Alpha KG Research Articles

A novel alpha-ketoglutarate reductase activity of the serA-encoded 3-phosphoglycerate dehydrogenase of Escherichia coli K-12 and its possible implications for human 2-hydroxyglutaric aciduria

Escherichia coli serA-encoded 3-phosphoglycerate (3PG) dehydrogenase catalyzes the first step of the major phosphorylated pathway of L-serine (Ser) biosynthesis. The SerA enzyme is evolutionarily related to the pdxB gene product, 4-phosphoerythronate dehydrogenase, which catalyzes the second step in one branch of pyridoxal 5'-phosphate coenzyme biosynthesis. Both the Ser and pyridoxal 5'-phosphate biosynthetic pathways use the serC(pdxF)-encoded transaminase in their next steps. In an analysis of these parallel pathways, we attempted to couple the transaminase and dehydrogenase reactions in the reverse direction. Unexpectedly, we found that the SerA enzyme catalyzes a previously undetected reduction of alpha-ketoglutarate (alpha KG) to 2-hydroxyglutaric acid (HGA). Numerous criteria ruled out the possibility that this SerA alpha KG reductase activity was caused by contamination in the substrate or purified enzyme preparations. HGA was confirmed as the product of the SerA alpha KG reductase reaction by thin-layer chromatography and by enzyme assays showing that both the D- and L-isomers of HGA were substrates for the reverse (dehydrogenase) reaction. Detailed steady-state kinetic analyses showed that alpha KG reduction (apparent Michaelis-Menten constant [Km(app)] = 88 microM; apparent catalytic constant [kcat(app)] = 33.3 s-1) and 3-phosphohydroxypyruvate reduction (Km(app) = 3.2 microM; kcatapp = 27.8 s-1), which is the reverse reaction of 3PG oxidation, were the major in vitro activities of the SerA enzyme. The SerA alpha KG reductase was inhibited by Ser, D-HGA, 3PG, and glycine (Gly), whereas the D-HGA dehydrogenase was inhibited by Ser, alpha KG, 3-phosphohydroxypyruvate, and Gly. The implications of these findings for the regulation of Ser biosynthesis, the recycling of NADH, and the enzymology of 2-hydroxyacid dehydrogenases are discussed. Since the same pathway of Ser biosynthesis seems to be present in all organisms, these results suggest that a mutation in the human SerA homolog may contribute to the neurometabolic diseases D- and L-2-hydroxyglutaric aciduria, which lead to the accumulation of D-HGA and L-HGA, respectively.

Action of ornithine alpha-ketoglutarate, ornithine hydrochloride, and calcium alpha-ketoglutarate on plasma amino acid and hormonal patterns in healthy subjects.

Ornithine alpha-ketoglutarate (OKG) has been useful as an adjuvant of enteral and parenteral nutrition. However, its metabolism and mechanism of action remain unclear although it is known that alpha-ketoglutarate (alpha KG) and ornithine (ORN) follow, in part, common metabolic pathways. Six fasting healthy male subjects underwent three separate oral load tests: (i) they received 10 g of OKG (i.e., 3.6 g of alpha KG and 6.4 g of ORN); (ii) 6.4 g of ORN as ornithine hydrochloride, and (iii) 3.6 g of alpha KG as calcium alpha-ketoglutarate. Blood was drawn 15 times over a five-hour period for measurements of plasma amino acids, alpha KG, insulin, and glucagon. After OKG and ORN administration, plasma ORN peaked at 60-75 min (494 +/- 91 and 541 +/- 85 mumol/L). The increase in plasma alpha KG was very small. OKG, alpha KG, and ORN all increased glutamate concentrations at 60 min (mean: +43%, +68%, +68%, respectively, p less than 0.05 compared to basal values). However, only OKG increased proline and arginine levels at 60 min (mean: +35%, p less than 0.01 and mean: +41%, p less than 0.05). Furthermore, glutamate, proline, and arginine concentrations correlated linearly with ornithine levels at 60 min. Finally, OKG increased insulinemia and glucagonemia (mean: +24% at 15 min, p less than 0.05 and +30% at 60 min, p less than 0.01, respectively). These data provide evidence that the combination of ORN and alpha KG modifies amino acid metabolism in a way which is not observed when they are administered separately. In addition, the OKG-mediated increase in insulin levels probably does not appear to result from a direct action of ORN on pancreatic secretion.

Action of ornithine α ketoglutarate on DNA synthesis by human fibroblasts

Ornithine alpha ketoglutarate (OKG) is largely used in clinical nutrition for its anabolic effects. However, the mechanism of its action remains questionable. We investigated the effect of OKG on the rate of DNA synthesis in human fibroblasts. The in vitro experimental procedure required to demonstrate in cell culture the anabolic effects of OKG observed in vivo was found to be glutamine-free and serum-poor medium with sparse cells. In these conditions, OKG induced a significant increase in [3H]thymidine incorporation compared to untreated control cells. This effect was dose-dependent and was observed in all the cultures tested. Taken individually, the two constituents of OKG, i.e. alpha KG and Orn, also showed a stimulatory effect, but did not demonstrate a dose-dependent response. Concomitant analysis of extracellular aminoacids showed in alpha KG-treated cultures an increase in glutamate and a decrease in aspartate, suggesting a cellular transamination of alpha KG. Glutamine, which is the preferential energetic substrate of fibroblasts, can be produced from glutamate and might play a role in the action of OKG. Moreover, OKG induced a rise in the cellular polyamine content. This, in association with the inhibitory effect on OKG action of difluoromethylornithine, a specific inhibitor of ornithine decarboxylase, suggests a link between the polyamine biosynthesis pathway and the anabolic effect of OKG.

Regulation of rat kidney mitochondrial metabolism in acute acidosis.

Proximal tubular ammoniagenesis is amplified under conditions of acute and chronic metabolic acidosis. Current hypotheses postulate that alterations in intracellular pH (pHi) or in the pH gradient across the inner mitochondrial membrane (delta pHm) influence mitochondrial glutamine metabolism. Enhanced glutamine transport across the inner mitochondrial membrane might constitute a key regulatory factor in acidosis. To examine changes in delta pHm, a technique was used to determine pHi and intramitochondrial pH (pHm) simultaneously. Regulation of the enzyme alpha ketoglutarate dehydrogenase (alpha KGDH) was assessed by evaluating enzyme activity at varied levels of medium pH, Ca++, and adenosine diphosphate (ADP). The results indicate that pHi decreased with an acid external pH. A fall in pHi correlated to increase activity of alpha KGDH associated with increased affinity for the substrate, alpha KG. Increments in either buffer Ca++ or ADP concentration increased enzyme affinity for alpha KG at pH 7.6 but not at pH 6.8. These results, compatible with previous reports, indicate that pH, Ca++, and ADP are effectors of the enzyme alpha KGDH. Alterations in pH across the inner mitochondrial membrane might augment flux through alpha KG by accelerating glutamine metabolism. Increased alpha KG oxidation over the range of 10 to 500 mumol/L Ca++ concentration is compatable with data for Ca++ regulation reported for the solubilized enzyme. These studies provide evidence that the above factors, through enhancing alpha KGDH activity, participate in regulation of ammoniagenesis during states of acidosis.

Influence of pyruvate on ammonia metabolism by renal cortical mitochondria

The effect of the tricarboxylic acid (TCA) cycle precursor, pyruvate, on glutamine metabolism by isolated renal cortical mitochondria was assessed by quantitating its key nitrogen and carbon metabolites. When mitochondria from normal rats were incubated at pH 7.4, pyruvate (2 mM) inhibited ammonia production by almost completely erradicating glutamate deamination and by diminishing glutamine deamidation but to a lesser extent. Alpha KG, citrate, and malate accumulation in the incubation medium were increased dramatically reflecting the increased flux of pyruvate through the TCA cycle; the intramitochondrial concentrations of both Alpha KG and glutamate were increased. Thus, pyruvate primarily inhibits flux through glutamate dehydrogenase as a result either of an increase in Alpha KG concentration and/or a decrease in the redox (NAD/NADH) potential secondary to enhanced flux through the TCA cycle. Glutamine deamidation is secondarily inhibited, presumably due to the increased intramitochondrial concentration of glutamate. Citrate (2 mM) produced changes comparable to those observed with pyruvate. Mitochondria from normal rats incubated at pH 7.0 as well as mitochondria from rats with chronic metabolic acidosis responded to pyruvate in a fashion qualitatively similar to normal mitochondria incubated at pH 7.4. Glutamate deamination was inhibited significantly, but a high rate persisted with chronic acidosis despite the presence of pyruvate. Nevertheless, when glutamine metabolism was contrasted with normal mitochondria incubated at pH 7.4, the response to in vitro incubation in an acid pH as well as to chronic metabolic acidosis was similar quantitatively regardless of whether glutamine alone or in combination with pyruvate was present in the incubation medium.

Changes of plasma amino acids in cirrhotics treated with ornithine salts

The plasma amino acid pattern of cirrhotic patients was determined before and after 24 h of continuous infusions of glucose, ornithine alpha-ketoglutarate (O alpha KG), ornithine chlorhydrate (ORN HCl) and sodium ketoglutarate (alpha CGNa). Before treatment, leucine, isoleucine, valine and glutamine levels were low. Tyrosine and methionine levels were high. (See formula in text) was low. Glucose infusions had no effect. O alpha KG increased levels of leucine, isoleucine, valine, alanine and arginine. Threonine, serine, glycine, aspartic acid, methionine, hemicystine, tyrosine and phenylalanine were significantly lowered. (See formula in text) increased. ORN HCl and alpha CGNa did not induce similar changes.