Characterization of Glutaminase from Triticale

The glutaminase (EC 3.5.1.2) isolated from seedlings of triticale (Triticalesp.) had a pH optimum of about 8, was inhibited with excess substrate (glutamine), and reaction products (glutamate and NH⁺ ₄). A monocharged anion (Cl^–) and a multicharged anion (phosphate) were shown to activate the glutaminase. Some features of the glutaminase from triticale were similar to those of animal glutaminase activated by phosphate and were different from features of the enzyme from Escherichia coli.     

Glutaminase II Pathway in Human Kidney

THE principal mechanism whereby excess hydrogen ions are excreted in man is by renal production of ammonia and subsequent urinary excretion as ammonium. The major and direct source of this renal ammoniagenesis is glutamine¹. Two distinct metabolic pathways of glutamine metabolism have been demonstrated in rat, guinea-pig and dog. The intramitochondrial glutaminase I isoenzymes which hydrolyse glutamine to ammonia and glutamic acid and its subsequent deamidation to ammonia and 2-oxo-glutarate constitute the major metabolic route in the rat². The extramitochondrial glutamine-aminotransferase-ω-amidase pathway (glutaminase II), however, has been shown to be important in the dog³. In man, whereas the glutaminase I pathway has been demonstrated⁴ there is no direct evidence for the latter metabolic pathway. We investigated this metabolic pathway using the alkyl substituted glutamine, L-γ-glutamylmethylamide. In contrast to glutamine, this substituted compound on deamidation yields methylamine⁵.     

Glutaminase catalyzes reaction of Glutamate to GABA

Here, for the first time, we report an NMR spectroscopy study of l-Glutamine (Gln) conversion by Glutaminase (Glnase), which shows that the reaction involves two distinct steps. In the first step, Glnase rapidly hydrolyzes Gln to Glutamate (Glu) (∼16.87 μmol of Gln/min/mg of Glnase) and in the second step, Glu generated in the first step is decarboxylated into gamma-amino butyric acid (GABA) with a much slower rate (∼0.185 μmol/min/mg). When Glnase was added to the sample containing l-Glu alone, it was also converted to GABA, at a similar rate as in the second step mentioned above. The rate of Glu decarboxylation into GABA by Glnase is about an order of magnitude lower than that by commonly known enzyme, Glutamate decarboxylase. Potential impact of these findings, on the mechanistic aspects of Gln–Glu shuttle in neuroscience and glutaminolysis in tumors, is discussed.     

Upregulation of the Glutaminase II Pathway Contributes to Glutamate Production upon Glutaminase 1 Inhibition in Pancreatic Cancer

The targeting of glutamine metabolism specifically via pharmacological inhibition of glutaminase 1 (GLS1) has been translated into clinical trials as a novel therapy for several cancers. The results, though encouraging, show room for improvement in terms of tumor reduction. In this study, the glutaminase II pathway is found to be upregulated for glutamate production upon GLS1 inhibition in pancreatic tumors. Moreover, genetic suppression of glutamine transaminase K (GTK), a key enzyme of the glutaminase II pathway, leads to the complete inhibition of pancreatic tumorigenesis in vivo unveiling GTK as a new metabolic target for cancer therapy. These results suggest that current trials using GLS1 inhibition as a therapeutic approach targeting glutamine metabolism in cancer should take into account the upregulation of other metabolic pathways that can lead to glutamate production; one such pathway is the glutaminase II pathway via GTK.     

Evidence for Compartmentation of Synaptosomal Phosphate-Activated Glutaminase

Abstract: Phosphate-activated glutaminase (EC 3.5.1.2) in synaptosomal preparations is inhibited 40–60% by the sulphydryl group reagent N -ethylmaleimide (NEM), forming the basis for distinction between NEM-sensitive and NEM-insensitive glutaminases. The NEM effect cannot be explained by differential effects on distinct glutaminases because other glutaminases have not been detected, and the synaptosomal glutaminase activity can be fully accounted for by the activity of phosphate-activated glutaminase. By fractionation of mitochondria isolated from synaptosomal preparations, which are preincubated with and without NEM, both NEM-sensitive and NEM-insensitive glutaminases are found to be localized to the inner mitochondrial membrane. Variations in pH (7.0–7.6) and the phosphate concentration (5–10 mM) affect chiefly NEM-sensitive glutaminase, demonstrating that this glutaminase may be subject to regulation by compounds in the cytosol having restricted permeability to the inner mitochondrial membrane. Since p -hydroxymercuribenzoate, which is known to be impermeable to the inner mitochondrial membrane, inhibits glutaminase similarly to NEM, phosphate-activated glutaminase is assumed to be compartmentalized within the inner mitochondrial membrane. Thus, NEM-sensitive glutaminase is localized to the outer face and NEM-insensitive glutaminase to the inner region of this membrane and probably also to the matrix region.     

Metformin Inhibits Glutaminase Activity and Protects against Hepatic Encephalopathy

Aim

To investigate the influence of metformin use on liver dysfunction and hepatic encephalopathy in a retrospective cohort of diabetic cirrhotic patients. To analyze the impact of metformin on glutaminase activity and ammonia production in vitro.

Methods

Eighty-two cirrhotic patients with type 2 diabetes were included. Forty-one patients were classified as insulin sensitizers experienced (metformin) and 41 as controls (cirrhotic patients with type 2 diabetes mellitus without metformin treatment). Baseline analysis included: insulin, glucose, glucagon, leptin, adiponectin, TNFr2, AST, ALT. HOMA-IR was calculated. Baseline HE risk was calculated according to minimal hepatic encephalopathy, oral glutamine challenge and mutations in glutaminase gene. We performed an experimental study in vitro including an enzymatic activity assay where glutaminase inhibition was measured according to different metformin concentrations. In Caco2 cells, glutaminase activity inhibition was evaluated by ammonia production at 24, 48 and 72 hours after metformina treatment.

Results

Hepatic encephalopathy was diagnosed during follow-up in 23.2% (19/82): 4.9% (2/41) in patients receiving metformin and 41.5% (17/41) in patients without metformin treatment (logRank 9.81; p = 0.002). In multivariate analysis, metformin use [H.R.11.4 (95% CI: 1.2–108.8); p = 0.034], age at diagnosis [H.R.1.12 (95% CI: 1.04–1.2); p = 0.002], female sex [H.R.10.4 (95% CI: 1.5–71.6); p = 0.017] and HE risk [H.R.21.3 (95% CI: 2.8–163.4); p = 0.003] were found independently associated with hepatic encephalopathy. In the enzymatic assay, glutaminase activity inhibition reached 68% with metformin 100 mM. In Caco2 cells, metformin (20 mM) decreased glutaminase activity up to 24% at 72 hours post-treatment (p<0.05).

Conclusions

Metformin was found independently related to overt hepatic encephalopathy in patients with type 2 diabetes mellitus and high risk of hepatic encephalopathy. Metformin inhibits glutaminase activity in vitro. Therefore, metformin use seems to be protective against hepatic encephalopathy in diabetic cirrhotic patients.     

Phosphate-Activated Glutaminase and Mitochondrial Glutamine Transport in the Brain

A review of the properties of purified and tissue bound phosphate activated glutaminase (PAG) in brain and kidney (pig and rat) is presented, based on kinetic, electron microscopic and immunocytochemical studies. PAG is a mitochondrial enzyme and two pools can be separated, a soluble and membrane associated one. Intact mitochondria appear to express PAG accessible only to the outer phase of the inner mitochondrial membrane. This PAG has properties similar to that of the membrane fraction and polymeric form of purified enzyme. PAG in the soluble fraction has properties similar to that of the monomeric form of purified enzyme and is assumed to be dormant due to the high matrix concentration of the inhibitor glutamate. A hypothetical model for the localization of PAG in the mitochondria is presented. The activity of PAG in vivo is assumed to be regulated by cytosolic glutamate and other compounds, that affect the activation by phosphate. Glutamine is transported into brain and kidney mitochondria by a protein catalyzed energy requiring process, which may be mediated by more than one protein. There is no correlation between glutamine hydrolysis and transport.     

Phosphate-Activated Glutaminase in Relation to Huntington''s Disease and Agonal State

Involvement of phosphate-activated glutaminase in Huntington's disease and agonal state was investigated in caudate nucleus and frontal cortex from postmortem brains. In Huntington's disease the activities of phosphate-activated glutaminase, glutamic acid decarboxylase, succinic dehydrogenase, choline acetyltransferase, and acetylcholinesterase were significantly reduced in the caudate nucleus, but not in the frontal cortex. The activity of phosphate-activated glutaminase, and to a lesser extent of glutamic acid decarboxylase, was reduced in cases of terminal illness, as compared with cases of sudden death. Succinic dehydrogenase and choline acetyltransferase were reduced only in the few cases of prolonged and severe terminal illness. Enzyme activities of the caudate nucleus were more affected by agonal state than were those of frontal cortex. Results indicate that phosphate-activated glutaminase could be a useful marker of neuronal damage due to agonal state, and that phosphate-activated glutaminase and succinic dehydrogenase are reduced in Huntington's disease.     

Deacetylation of Glutaminase by HDAC4 contributes to Lung Cancer Tumorigenesis

Inhibiting cancer metabolism via glutaminase (GAC) is a promising strategy to disrupt tumor progression. However, mechanism regarding GAC acetylation remains mostly unknown. In this study, we demonstrate that lysine acetylation is a vital post-translational modification that inhibits GAC activity in non-small cell lung cancer (NSCLC). We identify that Lys311 is the key acetylation site on GAC, which is deacetylated by HDAC4, a class II deacetylase. Lys311 acetylation stimulates the interaction between GAC and TRIM21, an E3 ubiquitin ligase of the tripartite motif (TRIM) family, therefore promoting GAC K63-linked ubiquitination and inhibiting GAC activity. Furthermore, GAC^K311Q mutation in A549 cells decreases cell proliferation and alleviates tumor malignancy. Our findings reveal a novel mechanism of GAC regulation by acetylation and ubiquitination that participates in non-small cell lung cancer tumorigenesis.