Covalent interaction of L-2-amino-4-oxo-5-chloropentanoic acid with rat renal phosphate-dependent glutaminase. Evidence for a specific glutamate binding site and of subunit heterogeneity

Rat renal phosphate-dependent glutaminase is rapidly inactivated by incubating with L-2-amino-4-oxo-5-chloropentanoic scid. Concentrations of phosphate, which increase the glutaminase activity, decrease the rate of inactivation by chloroketone. In addition, inactivation is not blocked by glutamine. Instead, glutamate was shown to specifically reduce the rate of chloroketone inactivation. Upon sodium lauryl sulfate-polyacrylamide gel electrophoresis, the purified glutaminase preparation exhibits at least five protein staining bands which range in molecular weight from 57,000 to 75,000. Studies with 14C-labeled chloroketone indicate that this reagent reacts with each of these peptides. The mean stoichiometry of binding was calculated to be 1.3 mol/mol of enzyme. Therefore, these results indicate that the glutaminase may contain a specific site for binding glutamate and that the purified enzyme consists of a series of related peptides which may have resulted from partial proteolysis.     

Inactivation of rat renal phosphate-dependent glutaminase with 6-diazo-5-oxo-L-norleucine. Evidence for interaction at the glutamine binding site.

Inactivation of rat renal phosphate-dependent glutaminase by 6-diazo-5-oxo-L-norleucine occurs only under conditions where the enzyme is catalytically active. The glutaminase activity and the rate of inactivation by the diazoketone exhibit very similar phosphate concentration-dependent activation profiles. Because of this phosphate dependency, it was not possible to differentiate an apparent protection by glutamine from the strong inhibition of inactivation caused by glutamate. The ability of glutamate to protect the glutaminase against inactivation is reversed by increasing concentrations of phosphate.The observed characteristics of inactivation by 6-diazo-5-oxo-L-norleucine differ considerably from those reported for the inactivation by L-2-amino-4-oxo-5-chloropentanoic acid. In addition, the presence of o-carbamoyl-L-serine was found to stimulate inactivation by 6-diazo-5-oxo-L-norleucine, but to protect the glutaminase against inactivation by the chloroketone. Preinactivation of the glutaminase by the diazoketone only slightly reduced the stoichiometry of binding of [5-14C]chloroketone. These observations suggest that 6-diazo-5-oxo-L-norleucine and L-2-amino-4-oxo-5-chloropentanoic acid interact with different sites on the glutaminase which are specific for binding glutamine and glutamate, respectively.     

Inhibition of Glutamine Transport in Rat Brain Mitochondria by Some Amino Acids and Tricarboxylic Acid Cycle Intermediates

Glutamine transport into rat brain synaptic and non-synaptic mitochondria has been monitored by the uptake of [³H]glutamine and by mitochondrial swelling. The concentration of glutamate in brain mitochondria is calculated to be high, 5–10 mM, indicating that phosphate activated glutaminase localized inside the mitochondria is likely to be dormant and the glutamine taken up not hydrolyzed. The uptake of [³H]glutamine is largely stereospecific. It is inhibited by glutamate, asparagine, aspartate, 2-oxoglutarate and succinate. Glutamate inhibits this uptake into synaptic and non-synaptic mitochondria by 95 and 85%, respectively. The inhibition by glutamate, asparagine, aspartate and succinate can be explained by binding to an inhibitory site whereas the inhibition by 2-oxoglutarate is counteracted by aminooxyacetic acid, which indicates that it is dependent on transamination. The glutamine-induced swelling, a measure of a very low affinity uptake, is inhibited by glutamate at a glutamine concentration of 100 mM, but this inhibition is abolished when the glutamine concentration is raised to 200 mM. This suggests that the very low affinity glutamine uptake is competitively inhibited by glutamate. Furthermore, glutamine-induced swelling is inhibited by 2-oxoglutarate, succinate and malate, similarly to that of the [³H]glutamine uptake. The properties of the mitochondrial glutamine transport are not identical with those of a recently purified renal glutamine carrier.     

Neuronal Glutamine Utilization: Glutamine/Glutamate Homeostasis in Synaptosomes

The synaptosomal metabolism of glutamine was studied under in vitro conditions that simulate depolarization in vivo. With [2-15N]glutamine as precursor, the [glutamine]i was diminished in the presence of veratridine or 50 mM KCl, but the total amounts of [15N]glutamate and [15N]aspartate formed were either equal to those of control incubations (veratridine) or higher (50 mM [KCl]). This suggests that depolarization decreases glutamine uptake and independently augments glutaminase activity. Omission of sodium from the medium was associated with low internal levels of glutamine which indicates that influx occurs as a charged Na(+)-amino acid complex. It is postulated that a reduction in membrane potential and a collapse of the Na+ gradient decrease the driving forces for glutamine accumulation and thus inhibit its uptake and enhance its release under depolarizing conditions. Inorganic phosphate stimulated glutaminase activity, particularly in the presence of calcium. At 2 mM or lower [phosphate] in the medium, calcium inhibited glutamine utilization and the production of glutamate, aspartate, and ammonia from glutamine. At a high (10 mM) medium [phosphate], calcium stimulated glutamine catabolism. It is suggested that a veratridine-induced increase in intrasynaptosomal inorganic phosphate is responsible for the enhancement of flux through glutaminase; calcium affects glutaminase indirectly by modulating the level of free intramitochondrial [phosphate]. Because phosphate also lowers the Km of glutaminase for glutamine, augmentation of the amino acid breakdown may occur even when depolarization lowers [glutamine]i. Reducing the intrasynaptosomal glutamate to 26 nmol/mg of protein had little effect on glutamine catabolism, but raising the pH to 7.9 markedly increased formation of glutamate and aspartate. It is concluded that phosphate and H+ are the major physiologic regulators of glutaminase activity.     

Evidence indicating that pig renal phosphate-activated glutaminase has a functionally predominant external localization in the inner mitochondrial membrane

Phosphate-activated glutaminase in intact pig renal mitochondria was inhibited 50-70% by the sulfhydryl reagents mersalyl and N-ethylmaleimide (0.3-1.0 mM), when assayed at pH 7.4 in the presence of no or low phosphate (10 mM) and glutamine (2 mM). However, sulfhydryl reagents added to intact mitochondria did not inhibit the SH-enzyme beta-hydroxybutyrate dehydrogenase (a marker of the inner face of the inner mitochondrial membrane), but did so upon addition to sonicated mitochondria. This indicates that the sulfhydryl reagents are impermeable to the inner membrane and that regulatory sulfhydryl groups for glutaminase have an external localization here. The inhibition observed when sulfhydryl reagents were added to intact mitochondria could not be attributed to an effect on a phosphate carrier, but evidence was obtained that pig renal mitochondria have also a glutamine transporter, which is inhibited only by mersalyl and not by N-ethylmaleimide. Mersalyl and N-ethylmaleimide showed nondistinguishable effects on the kinetics of glutamine hydrolysis, affecting only the apparent Vmax for glutamine and not the apparent Km calculated from linear Hanes-Woolf plots. Furthermore, both calcium (which activates glutamine hydrolysis), as well as alanine (which has no effect on the hydrolytic rate), inhibited glutamine transport into the mitochondria, indicating that transport of glutamine is not rate-limiting for the glutaminase reaction. Desenzitation to inhibition by mersalyl and N-ethylmaleimide occurred when the assay was performed under optimal conditions for phosphate activated glutaminase (i.e. in the presence of 150 mM phosphate, 20 mM glutamine and at pH 8.6). Desenzitation also occurred when the enzyme was incubated with low concentrations of Triton X-100 which did not affect the rate of glutamine hydrolysis. Following incubation with [14C]glutamine and correction for glutamate in contaminating subcellular particles, the specific activity of [14C]glutamate in the mitochondria was much lower than that of the surrounding incubation medium. This indicates that glutamine-derived glutamate is released from the mitochondria without being mixed with the endogenous pool of glutamate. The results suggest that phosphate-activated glutaminase has a functionally predominant external localization in the inner mitochondrial membrane.     

beta-2-Aminobicyclo-(2.2.1)-heptane-2-carboxylic acid. A new activator of glutaminase in intact rat liver mitochondria

beta-(+/-)-2-Aminobicyclo-(2.2.1)-heptane-2-carboxylic acid (BCH) stimulated, in a concentration-dependent manner, the formation of glutamate by mitochondria isolated from rat liver and incubated with 20 mM glutamine. Maximum enhancement was seen with 10 mM BCH while 5 mM leucine was without effect. The initial lag in the rate of glutamate formation was not eliminated by BCH. Preincubation of the mitochondria without glutamine also did not abolish the lag period; to the contrary, it resulted in a progressive deactivation of the glutaminase. The decrease in enzyme activity during the preincubation without glutamine was partially reversed by the addition of either 10 mM BCH or 1.4 mM NH4Cl and was essentially abolished by their combined action. The apparently sigmoid rise in the activity of glutaminase with increasing concentration of glutamine became hyperbolic in the presence of 1.4 mM NH4Cl. BCH stimulated the NH4Cl-activated glutaminase in the entire range of glutamine concentrations studied (2-40 mM) without changing the S50 value. In mitochondria disrupted by repeated cycles of freezing and thawing, the enzymatic activity was maximal even in the absence of BCH. It is postulated that BCH is a potent activator of mitochondrial glutaminase and that manifestation of its action requires intact organelle structure. In addition, it is concluded that BCH-induced stimulation of glutamine catabolism in isolated hepatocytes (Zaleski, J., Wilson, D. F., and Erecinska, M. (1986) J. Biol. Chem. 261, 14082-14090) is the consequence of activation of the mitochondrial glutaminase.     

Intracellular localization and properties of phosphate-dependent glutaminase in rat mesenteric lymph nodes.

Phosphate-dependent glutaminase was present at approximately similar activities in lymph nodes from mammals other than rat, and in thymus, spleen, Peyer's patches and bone marrow of the rat. This suggests that glutamine is important in all lymphoid tissues. Phosphate-dependent glutaminase activity was shown to be present primarily in the mitochondria of rat mesenteric lymph nodes, and most of the activity could be released by detergents. The properties of the enzyme in mitochondrial extracts were investigated. The pH optimum was 8.6 and the Km for glutamine was 2.0 mM. The enzyme was activated by phosphate, other phosphorylated compounds including phosphoenolpyruvate, and also leucine: 50% activation occurred at 5, 0.2 and 0.6 mM for phosphate, phosphoenolpyruvate and leucine respectively. The enzyme was inhibited by glutamate, 2-oxoglutarate, citrate and ammonia, and by N-ethylmaleimide and diazo-5-oxo-L-norleucine; 50% inhibition was observed at 0.7 and 0.1 mM for glutamate and 2-oxoglutarate respectively. Some of these properties may be important in the control of the enzyme activity in vivo.     

Response of rat liver glutaminase to pH. Mediation by phosphate and ammonium ions

The activity of rat liver glutaminase from sedimented fractions of freeze-thawed mitochondria is strongly affected by variation in pH over a physiologically relevant range at approximate physiological concentrations of activators. As pH increases from 7.1 to 7.7 at 0.7 mM ammonium and 10 mM phosphate, the S0.5 for glutamine decreases 3.5-fold, from 38 to 11 mM. This results in an 8-fold increase in reaction velocity at 10 mM glutamine. In addition, the M0.5 for phosphate activation decreases from 21 to 8.9 mM as pH increases from 7.1 to 7.7. This apparent effect of pH on the affinity of glutaminase for phosphate is similar to previous reports of the pH effect on activation by ammonium (Verhoeven, A. J., Van Iwaarden, J. F., Joseph, S. K., and Meijer, A. J. (1983) Eur. J. Biochem. 133, 241-244; McGivan, J. D., and Bradford, N. M. (1983) Biochim. Biophys. Acta 159, 296-302). Glutaminase does not respond to variation in pH between 7.1 and 7.7 when phosphate and ammonium are saturating. The effects of the two modifiers are additive. Each is still effective, as is pH, when the other is saturating. Therefore, it appears that the effects of pH on the apparent affinity of the enzyme for ammonium and phosphate account for the enzyme's response to pH. These results may help explain previous reports of minimal effects of pH on glutaminase at saturating concentrations of related substances (McGivan, J. D., Lacey, J. H., and Joseph, K. (1980) Biochim. J. 192, 537-542; Horowitz, M. L., and Knox, W. E. (1968) Enzymol. Biol. Clin. 9, 241-255; McGivan, J. D., and Bradford, N. M. (1983) Biochim. Biophys. Acta 759, 296-302). Glutaminase binds glutamine cooperatively with Hill coefficients ranging from 1.7 to 2.2, which suggests at least two and probably three or more interacting binding sites for glutamine. The strong response of liver glutaminase to pH and the fact that the reaction can supply metabolites for urea synthesis suggest a possible regulatory role of glutaminase in ureagenesis.     

Comparison of the phosphate-dependent glutaminase obtained from rat brain and kidney.

A phosphate-dependent glutaminase was purified 1200-fold from rat brain. In the absence of a polyvalent anion, the glutaminase exists as an inactive protomer which has an estimated Mr of 126000. The addition of 100mM-phosphate causes maximal activation and a dimerization (Mr 249000) of the glutaminase. The phosphate activation is sigmoidal, with a K0.5 of 25mM and a Hill coefficient (h) of 1.5 Glutamate inhibition is competitive with respect to glutamine and is decreased by increasing the concentration of phosphate. Phosphate also decreases the Km for glutamine. The purified glutaminase contains a predominant peptide (Mr 65000) and a minor peptide (Mr 68000) that are present in an approximate ratio of 4:1 respectively. The glutaminase immunoprecipitated from freshly solubilized brain tissue or from synaptosomal and non-synaptosomal brain mitochondria contains the same distribution of the two peptides. In contrast, the glutaminase purified from rat kidney contains five to seven peptides that range in Mr value from 59000 to 48000, and immunoprecipitates derived from freshly solubilized renal tissue contain only the Mr-65000 peptide. Partial proteolysis and size fractionation of the three immunoprecipitated peptides indicate that they are structurally related. The series of peptides characteristic of the purified renal glutaminase is generated on storage of the solubilized extract of kidney tissue. The glutaminase contained in the solubilized brain extract is not degraded unless a renal extract is added. Thus the difference in the pattern of peptides associated with the two purified enzymes is due to an endogenous renal proteinase that is not present in brain.     

Calcium stimulation of glutamine hydrolysis in synaptosomes from rat brain

Calcium stimulates the hydrolysis of glutamine in synaptosomes prepared from rat brain both by the sucrose- (12) and the Ficoll/sucrose-gradient techniques (13). The calcium activation is phosphate-dependent and maximal effect is obtained at a calcium concentration of 0.5-1.0 mM. It is reduced by increasing the numbers of synaptosomes in the incubation mixture, and abolished by the product inhibitors of glutaminase, glutamate and ammonia, but unaffected by the uncoupler 2,4-dinitrophenol which inhibits the mitochondrial proton pump. Moreover, since the hydrolysis of glutamine is mediated by glutaminase (EC 3.5.1.2), and calcium does not activate the purified enzyme, an indirect phosphate-dependent effect of calcium on glutaminase is most likely. Calcium activates preferentially the N-ethylmaleimide insensitive fraction of glutaminase. The calcium activation is not dependent on synaptosomal membranes as it is found in synaptosomes subject to previous freezing. It is also found in isolated synaptosomal mitochondria and is thus a property of nerve endings. The calcium activation of glutaminase is unaffected by potassium in depolarizing concentrations, and may not be directly involved in the neurotransmission processes, but possibly in replenishing depleted stores of transmitter glutamate.     

Response of rat liver glutaminase to pH, ammonium, and citrate. Possible regulatory role of glutaminase in ureagenesis

Liver glutaminase is stimulated by an increase in NH4+ concentration and NH4+ is an absolute requirement for activity at approximate physiological concentrations of phosphate and glutamine. Increases in the concentration of NH4+ cannot, however, overcome the inhibitory effect of a decrease in pH. In addition, the concentration of NH4+ required for half-maximal rate decreases as pH increases. This decrease is the result of two factors: a direct effect of pH on the apparent affinity of the enzyme for NH4+, and an indirect effect of pH brought about by an increase in the apparent affinity of the enzyme for phosphate which results in a further decrease in the M0.5 for NH4+. In addition, liver glutaminase responds strongly to the concentration of citrate over a physiologically relevant range at approximate physiological concentrations of NH4+, phosphate, and glutamine. An increase in citrate concentration stimulates glutaminase by increasing the affinity of the enzyme for glutamine. The apparent affinity of the enzyme for citrate increases as pH increases. The strong response of liver glutaminase to pH, NH4+, and citrate and the fact that the hydrolysis of glutamine can supply metabolites and effectors for urea synthesis suggest a possible regulatory role of glutaminase in ureagenesis.     

Glutamine metabolism in isolated perfused rat liver. The transamination pathway

In isolated perfused rat liver, added 4-methyl-thio-2-oxobutyrate and phenylpyruvate are rapidly transaminated to the corresponding amino acids with glutamine, the latter being supplied via the portal vein or by endogenous synthesis. With portal glutamine concentrations below 5mM and in the presence of a oxo-acid acceptor, the flux through glutamine transaminases exceeded the ammonium ion-stimulated glutaminase flux. 4-Methylthio-2-oxobutyrate-induced extra glutamine uptake was not dependent on the perfusate pH in the range of pH 7 to 8. During glutamine/4-methylthio-2-oxobutyrate transamination, the amide nitrogen of glutamine is fully recovered as glutamate, ammonia, urea and alanine. Oxoglutarate formed by omega-amidase activity is released as glutamate or oxidized by oxoglutarate dehydrogenase. alpha-Cyanocinnamate, the inhibitor of the monocarboxylate translocator in the mitochondrial membrane inhibited 4-methylthio-2-oxobutyrate-induced glutamine uptake and methionine release by about 30%. This might indicate that about 2/3 of glutamine transaminase flux is cytosolic. alpha-Cyanocinnamate inhibited 4-methylthio-2-oxobutyrate-induced glutamate efflux by about 90%. Stimulation of flux through glutamine transaminases is accompanied by a 70-80% inhibition of glutaminase flux. This is not explained by a direct inhibition of glutaminase by 4-methylthio-2-oxobutyrate but by a substrate competition between glutaminase and glutamine transaminases. 4-Methylthio-2-oxobutyrate decreases glutamine release by the liver due to withdrawal by transamination. The oxo acid itself is without effect on glutamine synthetase flux. With respect to hepatocyte heterogeneity there is no evidence for a zonal distribution of glutamine transaminase activities, as it has been shown for glutamine synthetase and glutaminase activities.     

Regulation of the activity of the Bacillus licheniformis A5 glutamine synthetase

The regulation of glutamine synthetase activity by positive and negative effectors of enzyme activity singularly and in combinations was studied by using a homogeneous enzyme preparation from Bacillus licheniformis A5. Phosphorylribosyl pyrophosphate at concentrations greater than 2mM stimulated glutamine synthetase activity by approximately 70%. The concentration of phosphorylribosyl pyrophosphate required for half-maximal stimulation of enzyme activity was 0.4 mM. Results obtained from studies of fractional inhibition of glutamine synthetase activity were consistent with the presence of one allosteric site for glutamine binding (apparent I0.5, 2.2mM) per active enzyme unit at a glutamate concentration of 50 mM. At a glutamate concentration of 30 mM or less, the data were consistent with the enzyme containing two binding sites for glutamine (one of which was an allosteric site with an apparent I0.5 of 0.4 mM). Bases on an analysis of the response of glutamine synthetase activity to positive and negative effectors in vitro and to the intracellular concentration of these effectors in vivo, the primary modulators of glutamine synthetase activity in B. licheniformis A5 appear to be glutamine and alanine (apparent I0.5, 5.2mM).     

Characteristics of Triticale glutaminase]

The glutaminase (EC 3.5.1.2) isolated from seedlings of triticale (Triticale sp.) had a pH optimum of about 8, was inhibited with excess substrate (glutamine), and reaction products (glutamate and NH4+). A monovalent anion (Cl-) and a multivalent 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.     

THE EFFECTS OF HYPERKETONEMIA ON GLUTAMATE AND GLUTAMINE METABOLISM IN DEVELOPING RAT BRAIN

Abstract— The effect of increased exposure to ketone bodies in the developing rat brain suggest that intrauterine and postnatal hyperketonemia lead to an altered metabolism of glutamine and glutamate. It is postulated that this effect is related to the delayed development of glutaminase ( l -glutamine amido-hydrolase EC 3.5.1.2) and glutamate dehydrogenase ( l -glutamate: NAD oxidoreductase EC 1.4.1.2).
The specific activities of glutamate dehydrogenase (GDH), glutaminase and glutamine synthetase ( l -glutamate: ammonia ligase EC 6.3.1.2) in the brains of newborn rats increased during early development. A positive correlation was observed between the specific activity of glutaminase and the concentration of glutamate in the brain as well as between the concentrations of blood and brain glutamine and glutamate in both control and hyperketonemic pups. This indicates a different degree of permeability and metabolism for glutamine and glutamate in the brain during the neonatal period, as compared to adulthood.
In hyperketonemic pups, glutamine and glutamate metabolism were found to differ from that in control animals. The concentrations of glutamate were higher, and glutamine lower, in both the blood and brain as compared to that in controls. The concentrations of α-ketoglutarate were also lower in their brain. In the brains of hyperketonemic and control pups, the concentration of malate was the same. During the first 3 weeks of life the increase of spec. act. of GDH and glutaminase was found to be suppressed in the brains of hyperketonemic pups. However, the spec. act. of glutamine synthetase was similar to that of the control pups.     

pH control of hepatic glutamine degradation. Role of transport

Glutamine uptake is decreased in isolated perfused rat liver when the extracellular pH is lowered. This is also observed in the presence of ammonia concentrations nearly 20-fold above that required for half-maximal stimulation of glutaminase, indicating that the effect is not explained by a submaximal ammonium activation of the enzyme. In livers perfused with a physiological glutamine concentration (0.6 mM), the tissue glutamine but not glutamate content is strongly dependent on the extracellular pH and increases from 2.9 mumol/g to 4.7 mumol/g liver when the extracellular pH is increased from 7.3 to 7.5. Subfractionation of the livers revealed that the mitochondrial glutamine concentration increases from about 15 mM to 50 mM, when the extracellular pH is raised from 7.3 to 7.7, whereas the cytosolic glutamine concentration increases only slightly. Simultaneously the cytosolic and mitochondrial pH values are largely unaffected, being 7.25 and 7.7 respectively. Thus, the pH gradient between mitochondria and cytosol remains unchanged when the extracellular pH varies. Amiloride (2 mM) inhibits glutamine uptake by the liver and abolishes the extra/intracellular pH gradient. With amiloride present, tissue glutamine levels are no longer dependent on extracellular pH and are only about 2 mumol/g liver. It is concluded that pH control of glutaminase flux is also mediated by variations of the mitochondrial glutamine concentration pointing to a regulatory role of the glutamine carrier in the mitochondrial membrane for hepatic glutamine breakdown.     

A role for bicarbonate in the regulation of mammalian glutamine metabolism.

1. The concentration of HCO3- (independent of any change of pH) exerts different effects on glutamine metabolism in rat kidney-cortex tubules, hepatocytes and enterocytes.2. In kidney tubules HCO3- (10.5-50 MM) has no effect on glutaminase (EC 3.5.1.2), whereas glutamate dehydrogenase (EC 1.4.1.3) is inhibited as HCO3- concentration is increased. The result is that flux through the entire glutamate-to-glucose pathway is inhibited by increasing HCO3- concentrations. A large proportion (more than 30%) of the glutamine removed undergoes complete oxidation. 3. In hepatocytes, and to a smaller extent in enterocytes, HCO3- is an accelerator of glutaminase. Synthesis of glucose and urea from glutamine in hepatocytes increases as HCO3- concentration is increased. Calculations show that fumarate, formed via aspartate aminotransferase and arginino-succinate lyase, is the precursor of the glucose. There is no complete oxidation of the carbon skeleton of glutamine in hepatocytes. 4. Leucine at near-physiological concentrations (0.1-1 mM) is an accelerator of glutaminase in hepatocytes, but not in kidney tubules or in enterocytes. 5. The results are discussed in relation to regulation of acid/base balance in vivo.     

Imidazole glycerol phosphate synthase from Thermotoga maritima. Quaternary structure, steady-state kinetics, and reaction mechanism of the bienzyme complex

Imidazole glycerol phosphate synthase, which links histidine and de novo purine biosynthesis, is a member of the glutamine amidotransferase family. In bacteria, imidazole glycerol phosphate synthase constitutes a bienzyme complex of the glutaminase subunit HisH and the synthase subunit HisF. Nascent ammonia produced by HisH reacts at the active site of HisF with N'-((5'-phosphoribulosyl)formimino)-5-aminoimidazole-4-carboxamide-ribonucleotide to yield the products imidazole glycerol phosphate and 5-aminoimidazole-4-carboxamide ribotide. In order to elucidate the interactions between HisH and HisF and the catalytic mechanism of the HisF reaction, the enzymes tHisH and tHisF from Thermotoga maritima were produced in Escherichia coli, purified, and characterized. Isolated tHisH showed no detectable glutaminase activity but was stimulated by complex formation with tHisF to which either the product imidazole glycerol phosphate or a substrate analogue were bound. Eight conserved amino acids at the putative active site of tHisF were exchanged by site-directed mutagenesis, and the purified variants were investigated by steady-state kinetics. Aspartate 11 appeared to be essential for the synthase activity both in vitro and in vivo, and aspartate 130 could be partially replaced only by glutamate. The carboxylate groups of these residues could provide general acid/base catalysis in the proposed catalytic mechanism of the synthase reaction.     

Regulatory effects of fatty acyl-coenzyme A derivatives on phosphate-activated pig brain and kidney glutaminase in vitro.

1. Fatty n-acyl-CoA derivatives in the concentration range 5muM-0.1mM and with 5-18 fatty acyl carbons have dual effects on phosphate-activated glutaminase from pig brain and kidney. Generally, fatty acyl-CoA derivatives in low concentrations activate the enzyme, but inhibit at higher concentrations; phosphate and citrate potentiate the activation, displaying positive co-operatively, and protect against inactivation. The fatty acyl-CoA derivatives affect glutaminase similarly to Bromothymol Blue, but differently from acetyl-CoA, which activates the enzyme only at very low phosphate or citrate concentrations. 2. Saturated fatty acyl-CoA derivatives, with 5-10 fatty acyl carbons, only activate the enzyme in the concentration range 0-0.1 mM. When the fatty acyl chain is elongated, the fatty acyl-CoA derivatives gradually become more powerful inhibitors of glutaminase at the expense of their activating capacity. In particular, palmitoyl-CoA and stearoyl-CoA are strong inhibitors at concentrations (10 muM) at which the corresponding free fatty acids and fatty acyl-carnitine derivatives have no effect. 3. The unsaturated fatty acyl-CoA derivatives, oleoyl-CoA and linoleoyl-CoA, behave as potent activators in the lower part of the concentration range tested (0-0.05mM), and as inhibitors in the upper part of this range (0.02-0.10mM). Oleic acid and linoleic acid have similar properties, but their activating capacity is less pronounced. 4. Phosphate both prevented and reversed the inhibition, but no restoration of activity was possible once the enzyme became inactivated. 5. By changing the pH from 7.0 to 8.0 the activating capacity of the fatty acyl-CoA derivatives is increased, as is their concentration range for activation. 6. The fatty acyl-CoA derivatives are somewhat more potent activator for brain glutaminase, but otherwise they affect the two enzymes similarly.     

The orientation of phosphate-dependent glutaminase on the inner membrane of rat renal mitochondria

Phosphate-dependent glutaminase is associated with the inner membrane of rat renal mitochondria. The orientation of this enzyme was characterized by comparing its sensitivity in isolated mitochondria and in mitoplasts to two membrane impermeable inhibitors. Mitoplasts were prepared by repeated swelling of mitochondria in a hypotonic phosphate solution. This procedure released greater than 70% of the adenylate kinase from the intermembrane space, but less than 10 and 25% of the marker activities characteristic of the inner membrane and matrix compartments, respectively. The addition of 20 microM p-chloromercuriphenylsulfonate (pCMPS) caused a rapid inactivation of the purified glutaminase. In contrast, the glutaminase contained in isolated mitochondria and mitoplasts was only slightly affected by the addition of up to 2 mM pCMPS. Similarly, the activity in mitochondria and mitoplasts was not inhibited by the addition of an excess of inactivating Fab antibodies. However, a similar extent of inactivation occurred when either membrane fraction was incubated with concentrations of octylglucoside greater than 0.35%. Mitochondria were also treated with increasing concentrations of digitonin. At 0.4 mg digitonin/mg protein, all of the adenylate kinase was released but the glutaminase activity was either slightly inhibited or unaffected by the addition of pCMPS or the Fab antibodies, respectively. These studies establish that the glutaminase is localized on the inner surface of the inner membrane. Therefore, mitochondrial catabolism of glutamine must occur only within the matrix compartment.