The activity of glutaminase in the human placenta

Homogenates and extracts of human placenta are able to desamidate glutamine by means of an enzyme which has the properties of glutaminase. Placental glutaminase is activated by phosphate. Its pH optimum lies at 9.0.A method for its assay in placental homogenate is described. It was found that the glutaminase activity decreases toward the end of pregnancy. At this time, the activity, expressed as Q_NH3 (N), amounts to 23.7 ± 6.7.Some quantitative aspects of glutaminase activity in the human placenta and kidney are discussed.     

Glutaminase-containing microvesicles from HIV-1-infected macrophages and immune-activated microglia induce neurotoxicity

Background

HIV-1-infected and/or immune-activated microglia and macrophages are pivotal in the pathogenesis of HIV-1-associated neurocognitive disorders (HAND). Glutaminase, a metabolic enzyme that facilitates glutamate generation, is upregulated and may play a pathogenic role in HAND. Our previous studies have demonstrated that glutaminase is released to the extracellular fluid during HIV-1 infection and neuroinflammation. However, key molecular mechanisms that regulate glutaminase release remain unknown. Recent advances in understanding intercellular trafficking have identified microvesicles (MVs) as a novel means of shedding cellular contents. We posit that during HIV-1 infection and immune activation, microvesicles may mediate glutaminase release, generating excessive and neurotoxic levels of glutamate.

Results

MVs isolated through differential centrifugation from cell-free supernatants of monocyte-derived macrophages (MDM) and BV2 microglia cell lines were first confirmed in electron microscopy and immunoblotting. As expected, we found elevated number of MVs, glutaminase immunoreactivities, as well as glutaminase enzyme activity in the supernatants of HIV-1 infected MDM and lipopolysaccharide (LPS)-activated microglia when compared with controls. The elevated glutaminase was blocked by GW4869, a neutral sphingomyelinase inhibitor known to inhibit MVs release, suggesting a critical role of MVs in mediating glutaminase release. More importantly, MVs from HIV-1-infected MDM and LPS-activated microglia induced significant neuronal injury in rat cortical neuron cultures. The MV neurotoxicity was blocked by a glutaminase inhibitor or GW4869, suggesting that the neurotoxic potential of HIV-1-infected MDM and LPS-activated microglia is dependent on the glutaminase-containing MVs.

Conclusions

These findings support MVs as a potential pathway/mechanism of excessive glutamate generation and neurotoxicity in HAND and therefore MVs may serve as a novel therapeutic target.

     

Purification and characterization of a glutaminase enzyme accounting for the majority of glutaminase activity in Aspergillus sojae under solid-state culture

Glutaminase, an enzyme that hydrolyzes l-glutamine to l-glutamate, plays an important role in the production of fermented foods by enhancing the umami taste. In this study, we found ten glutaminase genes in the Aspergillus sojae genome by conducting a BLAST search of the characterized glutaminase sequence. We subsequently constructed glutaminase gene disruptants. The glutaminase activity of the gahB disruptant was decreased by approximately 90 % in A. sojae and Aspergillus oryzae, indicating that this enzyme (GahB) accounted for the majority of the glutaminase activity in Aspergillus species. Subsequently, GahB protein was purified from the AsgahB-overexpressing transformant and characterized. The molecular mass was estimated to be approximately 110 and 259 kDa by SDS-PAGE and gel filtration chromatography, respectively, indicating that the native form of AsGahB was a dimer. The optimal pH was 9.0, and the optimal temperature was 50 °C. Analysis of substrate specificity revealed that AsGahB had peptidoglutaminase-asparaginase activity, similar to AsGahA, but preferred free l-glutamine to free l-asparagine, C-terminal glutaminyl, and asparaginyl residues in peptides.     

Functional intracellular glutaminase activity in intact astrocytes

Numerous cellular metabolites such as glutamine, glutamate, phosphate, calcium, ammonia and acetyl derivatives are known to affect the phosphate-activated glutaminase activity in whole cell homogenates or extracts. Since measurements in extracts under non-physiological conditions may obscure the actual intracellular metabolic flux, the functional intracellular phosphate-activated glutaminase activity was measured by the formation of³H₂O froml-[2-³H]glutamine (Anal. Biochem. 127:134–142, 1982) in cultures of intact astrocytes, untreated and treated with dibutyryl c-AMP (DiBcAMP), in the presence of several potential effectors. These values were compared with enzyme levels determined in extracts from identical cells. The rate of¹⁴CO₂ release froml-[1-¹⁴C]glutamine was also measured in both untreated and DiBcAMP treated astrocytes. The intracellular activity of glutaminase for untreated cells assayed in MEM medium with 1mM radioactive glutamine was 88 nmol/mg protein/h and in DiBcAMP treated cells the rate was 153 nmol/mg protein/h. However, the enzymatic activity measured under optimal conditions in extracts from both untreated and treated cells was much higher, but essentially the same, about 1,750 nmol/mg protein/h. The rate of¹⁴CO₂ release froml-[1-¹⁴C]glutamine was 74 and 133 nmol/mg protein/h in untreated and DiBcAMP treated cells, respectively. This represents approximately 85% of the intracellular glutaminase activity. Furthermore, increasing the concentration of glutamine in the medium from 1 to 6.4 mM increased glutaminase intracellular activity about 3 fold in both untreated and treated cells. Addition of 250 M glutamate to the medium inhibited intracellular glutaminase activity by 70% under both treatment conditions. Deletion of glucose stimulated glutaminase activity. In contrast the removal of fetal bovine serum decreased activity by 35%. The addition of 10 mM phosphate and the alpha keto acids of isoleucine and valine marginally increased intracellular glutaminase activity. The addition of 0.4 mM ammonium chloride to the medium had no effect. An increase in media pH from 6.8 to 7.7 increased intracellular glutaminase activity almost 2 fold. These results provide evidence that phosphate-activated glutaminase activity in vivo is regulated by cellular metabolites, that its functional activity is 5–9% of the rate obtained using extracts, and this functional activity is sufficient to account for the rate of glutamine oxidation.Special issue dedicated to Dr. Elling Kvamme     

Variations in the kinetic response of several different phosphate-dependent glutaminase isozymes during acute metabolic acidosis

Summary We describe the kinetic modifications to mitochondrial-membrane-bound phosphate-dependent glutaminase in various types of rat tissue brought about by acute metabolic acidosis. The activity response of phosphate-dependent glutaminase to glutamine was sigmoidal, showing positive co-operativity, the Hill coefficients always being higher than 2. The enzyme from acidotic rats showed increased activity at subsaturating concentrations of glutamine in kidney tubules, as might be expected, but not in brain, intestine or liver tissues. Nevertheless, when brain and intestine from control rats were incubated in plasma from acutely acidotic rats enzyme activity increased at 1 mM glutamine in the same way as in kidney cortex. The enzyme from liver tissue remained unaltered. S_0.5 and n_H values decreased significantly in kidney tubules, enterocytes and brain slices preincubated in plasma from acidotic rats. The sigmoidal curves of phosphate-dependent glutaminase shifted to the left without any significant changes in V_max. The similar response of phosphate-dependent glutaminase to acute acidosis in the kidney, brain and intestine confirms the fact that enzymes from these tissues are kinetically identical and reaffirms the presence of an ammoniagenic factor in plasma, either produced or concentrated in the kidneys of rats with acute acidosis.Abbreviations Hepes 4-(2-hydroxyethyl)-1-piperazineethanesulphonic acid - EDTA NN-1,2-Ethane-diylbis [N-(carboxymethyl)glycyne] - Tris 2-amino-2-hydroxymethyl-1,3-propanediol - PDG phosphate dependent glutaminase Publication No. 145 from Drogas, Tóxicos Ambientales y Metabolismo Celular Research Group. Department of Biochemistry and Molecular Biology, University of Granada, Spain     

Digestion by serine proteases enhances salt tolerance of glutaminase in the marine bacterium <Emphasis Type="Italic">Micrococcus luteus</Emphasis> K-3

Salt-tolerant glutaminase (Micrococcus glutaminase, with an apparent molecular mass of 48.3 kDa, intact glutaminase) from the marine bacterium Micrococcus luteus K-3 was digested using protease derived from M. luteus K-3. The digestion products were a large fragment (apparent molecular mass of 38.5 kDa, the glutaminase fragment) and small fragments (apparent molecular mass of 8 kDa). The digestion was inhibited by phenylmethanesulfonyl fluoride (PMSF). Digestion of intact glutaminase by serine proteases including trypsin, elastase, lysyl endopeptidase, and arginylendopeptidase also produced the glutaminase fragment. The N-terminus of the glutaminase fragment was the same as that of intact glutaminase. The N-termini of two small fragments were Ala394 and Ala396, respectively. The enzymological and kinetic properties of the glutaminase fragment were almost the same as those of intact glutaminase except for salt-tolerant behavior. The glutaminase fragment was a higher salt-tolerant enzyme than the intact glutaminase, suggesting that Micrococcus glutaminase is digested in the C-terminal region by serine protease from M. luteus K-3 to confer salt tolerance on glutaminase.     

An Enzyme Hydrolyzing l-Theanine in Tea Leaves

Theanine hydrolase activity in tea leaves was assayed by measuring enzymatically released ethylamine from l-theanine. The o-phthalaldehyde derivative of ethylamine was measured by reverse phase HPLC recorded with a spectrofluorometric detector.

Theanine hydrolase activity was purified about 4.6-fold by DEAE-cellulose column chromatography. Although this active fraction also had glutaminase activity, the yield of the glutaminase activity was about 50% of that of theanine hydrolytic activity. The theanine hydrolytic activity was inhibited by acidic amino acid and l-alanine, and stimulated by l-malic acid. The purified enzyme solution hydrolyzed not only theanine but also γ-glutamylmethylamide, γ-glutamyl-n-propylamide, γ-glutamyl-n-butylamide, γ-glutamyl-iso-butylamide, and γ-glutamyl-n-amylamide, which were synthesized from l-pyroglutamic acid and corresponding alkylamines. However, N-methylpropionamide and N-ethylpropionamide were not hydrolyzed. The theanine hydrolase activity and glutaminase in tea leaves showed the same pH optimum (8.5).

The activity of theanine hydrolase in tea leaves increased during the first lOhr after plucking but then decreased gradually, while that of glutaminase decreased constantly and was almost lost     

Physiological role of glutaminase activity in Saccharomyces cerevisiae

The participation of glutaminase activity in glutamine degradation was studied in a wild-type strain (S288C) of Saccharomyces cerevisiae. Evidence is presented that this strain has two glutaminase activities, a readily extractable form (glutaminase B) and a membrane-bound enzyme (glutaminase A). Glutaminase A and B activities could also be distinguished by their thermostability, pyruvate sensitivity and pH optimum. Glutaminase B activity was negatively modulated by some 2-oxo acids, and in vivo pyruvate accumulation inhibited this activity. A mutant strain (CN10) with an altered glutaminase B activity was isolated and partially characterized. Its glutaminase B activity was more sensitive to inhibition by pyruvate and 2-oxoglutarate than the wild type, thus resulting in inactivation of this enzyme in vivo. The physiological role of glutaminase activity is discussed with regard to the phenotype shown by the mutant strain.     

Properties of rat renal phosphate-dependent glutaminase coupled to Sepharose. Evidence that dimerization is essential for activation.

In the absence of phosphate, purified rat renal phosphate-dependent glutaminase exists as a catalytically inactive protomer. The addition of phosphate results in both dimerization and activation of the glutaminase. Covalent attachment of the dimeric form of the glutaminase to CNBr-activated Sepharose was achieved with 84% retention of activity. At least 70% of the bound glutaminase activity was expressed even in the absence of added phosphate. In addition, 6-diazo-5-oxo-L-norleucine, which interacts only with the catalytically active form of the glutaminase, inactivates the bound dimeric form of glutaminase at the same rate in either the absence or the presence of added phosphate. Therefore retention of dimeric structure is apparently sufficient to maintain glutaminase activity. In contrast, the coupling of the protomeric form of the enzyme to Sepharose resulted in retention of only 3% of the phosphate-induced glutaminase activity. However, up to 48% of this activity could be reconstituted by addition of soluble glutaminase under conditions that promote dimerization. These results indicate that the monomeric form of the glutaminase has minimal inherent activity and that dimerization is an essential step in the phosphate-induced activation of the glutaminase.     

Aspartate aminotransferase and glutaminase activities in rat olfactory bulb and cochlear nucleus; Comparisons with retina and with concentrations of substrate and product amino acids

The quantitative distributions of aspartate aminotransferase and glutaminase were mapped in subregions of olfactory bulb and cochlear nucleus of rat, and were compared with similar data for retina and with the distributions of their substrate and product amino acids aspartate, glutamate, and glutamine. The distributions of both enzymes paralleled that of aspartate in the olfactory bulb and that of glutamate in the cochlear nucleus. In retina (excluding inner segments), there were similarities between aspartate aminotransferase and both glutamate and aspartate distributions. The distribution of -aminobutyrate (GABA) was similar to those of both enzymes in olfactory bulb, to aspartate aminotransferase in cochlear nucleus, and to glutaminase in retina (excluding inner segments). The results are consistent with significant involvement of aspartate aminotransferase, especially the cytosolic isoenzyme, and glutaminase in accumulation of the neurotransmitter amino acids glutamate, aspartate, and GABA, although with preferential accumulation of different amino acids in different brain regions.     

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.     

Properties of phosphate activated glutaminase in astrocytes cultured from mouse brain

Astrocytes in primary cultures contain a relatively high activity, of phosphate activated glutaminase, although it is significantly lower than that of synaptosomal enriched preparations. The relatively high glutaminase activity in the astrocytes appears not to be caused by substrate induction, since a 10-fold variation in the glutamine concentration of the culture medium does not affect the activity. Of the reaction products, only glutamate inhibits astrocytic glutaminase whereas that of synaptosomal enriched preparations is inhibited by both glutamate and ammonia. Similar to the synaptosomal enzyme, glutaminase in astrocytes is inhibited about 50% by N-ethylmaleimide, indicating N-ethylmaleimide-sensitive and-insensitive compartments of the enzyme. Calcium activates glutaminase in astrocytes as in synaptosomes, by promoting phosphate activation. Except for the lower activity and the lack of effect of ammonia, the properties of the astroglial glutaminase has been found to be no different from that of the synaptosomal one. The relatively unrestrained astroglial glutaminase may, however, argue against the concept of a glutamine cycle operating in a stoichiometric manner.Abbreviations NEM N-ethylmaleimide - PAG Phosphate-activated glutaminase - PMB p-mercuribenzoate     

Phosphate-dependent glutaminase from rat kidney. Cause of increased activity in response to acidosis and identity with glutaminase from other tissues.

Immune serum was prepared against phosphate-dependent glutaminase purified from rat kidney and was used to investigate the cause of increased renal glutaminase activity in acidotic rats. Crude kidney homogenates from acidotic rats exhibited a fourfold greater specific activity for phosphate-dependent glutaminase. The glutaminase was solubilized initially by lyophilization of borate treated mitochondria with a 40–60% recovery and with maintenance of threefold difference in specific activity. Both preparations showed the same equivalence point in a quantitative precipitin experiment. To confirm these results, phosphate-dependent glutaminase was also solubilized by treatment of mitochondria isolated from normal and acidotic rat kidney cortex with 1% Triton X-100. The two preparations exhibited a fivefold difference in specific activity and again showed the same equivalence point in a quantitative precipitin experiment. These results indicate that the cause of increased phosphate-dependent glutaminase activity during acidosis is due to the presence of an increased amount of this enzyme. The antiserum prepared against the kidney phosphate-dependent glutaminase did not crossreact with glutaminase solubilized from rat liver mitochondria. But, rat brain mitochondria do contain a phosphate-dependent glutaminase that is immunologically identical to the enzyme from rat kidney.     

Isolation and characterization of salt-tolerant glutaminases from marine Micrococcus luteus K-3

Marine Micrococcus luteus K-3 constitutively produced two salt-tolerant glutaminases, designated glutaminase I and II. Glutaminase I was homogeneously purified about approximately, 1620-fold with a 4% yield, and was a dimer with a molecular weight of about 86,000. Glutaminase II was partially purified about 190-fold with a 0.04% yield. The molecular weight of glutaminase II was also 86,000. Maximum activity of glutaminase I was observed at pH 8.0, 50°C and 8–16% NaCl. The optimal pH and temperature of glutaminase II were 8.5 and 50°C. The activity of glutaminase II was not affected by the presence of 8 to 16% NaCl. The presence of 10% NaCl enhanced thermal stability of glutaminase I. Both enzymes catalyzed the hydrolysis of l-glutamine, but not its hydroxylaminolysis. The K_m values for l-glutamine were 4.4 (glutaminase I) and 6.5 mM (glutaminase II). Neither of the glutaminases were activated by the addition of 2 mM phosphate or 2 mM sulfate. p-Chloromercuribenzoate (0.01 mM) significantly inhibited glutaminase I, but not glutaminase II. The conserved sequences LA**V and V**GGT*A were observed in the N-terminal amino acid sequences of glutaminase I, similar to that for other glutaminases.     

Microbial glutaminase: biochemistry, molecular approaches and applications in the food industry

Glutaminase is widely distributed in microorganisms including bacteria, yeast and fungi. The enzyme mainly catalyzes the hydrolysis of γ-amido bond of -glutamine. In addition, some enzymes also catalyze γ-glutamyl transfer reaction. A highly savory amino acid, -glutamic acid and a taste-enhancing amino acid of infused green tea, theanine can be synthesized by employing hydrolytic or transfer reaction catalyzed by glutaminase. Therefore, glutaminase is one of the most important flavor-enhancing enzymes in food industries. In this review, subsequent to a discussion on the definition of glutaminase, the enzymatic properties, applications of glutaminase in the food industry, and occurrence and distribution of the enzyme are described. We then illustrate the gene cloning, primary structure, and 3D-structure of glutaminase. Finally, to facilitate the future applications of glutaminase in food fermentations, the mechanisms of action of salt-tolerant glutaminase are briefly discussed.     

Phosphate-independent glutaminase from rat kidney. Partial purification and identity with gamma-glutamyltranspeptidase.

Phosphate-independent glutaminase can be quantitatively solubilized from a microsomal preparation of rat kidney by treatment with papain. Subsequent gel filtration and chromatography on quaternary aminoethyl (QAE)-Sephadex and hydroxylapatite yield a 200-fold purified preparation of this glutaminase. The purified enzyme also hydrolyzes gamma-glutamylhydroxamate and exhibits substrate inhibition at high concentrations of either glutamine or gamma-glutamyhydroxamate, which is partially relieved by increasing concentrations of maleate. Rat kidney phosphate-independent glutaminase reaction is catalyzed by the same enzyme which catalyzes the gamma-glutamyltranspeptidase reaction. The ratio of glutaminase to transpeptidase activities remained constant throughout a 200-fold purification of this enzyme. The observation that the phosphate0independent glutaminase and gamma-glutamyltranspeptidase activities exhibit coincident mobilities during electrophoresis, both before and after extensive treatment with neuraminidase, strongly suggests that both reactions are catalyzed by the same enzyme. This conclusion is strengthened by the observation that maleate and various amino acids have reciprocal effects on the two activities. Maleate increases glutaminase activity and blocks transpeptidation, whereas amino acids activate the transpeptidase but inhibit glutaminase activity. In contrast, the addition of both maleate and alanine resulted in a strong inhibition of both activities. Both activities exhibit a similar distribution in the various regions of the kidney. Recovery of maximal activities in the outer stripe region of the medulla is consistent with previous quantitative microanalysis which indicated that this glutaminase activity is localized primarily in the proximal straight tubule cells. The glutaminase and transpeptidase activities have different pH optima. Examination of the product specificity suggests that decreasing pH also promotes glutaminase activity and that below pH 6.0, this enzyme functions strictly as a glutaminase. Because of the localization of this activity on the brush border membrane, these resuts are consistent with the possibility that the physiological conditions induced by metabolic acidosis could convert this enzyme from a broad specificity transpeptidase to a glutaminase. Therefore, this enzyme could contribute to the increased renal synthesis of ammonia from glutamine which is observed during metabolic acidosis.     

Regulation of glutaminase levels in Escherichia coli.

Nitrogenous metabolites, cyclic adenosine 3':5'-monophosphate (cAMP), and the stage of culture growth all influence the levels of glutaminase A in Escherichia coli, but no variables in culture conditions alter the levels of glutaminase B. Growth of E. coli on culture media containing glucose and excess ammonia results in a rise in the level of glutaminase A as the cultures enter stationary phase; this rise is abolished by ammonia limitation. cAMP or glycerol reduce the level of glutaminase A. In mutants deficient in cAMP receptor protein, glutaminase A levels are unchanged by cAMP, but they are still susceptible to regulation by ammonia. We consider glutaminase B to be a constitutive enzyme, since its levels appear independent of nutritional conditions.     

Crystal structure of imidazole glycerol phosphate synthase: a tunnel through a (beta/alpha)8 barrel joins two active sites

BACKGROUND: Imidazole glycerol phosphate synthase catalyzes a two-step reaction of histidine biosynthesis at the bifurcation point with the purine de novo pathway. The enzyme is a new example of intermediate channeling by glutamine amidotransferases in which ammonia generated by hydrolysis of glutamine is channeled to a second active site where it acts as a nucleophile. In this case, ammonia reacts in a cyclase domain to produce imidazole glycerol phosphate and an intermediate of purine biosynthesis. The enzyme is also a potential target for drug and herbicide development since the histidine pathway does not occur in mammals. RESULTS: The 2.1 A crystal structure of imidazole glycerol phosphate synthase from yeast reveals extensive interaction of the glutaminase and cyclase catalytic domains. At the domain interface, the glutaminase active site points into the bottom of the (beta/alpha)(8) barrel of the cyclase domain. An ammonia tunnel through the (beta/alpha)(8) barrel connects the glutaminase docking site at the bottom to the cyclase active site at the top. A conserved "gate" of four charged residues controls access to the tunnel. CONCLUSIONS: This is the first structure in which all the components of the ubiquitous (beta/alpha)(8) barrel fold, top, bottom, and interior, take part in enzymatic function. Intimate contacts between the barrel domain and the glutaminase active site appear to be poised for crosstalk between catalytic centers in response to substrate binding at the cyclase active site. The structure provides a number of potential sites for inhibitor development in the active sites and in a conserved interdomain cavity.     

Identification of two glutaminases inRhizobium etli

We present evidence thatRhizobium etli has two glutaminases differentiated by their thermostability and electrophoretic mobility. The thermostable glutaminase (B) is constitutive, in contrast with the thermolabile glutaminase (A), which is positively regulated by glutamine and negatively regulated by ammonium and by the carbon source. In distinction to glutaminase A, glutaminase B plays a minor role in the utilization of glutamine as a carbon source, but it may play a role in maintaining the balance of glutamine and glutamate. By complementation of theRhizobium etli LM16 mutant that lacks glutaminase A, we have cloned the gene that codes for this enzyme.     

Studies on the mechanism of the glutamine-dependent reaction catalyzed by asparagine synthetase from mouse pancreas

Initial velocity and product inhibition studies were conducted with the glutamine-dependent reaction of asparagine synthetase from mouse pancreas. Double reciprocal plots of glutamine versus either aspartate or ATP were parallel, while aspartate versus ATP gave intersecting patterns. These patterns are indicative of a hybrid ping-pong mechanism consisting of a glutaminase partial reaction and a sequential catalysis involving aspartate and ATP. Inhibition patterns of the four products, glutamate, AMP, PPi, and asparagine, versus each of the three substrates are consistent with a hybrid Uni Uni Bi Ter Ping Pong Theorell-Chance mechanism where the glutaminase reaction occurs first and aspartate binds to the enzyme before ATP in the sequential segment. PPi is the first product released in the Theorell-Chance reaction, which is followed by the ordered release of AMP and asparagine. Product inhibition patterns also indicate the formation of E . NH3 . Asn and E . NH3 . Asp . AMP abortive complexes. Although an amide site (for glutamine and asparagine), presumably responsible for the glutaminase reaction, an acid site (for glutamate and aspartate), and a nucleotide site are involved in the overall catalysis, the "two-site" ping-pong mechanism is incompatible with the experimentally observed product inhibition patterns.