Mycobacterium tuberculosis lipoamide dehydrogenase occurs at the EH4 level

Lipoamide dehydrogenase catalyzes the reversible NAD(+)-dependent oxidation of the dihydrolipoyl cofactors that are covalently attached to the acyltransferase components of the pyruvate dehydrogenase, alpha-ketoglutarate dehydrogenase, and glycine reductase multienzyme complexes. It contains two redox centers: a tightly, but noncovalently, bound FAD and an enzymic disulfide, each of which can accommodate two electrons. In the two-electron-reduced enzyme (EH(2)), the disulfide is reduced while the FAD cofactor is oxidized. In the four-electron-reduced enzyme (EH(4)), both redox centers are reduced. Lipoamide dehydrogenase can also catalyze the NADH-dependent reduction of alternative electron acceptors such as 2,6-dichlorophenolindophenol, ferricyanide, quinones, and molecular oxygen (O(2)). To determine the mechanism of these "diaphorase" reactions, we generated the EH(2) and EH(4) forms of Mycobacterium tuberculosis lipoamide dehydrogenase and rapidly mixed these enzyme forms with d,l-lipoylpentanoate, 2,6-dimethyl-1,4-benzoquinone, and O(2), in a stopped-flow spectrophotometer at pH 7.5 and 4 degrees C. EH(2) reduced d,l-lipoylpentanoate >/=100 times faster than EH(4) did. Conversely, EH(4) reduced 2,6-dimethyl-1,4-benzoquinone and molecular oxygen 90 and 40 times faster than EH(2), respectively. Comparison of the rates of reduction of the above substrates by EH(2) and EH(4) with their corresponding steady-state kinetic parameters for kinetic competence leads to the conclusion that reduction of lipoyl substrates occurs with EH(2) while reduction of diaphorase substrates occurs with EH(4).     

Effect of corticotropin on the dehydrogenase activity of cells and tissues

The authors studied the effect of native ACTH on dehydrogenase activity of isolated strips of rat diaphragm and suspension of E. coli cells, serotype O III:B4, grown on beef extract agar in a medium with different dehydrogenation substrates. ACTH activated dehydrogenase of rat diaphragm in a medium containing pyruvate, alpha-ketoglutarate, malate, beta-hydroxybutyrate, D-aspartic acid and did not alter it in a medium containing succinate. In contradistinction to rat diaphragm, ACTH activated dehydrogenase of E. coli cells whatever the substrates used (oxaloacetate, isocitrate, alpha-ketoglutarate, succinate, fumarate, malate, pyruvate, lactate, beta-hydroxybutyrate, glucose, D-aspartic acid. Synacthen (ACTH1-24) exerted a similar effect. It is suggested that the effects of ACTH are mediated via its influence on adenylate cyclase in the absence of receptors.     

alpha-Ketoglutarate dehydrogenase mutant of Rhizobium meliloti.

A mutant of Rhizobium meliloti selected as unable to grow on L-arabinose also failed to grow on acetate or pyruvate. It grew, but slower than the parental strain, on many other carbon sources. Assay showed it to lack alpha-ketoglutarate dehydrogenase (kgd) activity, and revertants of normal growth phenotype contained the activity again. Other enzymes of the tricarboxylic acid cycle and of the glyoxylate cycle were present in both mutant and parent strains. Enzymes of pyruvate metabolism were also assayed. L-Arabinose degradation in R. meliloti was found to differ from the known pathway in R. japonicum, since the former strain lacked 2-keto-o-deoxy-L-arabonate aldolase but contained alpha-ketoglutarate semialdehyde dehydrogenase; thus, it is likely that R. meliloti has the L-arabinose pathway leading to alpha-ketoglutarate rather than the one to glycolaldehyde and pyruvate. This finding accounts for the L-arabinose negativity of the mutant. Resting cells of the mutant were able to metabolize the three substrates which did not allow growth.