N3-haloacetyl derivatives of L-2,3-diaminopropanoic acid: novel inactivators of glucosamine-6-phosphate synthase.

N3-Haloacetyl derivatives of L-2,3-diaminopropanoic acid, novel glutamine analogs, were shown to be strong inhibitors of glucosamine-6-phosphate synthase from bacteria and Candida albicans. The inhibition was competitive with respect to glutamine and non-competitive with respect to D-fructose-6-phosphate. In the absence of glutamine, the tested compounds inactivated glucosamine-6-phosphate synthase from C. albicans with Kinact = 0.5 microM, 0.55 microM and 18.5 microM for bromoacetyl-, iodoacetyl- and chloroacetyl derivatives of L-2,3-diaminopropanoic acid, respectively. The inactivation obeyed the criteria for active site-directed modification.     

Cytotoxic mechanisms of glutamine antagonists in mouse L1210 leukemia

The glutamine antagonists, acivicin (NSC 163501), azaserine (NSC 742), and 6-diazo-5-oxo-L-norleucine (DON) (NSC 7365), are potent inhibitors of many glutamine-dependent amidotransferases in vitro. Experiments performed with mouse L1210 leukemia growing in culture show that each antagonist has different sites of inhibition in nucleotide biosynthesis. Acivicin is a potent inhibitor of CTP and GMP synthetases and partially inhibits N-formylglycineamidine ribotide (FGAM) synthetase of purine biosynthesis. DON inhibits FGAM synthetase, CTP synthetase, and glucosamine-6-phosphate isomerase. Azaserine inhibits FGAM synthetase and glucosamine-6-phosphate isomerase. Large accumulations of FGAR and its di- and triphosphate derivatives were observed for all three antagonists which could interfere with the biosynthesis of nucleic acids, providing another mechanism of cytotoxicity. Acivicin, azaserine, and DON are not potent inhibitors of carbamyl phosphate synthetase II (glutamine-hydrolyzing) and amidophosphoribosyltransferase in leukemia cells growing in culture although there are reports of such inhibitions in vitro. Blockade of de novo purine biosynthesis by these three antagonists results in a "complementary stimulation" of de novo pyrimidine biosynthesis.     

Glucosamine-6-phosphate synthase, a novel target for antifungal agents. Molecular modelling studies in drug design

Fungal infections are a growing problem in contemporary medicine, yet only a few antifungal agents are used in clinical practice. In our laboratory we proposed the enzyme L-glutamine: D-fructose-6-phosphate amidotransferase (EC 2.6.1.16) as a new target for antifungals. The structure of this enzyme consists of two domains, N-terminal and C-terminal ones, catalysing glutamine hydrolysis and sugar-phosphate isomerisation, respectively. In our laboratory a series of potent selective inhibitors of GlcN-6-P synthase have been designed and synthesised. One group of these compounds, including the most studied N3-(4-methoxyfumaroyl)-l-2,3-diaminopropanoic acid (FMDP), behave like glutamine analogs acting as active-site-directed inactivators, blocking the N-terminal, glutamine-binding domain of the enzyme. The second group of GlcN-6-P synthase inhibitors mimic the transition state of the reaction taking place in the C-terminal sugar isomerising domain. Surprisingly, in spite of the fact that glutamine is the source of nitrogen for a number of enzymes it turned out that the glutamine analogue FMDP and its derivatives are selective against GlcN-6-P synthase and they do not block other enzymes, even belonging to the same family of glutamine amidotransferases. Our molecular modelling studies of this phenomenon revealed that even within the family of related enzymes substantial differences may exist in the geometry of the active site. In the case of the glutamine amidotransferase family the glutamine binding site of GlcN-6-P synthase fits a different region of the glutamine conformational space than other amidotransferases. Detailed analysis of the interaction pattern for the best known, so far, inhibitor of the sugar isomerising domain, namely 2-amino-2-deoxy-D-glucitol-6-phosphate (ADGP), allowed us to suggest changes in the structure of the inhibitor that should improve the interaction pattern. The novel ligand was designed and synthesised. Biological experiments confirmed our predictions. The new compound named ADMP is a much better inhibitor of glucosamine-6-phosphate synthase than ADGP.     

Amide and ester derivatives of N3-(4-methoxyfumaroyl)-(S)-2,3-diaminopropanoic acid: the selective inhibitor of glucosamine-6-phosphate synthase

Several amide and ester derivatives of a glutamine analogue, N3-(4-methoxyfumaroyl)-(S)-2,3-diaminopropanoic acid (FMDP) (1-8), were synthesized and evaluated for the inhibitory activity in regard to glucosamine-6-phosphate synthase from Candida albicans. The syntheses were accomplished by the reaction of N2-tert-butoxycarbonyl-N3-(4-methoxyfumaroyl)-(S)-2,3-diaminopropanoic acid (BocFMDP) with the corresponding amines to give the FMDP amides (1-4) or with alkyl halides to give corresponding esters of FMDP (5-8). Among the synthesized compounds, the acetoxymethyl ester of FMDP was the most active inhibitor of the enzyme. Its IC50 value compared to that of FMDP (4 microM) was equal to 11.5 microM. The methyl and allyl esters and the N-hexyl-N-methyl-amide of FMDP exhibited a moderate enzyme inhibitory activity.     

Discovery of a metabolic pathway mediating glucose-induced desensitization of the glucose transport system. Role of hexosamine biosynthesis in the induction of insulin resistance

Based on our previous finding that desensitization of the insulin-responsive glucose transport system (GTS) requires three components, glucose, insulin, and glutamine, we postulated that the routing of incoming glucose through the hexosamine biosynthesis pathway plays a key role in the development of insulin resistance in primary cultured adipocytes. Two approaches were used to test this hypothesis. First, we assessed whether glucose-induced desensitization of the GTS could be prevented by glutamine analogs that irreversibly inactivate glutamine-requiring enzymes, such as glutamine:fructose-6-phosphate amidotransferase (GFAT) the first and the rate-limiting enzyme in hexosamine biosynthesis. Both O-diazoacetyl-L-serine (azaserine) and 6-diazo-5-oxonorleucine inhibited desensitization in 18-h treated cells without affecting maximal insulin responsiveness in control cells. Moreover, close agreement was seen between the ability of azaserine to prevent desensitization of the GTS in intact adipocytes (70% inhibition, ED50 = 1.1 microM), its ability to inactivate GFAT in intact adipocytes (64% inhibition, ED50 = 1.0 microM) and its ability to inactivate GFAT activity in a cytosolic adipocyte preparation (ED50 = 1.3 microM). From these results we concluded that a glutamine amidotransferase is involved in the induction of insulin resistance. As a second approach, we determined whether glucosamine, an agent known to preferentially enter the hexosamine pathway at a point distal to enzymatic amidation by GFAT, could induce cellular insulin resistance. When adipocytes were exposed to various concentrations of glucosamine for 5 h, progressive desensitization of the GTS was observed (ED50 = 0.36 mM) that culminated in a 40-50% loss of insulin responsiveness. Moreover, we estimated that glucosamine is at least 40 times more potent than glucose in mediating desensitization, since glucosamine entered adipocytes at only one-quarter of the glucose uptake rate, yet induced desensitization at an extra-cellular dose 10 times lower than glucose. In addition, we found that glucosamine-induced desensitization did not require glutamine and was unaffected by azaserine treatment. Thus, we conclude that glucosamine enters the hexosamine-desensitization pathway at a point distal to GFAT amidation. Overall, these studies indicate that a unique metabolic pathway exists in adipocytes that mediates desensitization of the insulin-responsive GTS, and reveal that an early step in this pathway involves the conversion of fructose 6-phosphate to glucosamine 6-phosphate by the first and rate-limiting enzyme of the hexosamine pathway, glutamine:fructose-6-phosphate amidotransferase.     

Facilitated diffusion of glucosamine-6-phosphate synthase inhibitors enhances their antifungal activity

N3-(4-Methoxyfumaroyl)-L-2,3-diaminopropanoic acid (FMDP) and 2-amino-2-deoxy-D-glucitol-6-phosphate (ADGP) are strong inhibitors of the essential fungal enzyme, glucosamine-6-phosphate synthase, but their antifungal activity is poor, due to slow penetration of these agents through the cytoplasmic membrane. In the present studies we have exploited the possibility of enhancement of ADGP and FMDP antifungal activity by improving their transport properties. It has been found that membrane-permeabilising polyene macrolides amphotericin B (AMB) and its N-methyl-N-fructosyl methyl ester derivative (MF-AME), at subinhibitory concentrations, facilitate diffusion of ADGP through the fungal cell membrane, thus allowing a decrease of its minimal inhibitory concentration (MIC). Synergistic effects have been observed for combinations of ADGP with AMB or MF-AME. Fractional inhibitory concentration (FIC) indexes, determined against a number of Candida spp., have been in the 0.18-0.81 range. Weak antifungal synergistic effects have been found for combinations of FMDP with AMB or MF-AME. ADGP can be easily encapsulated into unilamellar lipid vesicles. Liposomal preparations of ADGP demonstrated stronger antifungal activity against some fungal strains than free ADGP.     

The hexosamine biosynthetic pathway and glucose-induced down regulation of glucose transport in L6 myotubes

Based on experiments in cultured adipocytes, it has been proposed that glucose-induced down regulation of glucose transport is mediated by the conversion of fructose-6-phosphate to glucosamine-6-phosphate via the first and rate-determining enzyme of the hexasamine biosynthetic pathway, glutamine: fructose-6-phosphate amidotransferase (glutamine hexosephosphate aminotransferase). Evidence for this assertion was: (a) l-glutamine, the provider group for the aminotransferase was essential; (b) two inhibitors of glutamine hexosephosphate aminotransferase, 6-diazo-5-oxonorleucine (l form) and azaserine, blocked glucose-induced down regulation of glucose transport; (c) azaserine inhibited the activity of the aminotransferase, (d) glucosamine, which enters the hexosamine pathway distal to this enzyme was 40-times more potent than glucose; and (e) azaserine was unable to block the effect of glucosmaine. Since muscle is quantitatively much more important than adipose tissue for whole body glucose utilization, we sought to determine if the hexosamine pathway was involved in glucose-induced down regulation of glucose transport in L₆ myotubes. Glucose was effective, both in the presence and absence of glutamine in the incubation media. Glucosamine was also effective but was as equipotent as glucose. Small amounts of glutamine hexosephosphate aminotransferase were present in the L₆ myotubes and although the leucine derivative (20 μM)_ inhibited the enzyme, it did not impair glucose-induced down regulation of glucose transport. Total GLUT-1 levels were similar when the cells were incubated in the absence or presence of 5 mM glucose or glucosamine although glucosamine was associated with a marked increase in a lower molecular weight band. These results do not suggest that the hexosamine biosynthetic pathway is involved in glucose-induced down regulation of glucose transport in L₆ myotubes. Thus, this phenomenon is regulated differently in muscle and fat.     

Regulation of glutamine synthetase activity in the unicellular cyanobacterium Synechocystis sp. strain PCC 6803 by the nitrogen source: effect of ammonium.

Glutamine synthetase activity from Synechocystis sp. strain PCC 6803 is regulated as a function of the nitrogen source available in the medium. Addition of 0.25 mM NH4Cl to nitrate-grown cells promotes a clear short-term inactivation of glutamine synthetase, whose enzyme activity decreases to 5 to 10% of the initial value in 25 min. The intracellular levels of glutamine, determined under various conditions, taken together with the results obtained with azaserine (an inhibitor of transamidases), rule out the possibility that glutamine per se is responsible for glutamine synthetase inactivation. Nitrogen starvation attenuates the ammonium-mediated glutamine synthetase inactivation, indicating that glutamine synthetase regulation is modulated through the internal balance between carbon-nitrogen compounds and carbon compounds. The parallelism observed between the glutamine synthetase activity and the internal concentration of alpha-ketoglutarate suggests that this metabolite could play a role as a positive effector of glutamine synthetase activity in Synechocystis sp. Despite the similarities of this physiological system to that described for enterobacteria, the lack of in vivo 32P labeling of glutamine synthetase during the inactivation process excludes the existence of an adenylylation-deadenylylation system in this cyanobacterium.     

Synthesis of N3-fumaramoyl-L-2,3-diaminopropanoic acid analogues, the irreversible inhibitors of glucosamine synthetase

Several analogues of N3-fumaramoyl-L-2,3-diaminopropanoic acid were synthesized and evaluated for inhibition of glucosamine-6-phosphate synthetase activity. The syntheses were accomplished by acylation reaction of N2-tert.-butoxycarbonyl-L-2,3-diaminopropanoic acid (Boc-A2pr) or N2-tert.-butoxycarbonyl-L-2,4-diaminobutanoic acid (Boc-A2-bu) with the N-succinimidoyl esters of several derivatives of alpha, beta-unsaturated acids 2a-d followed by deprotection of the Boc groups. The obtained compounds were tested for inhibition of glucosamine synthetase isolated from Salmonella typhimurium and Saccharomyces cerevisiae. The results indicated that among the synthesized compounds, N3-4-methoxyfumaroyl-L-2,3-diaminopropanoic acid (FMDP) was the most powerful inhibitor of glucosamine synthetase.     

The distinction between γ-glutamylhydroxamate synthetase and l-glutamine–hydroxylamine glutamyltransferase activities in rat tissues. Studies in vitro

Two common ways of measuring the potential for glutamine synthesis in a tissue are the rates of formation of gamma-glutamylhydroxamate either by synthesis from glutamate (the glutamylhydroxamate synthetase reaction) or by transfer from glutamine (the glutamyltransferase reaction); it has not been established, however, that either reaction is a specific measure of glutamine synthetase. By differential extraction of glutamylhydroxamate synthetase and glutamyltransferase activities from water homogenates of several rat tissues I obtained an extract, rich in glutamylhydroxamate synthetase activity but nearly devoid of glutamyltransferase activity, and a fraction, solubilized by deoxycholate from the pellet, which contained virtually no glutamylhydroxamate synthetase activity but most of the original glutamyltransferase activity. Synthesis of glutamine, quantitatively similar to the gamma-glutamylhydroxamate formed by glutamylhydroxamate synthetase, is catalysed in the water extract but not in the particulate fraction. gamma-Glutamylhydroxamate formation by glutamylhydroxamate synthetase and glutamyltransferase shows discrepant substrate and metal specificities and can be differentially inhibited by l-methionine sulphoximine, phosphate and adenine nucleotides. The concordance between the formation of glutamine and gamma-glutamylhydroxamate by glutamylhydroxamate synthetase but not by glutamyltransferase and the different solubilities of the glutamylhydroxamate synthetase and glutamyltransferase enzyme activities demonstrate that these two activities are not inextricably associated; they therefore cannot be catalysed exclusively by the same protein.     

Inactivation of gamma-glutamylcysteine synthetase, but not of glutamine synthetase, by S-sulfocysteine and S-sulfohomocysteine

The reactions catalyzed by gamma-glutamylcysteine synthetase and glutamine synthetase are thought to proceed via enzyme-bound gamma-glutamyl phosphate intermediates. We investigated the possibility that S-sulfocysteine and S-sulfohomocysteine might act as analogs of gamma-glutamyl phosphate or of the associated putative tetrahedral intermediates. The D- and L-enantiomers of S-sulfocysteine and S-sulfohomocysteine were found to rapidly inactivate rat kidney gamma-glutamylcysteine synthetase but to be reversible inhibitors of sheep brain glutamine synthetase. Inactivation of gamma-glutamylcysteine synthetase does not require ATP and is associated with noncovalent binding of close to 1 mol of inactivator/mol of enzyme. The findings indicate that the S-sulfo amino acids are transition-state analogs, and that binding of S-sulfo amino acid to the enzyme induces formation of a very stable enzyme-inactivator complex. The data suggest that stabilization of the enzyme-inactivator complex results from interactions involving the sulfenyl sulfur atom of the S-sulfo amino acid and the active site thiol group of the enzyme.     

6-diazo-5-oxo-L-norleucine and azaserine as affinity inhibitors of glutamin(asparagin)ase

Incubation of homogeneous glutamin(asparagin)ase from Pseudomonas aurantiaca with 6-diazo-5-oxo-L-norleucine (DON) and azaserine leads to an almost complete inactivation of the enzyme. The inactivation process in both cases involves the step of reversible binding of the enzyme with the inhibitor into a complex and subsequent modification of the enzyme within this complex. The data on saturation of the enzyme by low concentrations of inhibitors, the protective effect of substrate and its analogs as well as of the competitive inhibitor and product of the enzymatic reaction, L-aspartate, suggest that the modification of functional groups takes place in the enzyme active site. The presence of essential threonine hydroxyl groups in/or near the enzyme active site is surmised.     

Affinity labeling of the active site of Escherichia coli glutamine synthetase by 5'-p-fluorosulfonylbenzoyladenosine

The interaction of Escherichia coli glutamine synthetase with the adenosine 5'-triphosphate analogue, 5'-p-fluorosulfonylbenzoyladenosine (5'-FSO2BzAdo), has been studied. This interaction results in the covalent attachment of the 5'-FSO2BzAdo to the enzyme with concomitant loss of catalytic activity. Although adenine nucleotides interact with glutamine synthetase at three distinct sites--a noncovalent AMP effector site, a regulatory site of covalent adenylylation, and the catalytic ATP/ADP binding site--our studies suggest that reaction with 5'-FSO2BzAdo occurs only at the active center. When glutamine synthetase was incubated with 5'-FSO2BzAdo, the decrease in catalytic activity obeyed pseudo-first order kinetics. The plot of the observed rate constant of inactivation versus the concentration of 5'-FSO2BzAdo was hyperbolic, consistent with reversible binding of the analogue to the enzyme prior to covalent attachment. Protection against inactivation was afforded by ATP and ADP; L-glutamate did not protect the enzyme against inactivation, but rather enhanced the rate of inactivation, consistent with the observations of others (Timmons, R. B., Rhee, S. G., Luterman, D. L., and Chock, P. B. (1974) Biochemistry 13, 4479-4485) that there is synergism in the binding of the two substrates to the enzyme. The incorporation of approximately 1.09 mol of the 5'-FSO2BzAdo/mol of glutamine synthetase subunit resulted in the total loss of enzymatic activity. The results suggest that 5'-FSO2BzAdo occupies the ATP binding site at the active center of glutamine synthetase and binds covalently to an amino acid residue nearby.     

Characterization of the glutamine site of Escherichia coli guanosine 5'-monophosphate synthetase.

Alkylation of guanosine 5'-monophosphate (GMP) synthetase with the glutamine analogs L-2-amino-4-oxo-5-chloropentanoic acid (chloroketon) and 6-diazo-5-oxonorleucine (DON) inactivated glutamine- and NH3-dependent GMP synthetase. Inactivation exhibited second order kinetics. Complete inactivation was accompanied by covalent attachment of 0.4 to 0.5 equivalent of chloroketon/subunit. Alkylation of GMP synthetase with iodacetamide selectively inactivated glutamine-dependent activity. The NH3-dependent activity was relatively unaffected. Approximately 1 equivalent of carboxamidomethyl group was incorporated per subunit. Carboxymethylcysteine was the only modified amino acid hydrolysis. Prior treatment with chloroketone decreased the capacity for alkylation by iodacetamide, suggesting that both reagents alkylate the same residue. GMP synthetase exhibits glutaminase activity when ATP is replaced by adenosine plus PPi. Iodoacetamide inactivates glutaminase concomitant with glutamine-dependent GMP synthetase. Analysis of pH versus velocity and Km data indicates that the amide of glutamine remains enzyme bound and does not mix with exogenous NH3 in the synthesis of GMP.     

Isolation and characterization of the GFA1 gene encoding the glutamine:fructose-6-phosphate amidotransferase of Candida albicans.

Glutamine:fructose-6-phosphate amidotransferase (glucosamine-6-phosphate synthase) catalyzes the first step of the hexosamine pathway required for the biosynthesis of cell wall precursors. The Candida albicans GFA1 gene was cloned by complementing a gfa1 mutation of Saccharomyces cerevisiae (previously known as gcn1-1; W. L. Whelan and C. E. Ballou, J. Bacteriol. 124:1545-1557, 1975). GFA1 encodes a predicted protein of 713 amino acids and is homologous to the corresponding gene from S. cerevisiae (72% identity at the nucleotide sequence level) as well as to the genes encoding glucosamine-6-phosphate synthases in bacteria and vertebrates. In cell extracts, the C. albicans enzyme was 4-fold more sensitive than the S. cerevisiae enzyme to UDP-N-acetylglucosamine (an inhibitor of the mammalian enzyme) and 2.5-fold more sensitive to N3-(4-methoxyfumaroyl)-L-2,3-diaminopropanoic acid (a glutamine analog and specific inhibitor of glucosamine-6-phosphate synthase). Cell extracts from the S. cerevisiae gfa1 strain transformed with the C. albicans GFA1 gene exhibited sensitivities to glucosamine-6-phosphate synthase inhibitors that were similar to those shown by the C. albicans enzyme. Southern hybridization indicated that a single GFA1 locus exists in the C. albicans genome. Quantitative Northern (RNA) analysis showed that the expression of GFA1 in C. albicans is regulated during growth: maximum mRNA levels were detected during early log phase. GFA1 mRNA levels increased following induction of the yeast-to-hyphal-form transition, but this was a response to fresh medium rather than to the morphological change.     

The inactivation of glucosamine synthetase from bacteria by anticapsin, the C-terminal epoxyamino acid of the antibiotic tetaine

Incubation of anticapsin with the purified glucosamine synthetase (2-amino-2-deoxy-D-glucose-6-phosphate ketol-isomerase, amino transferring, EC 5.3.1.19) from Escherichia coli, Pseudomonas aeruginosa, Arthrobacter aurescens and Bacillus thuringiensis led to the formation of an inactive enzyme irreversibly modified. The inactivation reaction followed pseudo-first-order kinetics. The rate of the inactivation reaction at various concentrations of anticapsin exhibited saturation kinetics, implying that anticapsin binds reversibly to the enzyme prior to inactivation. The determined Kinact is in the range of 10(-5) M (B. thuringiensis) and 10(-6) M (E. coli, P. aeruginosa, A. aurescens ). The addition of glutamine protected the amidotransferase from inactivation by anticapsin . The anticapsin was demonstrated to be a mixed type or competitive inhibitor with respect to glutamine with a Ki value of 10(-6) to 10(-7) M. Reaction of anticapsin with the enzyme exhibits the characteristics of affinity labelling of the glutamine binding site. Chemical modification of the enzyme thiol group with various reagents, 5,5'-dithiobis-(2-nitrobenzoic) acid, 6,6'- dithiodinicotinic acid, 1,1'- dithiodiformamidine , N-ethylmaleimide and iodoacetamide, resulted in an inactive enzyme.     

Utilization of -glutamine in intercellular adhesion: Ascites tumor and embryonic cells

-Glutamine is required for the synthesis of complex carbohydrates required for the intercellular adhesion of mouse teratoma cells. It remained to be seen if these pathways were of general importance in the adhesion of other cell types. In this study, using an electronic particle counter assay to measure cell adhesion, Ehrlich ascites, Sarcoma 180 and Taper liver ascites tumor cells require exogenous -glutamine to aggregate. This effect is concentration dependent and the amino sugar, -glucosamine, replaces the glutamine requirement. Structural analogs of the active compounds are substantially less effective and metabolic inhibitors block the activity of the effective compounds. Two specific glutamine antagonists, DON (6-diazo-5-oxo- -norleucine) and azaserine (O-diazoacetyl-serine) decrease the action of -glutamine but not of -glucosamine. Trypsin dissociated six day old chick embryo neural retina cells do not require -glutamine to reaggregate, though the rate of aggregation is enhanced after preincubation with glutamine. Dissociation of small clumps of neural retina and inhibition of reaggregation of these cells are facilitated by preincubation with azaserine for 3–5 h. -Glutamine reduces the effect of azaserine on retina cells. These results are consistent with known metabolic pathways and suggest that -glutamine is involved in the synthesis of complex carbohydrates necessary for adhesion in a variety of cell types. The defective adhesion of the tumor cells examined may result from inability to produce glutamine synthetase, or effectively store cr transport -glutamine.     

Bovine pancreatic asparagine synthetase explored with substrate analogs and specific monoclonal antibodies.

Several substrate analogs were tested for their ability to inhibit bovine pancreatic asparagine synthetase. Of the substrate analogs tested both 6-diazo-5-oxo-L-norleucine (DON) and 5-chloro-4-oxo-L-norvaline (CONV) were shown to inhibit the enzyme strongly. DON inhibited the glutaminase and glutamine-dependent asparagine synthetase activities and CONV inhibited the ammonia-dependent activity as well. Both of these inhibitors appeared to be relatively tight binding since desalting failed to remove the inhibition. The inactivation of bovine pancreatic asparagine synthetase by DON is accompanied by a shift from a 47,000 molecular weight monomer to a 96,000 molecular weight dimer as observed by HPLC gel filtration chromatography. This DON-induced shift is prevented by the presence of the substrate glutamine. A monoclonal antibody known to inhibit specifically the ammonia-dependent and glutamine-dependent asparagine synthetase activities but not glutaminase (monoclonal antibody 2B4) binds to both the monomer and the dimer forms of untreated enzyme, as well as to the dimer form of the DON-inactivated enzyme. On the other hand, a monoclonal antibody known to inhibit specifically the glutaminase and glutamine-dependent activities and not the ammonia-dependent asparagine synthetase (monoclonal antibody 5A6) binds to both forms of untreated enzyme but cannot bind to the DON-inactivated enzyme. These data are used to describe the relation of regions of the active site of asparagine synthetase in relation to antibody binding sites.     

Possible involvement of Lys603 from Escherichia coli glucosamine-6-phosphate synthase in the binding of its substrate fructose 6-phosphate

Pyridoxal 5'-phosphate is a competitive inhibitor of glucosamine-6-phosphate synthase with respect to the substrate fructose 6-phosphate. Irreversible inactivation of pyridoxal-5'-phosphate-treated enzyme with [14C]-cyanide resulted in covalent incorporation of close to 1 mol pyridoxal 5'-phosphate/mol enzyme subunit. The enzyme-pyridoxal-5'-phosphate complex could also be inactivated by reduction with NaBH3CN. Sequence analysis of the unique radioactively labelled tryptic peptide, resulting from inactivation with [3H]NaBH3CN, identified the C-terminal nonapeptide encompassing the modified Lys603. The presence of fructose 6-phosphate protected this residue from pyridoxylation. Direct evidence that a lysine residue is involved in the binding of the substrate as a Schiff base came from the isolation at 4 degrees C of a enzyme-fructose-6-phosphate complex in a 1:1 molar ratio. Treatment of the enzyme-[14C]fructose-6-phosphate complex with NaBH3CN revealed one site of modification in the tryptic peptide map. In contrast, trapping the same complex with potassium cyanide resulted in the isolation of several radiolabelled peptides containing lysines which could potentially bind fructose 6-phosphate. However, since the radioactivity was not specifically associated with the lysine residues, it is suggested that these 14C-labelled peptides resulted from the decomposition of an unstable alpha,alpha'-dihydroxyaminonitrile adduct rather than from a lack of specificity of fructose 6-phosphate fixation. Lys603 is then the candidate of choice for fructose 6-phosphate binding since it lies at or near the active site as demonstrated by the trapping experiments with pyridoxal 5'-phosphate described above, and among the lysines which belong to the sugar-binding domain this is the only one conserved between the three members of the purF, glutamine-dependent, amidotransferase subfamily which include the glucosamine-6-phosphate synthase from Escherichia coli, Saccharomyces cerevisiae and the Rhizobium nodulation protein NodM.     

Glutamine synthetase of Bacillus stearothermophilus. Regulation, site interactions, and functional information.

The action of various feedback modifiers on Bacillus stearothermophilus glutamine synthetase has been investigated by initial velocity kinetics, using the Mn2+-stimulated biosynthetic assay at 55 degrees C. The most potent inhibitors, used singly, are AMP, L-glutamine, and L-alanine. Other modifiers of significance include glycine, CTP, L-histidine, glucosamine 6-phosphate, and GDP. Marked synergism of action is observed for AMP in the presence of L-glutamine, L-histidine, ADP, or glucosamine 6-phosphate (glucosamine-6-P), and for CTP with ADP or GDP. Inhibition by saturating levels of many modifiers is either less than 100%, or is not overcome by elevated substrate levels, or both. This argues for modifier binding sites separate from substrate sites, notably in the cases of AMP, L-glutamine, glycine, L-alanine, glucosamine-6-P, and CTP. Glycine and L-alanine are Vmax inhibitors, whereas L-glutamine, glucosamine-6-P, GDP, and CTP alter the binding of L-glutamate. ADP and L-histidine apparently can compete directly with MnATP, but AMP alters Mn-ATP binding from a separate site. The action of several modifiers requires or is enhanced by bound substrates. Considerable antagonistic interaction is observed in experiments with modifier pairs, but the most potent inhibitors show synergistic or cumulative (independent) interactions. One may interpret antagonistic effects as due to (a) overlapping modifier domains, or (b) separate but antagonistically interacting sites. Either interpretation leads to a scheme for modifier-substrate and modifier-modifier site interactions in which the thermophilic enzyme must maintain and stabilize a great deal of complex functional information under extreme environmental conditions.