Inhibition of Escherichia coliGlucosamine-6-phosphate Synthase by Reactive Intermediate Analogues

Glucosamine-6-phosphate synthase (GlmS) catalyzes the formation of d-glucosamine 6-phosphate fromd-fructose 6-phosphate using l-glutamine as the ammonia source. Because N-acetylglucosamine is an essential building block of both bacterial cell walls and fungal cell wall chitin, the enzyme is a potential target for antibacterial and antifungal agents. The most potent carbohydrate-based inhibitor of GlmS reported to date is 2-amino-2-deoxy-d-glucitol 6-phosphate, an analogue of the putative cis-enolamine intermediate formed during catalysis. The interaction of a series of structurally related cis-enolamine intermediate analogues with GlmS is described. Although arabinose oxime 5-phosphate is identified as a good competitive inhibitor of GlmS with an inhibition constant equal to 1.2 (±0.3) mm, the presence of the amino function at the 2-position is shown to be important for potent inhibition. Comparison of the binding affinities of 2-deoxy-d-glucitol 6-phosphate and 2-amino-2-deoxy-d-glucitol 6-phosphate indicates that the amino function contributes −4.1 (±0.1) kcal/mol to the free energy of inhibitor binding. Similarly, comparison of the binding affinities of 2-deoxy-d-glucose 6-phosphate andd-glucosamine 6-phosphate indicates that the amino function contributes −3.0 (±0.1) kcal/mol to the free energy of product binding. Interactions between GlmS and the 2-amino function of its ligands contribute to the uniform binding of the product and thecis-enolamine intermediate as evidenced by the similar contribution of the amino group to the free energy of binding ofd-glucosamine 6-phosphate and 2-amino-2-deoxy-d-glucitol 6-phosphate, respectively. Glucosamine-6-phosphate synthase (GlmS) catalyzes the formation of d-glucosamine 6-phosphate fromd-fructose 6-phosphate using l-glutamine as the ammonia source. Because N-acetylglucosamine is an essential building block of both bacterial cell walls and fungal cell wall chitin, the enzyme is a potential target for antibacterial and antifungal agents. The most potent carbohydrate-based inhibitor of GlmS reported to date is 2-amino-2-deoxy-d-glucitol 6-phosphate, an analogue of the putative cis-enolamine intermediate formed during catalysis. The interaction of a series of structurally related cis-enolamine intermediate analogues with GlmS is described. Although arabinose oxime 5-phosphate is identified as a good competitive inhibitor of GlmS with an inhibition constant equal to 1.2 (±0.3) mm, the presence of the amino function at the 2-position is shown to be important for potent inhibition. Comparison of the binding affinities of 2-deoxy-d-glucitol 6-phosphate and 2-amino-2-deoxy-d-glucitol 6-phosphate indicates that the amino function contributes −4.1 (±0.1) kcal/mol to the free energy of inhibitor binding. Similarly, comparison of the binding affinities of 2-deoxy-d-glucose 6-phosphate andd-glucosamine 6-phosphate indicates that the amino function contributes −3.0 (±0.1) kcal/mol to the free energy of product binding. Interactions between GlmS and the 2-amino function of its ligands contribute to the uniform binding of the product and thecis-enolamine intermediate as evidenced by the similar contribution of the amino group to the free energy of binding ofd-glucosamine 6-phosphate and 2-amino-2-deoxy-d-glucitol 6-phosphate, respectively. glucosamine-6-phosphate synthase d-fructose 6-phosphate d-glucosamine 6-phosphate 2-amino-2-deoxy-d-glucitol 6-phosphate 2-deoxy-d-glucitol 6-phosphate 2-deoxy-d-glucose 6-phosphate Glucosamine-6-phosphate synthase (l-glutamine:d-fructose-6-phosphate aminotransferase (GlmS,1 EC 2.6.1.16)) catalyzes the first step in hexosamine biosynthesis, convertingd-fructose 6-phosphate (Fru-6-P) intod-glucosamine 6-phosphate (GlcN-6-P) using glutamine as the ammonia source (Scheme 1) (1Ghosh S. Blumenthal H.J. Davidson E. Roseman S. J. Biol. Chem. 1960; 235: 1265-1273Abstract Full Text PDF PubMed Google Scholar, 2Badet-Denisot M.-A. René L. Badet B. Bull. Soc. Chim. Fr. 1993; 130: 249-255Google Scholar, 3Massière F. Badet-Denisot M.-A. Cell. Mol. Life Sci. 1998; 54: 205-222Crossref PubMed Scopus (168) Google Scholar). GlcN-6-P is a precursor of uridine diphospho-N-acetylglucosamine from which other amino sugar-containing molecules are derived. One of these products,N-acetylglucosamine, is an important constituent of the peptidoglycan layer of bacterial cell walls and fungal cell wall chitin. Accordingly, GlmS offers a potential target for antibacterial and antifungal agents and has attracted the interest of several research groups (2Badet-Denisot M.-A. René L. Badet B. Bull. Soc. Chim. Fr. 1993; 130: 249-255Google Scholar). GlmS catalyzes two coupled enzymatic reactions. The first is the hydrolysis of glutamine to yield glutamate and nascent ammonia, which is transferred to Fru-6-P. The second reaction is the isomerization of Fru-6-P from a ketose to an aldose, corresponding to a Heyns rearrangement (4Kort M.J. Adv. Carbohydr. Chem. Biochem. 1970; 25: 311-349Crossref Scopus (79) Google Scholar, 5Golinelli-Pimpaneau B. Le Goffic F. Badet B. J. Am. Chem. Soc. 1989; 111: 3029-3034Crossref Scopus (45) Google Scholar). Like other amidotransferases, GlmS is organized into two domains: the NH2-terminal glutamine amidotransferase domain, which catalyzes the hydrolysis of glutamine, and the COOH-terminal synthase domain, which catalyzes the isomerization (3Massière F. Badet-Denisot M.-A. Cell. Mol. Life Sci. 1998; 54: 205-222Crossref PubMed Scopus (168) Google Scholar, 6Mei B. Zalkin H. J. Bacteriol. 1990; 172: 3512-3514Crossref PubMed Google Scholar, 7Zalkin H. Adv. Enzymol. 1993; 66: 203-309PubMed Google Scholar, 8Denisot M.-A. Le Goffic F. Badet B. Arch. Biochem. Biophys. 1991; 288: 225-230Crossref PubMed Scopus (42) Google Scholar). The glutamine hydrolysis reaction has been studied extensively and utilizes the NH2-terminal cysteine thiol, which forms a γ-glutamyl thioester intermediate during the reaction. This catalytic role was confirmed by conversion of the NH2-terminal cysteine to alanine using site-directed mutagenesis which abolished enzymatic activity (2Badet-Denisot M.-A. René L. Badet B. Bull. Soc. Chim. Fr. 1993; 130: 249-255Google Scholar). In general, glutamine amidotransferases are inactivated by glutamine affinity analogues such as 6-diazo-5-oxo-l-norleucine and 6-chloro-5-oxo-l-norleucine (chloroketone), which alkylate the essential cysteine residue (3Massière F. Badet-Denisot M.-A. Cell. Mol. Life Sci. 1998; 54: 205-222Crossref PubMed Scopus (168) Google Scholar, 7Zalkin H. Adv. Enzymol. 1993; 66: 203-309PubMed Google Scholar, 9Buchanan J.M. Adv. Enzymol. 1973; 39: 91-183PubMed Google Scholar). Indeed, many of the active site-directed irreversible inactivators developed for GlmS contain an electrophilic function at the γ-position of glutamate and react irreversibly with the NH2-terminal cysteine residue. More recently, attempts to develop carbohydrate-based inhibitors have been made with the hope of developing more specificity (10Corizzi V. Badet B. Badet-Denisot M.-A. J. Chem. Soc. Chem. Commun. 1992; : 189-190Crossref Google Scholar, 11Badet-Denisot M.-A. Leriche C. Massière F. Badet B. Bioorg. Med. Chem. Lett. 1995; 5: 815-820Crossref Scopus (35) Google Scholar, 12Bearne S.L. J. Biol. Chem. 1996; 271: 3052-3057PubMed Google Scholar, 13Leriche C. Badet-Denisot M.-A. Badet B. Eur. J. Biochem. 1997; 245: 418-422Crossref PubMed Scopus (17) Google Scholar). The ketose/aldose isomerase activity of the enzyme proceeds by abstraction of the C1 pro-R hydrogen of a putative fructosimine 6-phosphate intermediate to form acis-enolamine reactive intermediate that, upon reprotonation at the Re face of C2, gives rise to GlcN-6-P (Scheme 2) (5Golinelli-Pimpaneau B. Le Goffic F. Badet B. J. Am. Chem. Soc. 1989; 111: 3029-3034Crossref Scopus (45) Google Scholar). In accord with this mechanism, Badet and co-workers (14Leriche C. Badet-Denisot M.-A. Badet B. J. Am. Chem. Soc. 1996; 118: 1797-1798Crossref Scopus (29) Google Scholar) have shown that GlmS, in the absence of glutamine, displays a low phosphoglucoisomerase activity. Analogues of the cis-enolamine reactive intermediate are expected to be potent inhibitors of the enzyme (15Wolfenden R. Acc. Chem. Res. 1972; 5: 10-18Crossref Scopus (546) Google Scholar, 16Wolfenden R. Mol. Cell. Biochem. 1974; 3: 111-207Crossref Scopus (87) Google Scholar, 17Wolfenden R. Annu. Rev. Biophys. Bioeng. 1976; 5: 271-306Crossref PubMed Scopus (381) Google Scholar, 18Wolfenden R. Frick L. Page M.I. Williams A. Enzyme Mechanisms. Royal Society of Chemistry, London1987: 97-122Google Scholar) and indeed, 2-amino-2-deoxy-d-glucitol 6-phosphate (GlcNol-6-P) is the most potent carbohydrate-based inhibitor reported to date (11Badet-Denisot M.-A. Leriche C. Massière F. Badet B. Bioorg. Med. Chem. Lett. 1995; 5: 815-820Crossref Scopus (35) Google Scholar, 12Bearne S.L. J. Biol. Chem. 1996; 271: 3052-3057PubMed Google Scholar). Identification of those structural elements necessary for tight binding is an important part of inhibitor design. This paper describes the inhibition of GlmS by several analogues of the cis-enolamine intermediate in an attempt to probe the structural requirements for potent inhibition of this enzyme. The energetic contribution of the 2-amino group to binding of the product and thecis-enolamine intermediate is determined. d-Arabinose, d-arabinose 5-phosphate, 2-amino-2-deoxy-d-glucose 6-phosphate (d-glucosamine 6-phosphate), 2-deoxy-d-glucose 6-phosphate (dGlc-6-P), d-Fru-6-P, and d-ribose 5-phosphate were purchased from Sigma Chemical Company. All other chemicals were analytical grade and used without further purification. NMR spectra (13C, 31P) were obtained using a Bruker AC 250F spectrometer. Chemical shifts (δ) for 13C and 31P spectra are reported relative to the deuterium lock signal and external H3PO4 (85% w/v in D2O), respectively. Elemental analyses were performed by Canadian Microanalytical Service Ltd., B. C. 2-Deoxy-d-glucitol 6-phosphate (dGlcol-6-P) was synthesized by reduction of 2-deoxy-d-glucose 6-phosphate (dGlc-6-P) with sodium borohydride and purified by ion-exchange chromatography following procedures similar to those outlined for the preparation of GlcNol-6-P (12Bearne S.L. J. Biol. Chem. 1996; 271: 3052-3057PubMed Google Scholar). dGlc-6-P (250 mg, 0.868 mmol) was dissolved in 10 ml of water and cooled on ice for 10 min. Sodium borohydride (0.750 g, 19.83 mmol) was added to the dGlc-6-P solution by small portions over 20 min. During the NaBH4 addition, the solution was stirred vigorously and held on ice. After the addition was complete, the solution was stirred for 1 h at room temperature. Reduction was complete as indicated by the inability of the solution to reduce Fehling's reagent. Undissolved NaBH4 was removed by filtration, and the filtrate was cooled on ice. The remaining NaBH4 was destroyed by dropwise addition of 6 m acetic acid over 30 min until the final pH was approximately 4. The solution (25 ml) was allowed to come to room temperature and stirred for 1 h. This solution was then filtered, and the filtrate was applied to a Dowex 50 (H+ form) column (1.5 × 47 cm) and eluted with water. Fractions containing product were identified by thin layer chromatography on cellulose (99% EtOH, n-BuOH, 0.15m sodium citrate buffer, pH 4.0; 10:1:6 v/v/v) developed with an iron-sulfosalicylic acid spray reagent (19Clark J.M. Switzer R.L. Experimental Biochemistry. 2nd Ed. W. H. Freeman and Co., San Francisco1977: 167Google Scholar) sensitive to phosphates. Fractions testing positive for phosphate were pooled and diluted with 0.50 volume of The was then removed using and of by with by of the and by The remaining was in 10 ml of water and applied to a Dowex 50 column (1.5 × 47 cm) eluted with water. Fractions containing were and a g, 13C NMR 31P NMR and coupled The was to the using Dowex 50 Elemental was as 1 oxime was by d-arabinose with as by and Carbohydr. Res. PubMed Scopus Google Scholar, J. Chem. Soc. : Scopus Google Scholar) and J. Am. Chem. Soc. Scopus Google Scholar, J. Am. Chem. Soc. Scopus Google Scholar). The oxime was then to its corresponding 5-phosphate using as by and Carbohydr. Res. PubMed Scopus Google Scholar). The 31P NMR of the synthesized and d-arabinose oxime 5-phosphate at and were to the coupled to in the oxime similar to NMR reported by and Carbohydr. Res. PubMed Scopus Google Scholar). were at and to The oxime 5-phosphate used in the inhibition was by of the NMR The at and from of the and forms were relative to an The of oxime using NMR was which is in with the reported of Carbohydr. Res. PubMed Scopus Google Scholar). GlmS was purified as (12Bearne S.L. J. Biol. Chem. 1996; 271: 3052-3057PubMed Google Scholar, S.L. R. Biochemistry. 1995; PubMed Scopus (42) Google Scholar). The activity of the final preparation was of GlcN-6-P was using a and the of the was as (12Bearne S.L. J. Biol. Chem. 1996; 271: 3052-3057PubMed Google Scholar, S.L. R. Biochemistry. 1995; PubMed Scopus (42) Google Scholar). and phosphate were to the procedures by and Enzymol. 3: Scopus Google Scholar). were in phosphate buffer, pH containing 1 The of inhibitors used in the were as d-arabinose 5-phosphate, and d-arabinose oxime 5-phosphate, and d-ribose 5-phosphate, and and and and In addition, the GlmS × l-glutamine at equal to and GlmS was to be to in and attempt was made to for in were at inhibitor using the of were by of the using the from The inhibition were in and the is The reported is the has been on glutamine analogues to GlmS activity with the of developing antibacterial and antifungal agents. these analogues an electrophilic function at the γ-position of which is to react irreversibly with the NH2-terminal cysteine residue in the glutamine amidotransferase The most inactivators have H. R. E. Biophys. Scopus Google Scholar, B. Le Goffic F. Biochemistry. PubMed Scopus Google Scholar, M.-A. Le Goffic F. Badet B. Biochemistry. 1990; PubMed Scopus Google Scholar, R. H. S. E. J. Res. PubMed Scopus Google Scholar) and S. Le Goffic F. Badet B. Bioorg. Chem. 1991; Scopus Google S. H. R. E. Biophys. 1992; PubMed Scopus Google Scholar). Although the enzyme is to an B. Le Goffic F. Biochemistry. PubMed Scopus Google inactivators and GlmS in the absence of the first Fru-6-P H. R. E. Biophys. Scopus Google B. Le Goffic F. Biochemistry. PubMed Scopus Google Scholar). has been an to carbohydrate-based inhibitors for GlmS (10Corizzi V. Badet B. Badet-Denisot M.-A. J. Chem. Soc. Chem. Commun. 1992; : 189-190Crossref Google Scholar, 11Badet-Denisot M.-A. Leriche C. Massière F. Badet B. Bioorg. Med. Chem. Lett. 1995; 5: 815-820Crossref Scopus (35) Google Scholar, 12Bearne S.L. J. Biol. Chem. 1996; 271: 3052-3057PubMed Google Scholar, 13Leriche C. Badet-Denisot M.-A. Badet B. Eur. J. Biochem. 1997; 245: 418-422Crossref PubMed Scopus (17) Google Scholar). One to developing potent enzyme inhibitors is to that are analogues of the reactive that are formed during (15Wolfenden R. Acc. Chem. Res. 1972; 5: 10-18Crossref Scopus (546) Google Scholar, 16Wolfenden R. Mol. Cell. Biochem. 1974; 3: 111-207Crossref Scopus (87) Google Scholar, 17Wolfenden R. Annu. Rev. Biophys. Bioeng. 1976; 5: 271-306Crossref PubMed Scopus (381) Google Scholar, 18Wolfenden R. Frick L. Page M.I. Williams A. Enzyme Mechanisms. Royal Society of Chemistry, London1987: 97-122Google Scholar). The of that the of an and a ketose have been shown to abstraction from to an intermediate Adv. Enzymol. Mol. Biol. Google Scholar, R. Soc. Sci. PubMed Scopus Google Scholar). of these activity in the absence of and have to hydrogen 1 R. Soc. Sci. PubMed Scopus Google Scholar, A. Biochemistry. 1989; PubMed Scopus Google Scholar). analogues of are potent inhibitors of such R. Frick L. Page M.I. Williams A. Enzyme Mechanisms. Royal Society of Chemistry, London1987: 97-122Google Scholar). isomerase is by and R. Biochemistry. 1970; PubMed Scopus Google Scholar, J. Biol. Chem. 1974; Full Text PDF PubMed Google isomerase is by J.M. J. Biol. Chem. Full Text PDF PubMed Google and both isomerase and are by R. J. Biol. Chem. Full Text PDF PubMed Google Scholar, C. J. Med. Chem. PubMed Scopus (35) Google Scholar). the that the for the step an these analogues be similar has been for both glucosamine-6-phosphate Biochemistry. PubMed Scopus Google Scholar) and GlmS (5Golinelli-Pimpaneau B. Le Goffic F. Badet B. J. Am. Chem. Soc. 1989; 111: 3029-3034Crossref Scopus (45) Google Scholar) abstraction from the an cis-enolamine intermediate (Scheme In accord with this mechanism, both the Biochemistry. PubMed Scopus Google Scholar) and the synthase (5Golinelli-Pimpaneau B. Le Goffic F. Badet B. J. Am. Chem. Soc. 1989; 111: 3029-3034Crossref Scopus (45) Google Scholar) have to hydrogen similar to the In addition, an analogue of thecis-enolamine is a potent inhibitor of both (11Badet-Denisot M.-A. Leriche C. Massière F. Badet B. Bioorg. Med. Chem. Lett. 1995; 5: 815-820Crossref Scopus (35) Google Scholar, 12Bearne S.L. J. Biol. Chem. 1996; 271: 3052-3057PubMed Google Scholar, Biochemistry. PubMed Scopus Google Scholar). In GlcNol-6-P is the binding carbohydrate-based inhibitor reported for The describes the inhibition of GlmS by several analogues of the cis-enolamine intermediate and the energetic contribution that the 2-amino function to binding. The of several analogues of the putative cis-enolamine intermediate and corresponding inhibition are shown in In the analogues were to be competitive inhibitors of GlmS activity with to Fru-6-P. The of the inhibition be of the for these inhibitors for GlcNol-6-P was shown to be similar to the constant of for this using (12Bearne S.L. J. Biol. Chem. 1996; 271: 3052-3057PubMed Google Scholar). The in the forms of arabinose 5-phosphate and 5-phosphate the in the the enzyme displays a low affinity for both of these relative to GlcNol-6-P indicates that the 2-amino and are important for tight binding of the at approximately a reduction in the binding of 5-phosphate relative to arabinose This is that such 1972; Scopus Google inhibition of GlmS by analogues of the constant from (12Bearne S.L. J. Biol. Chem. 1996; 271: 3052-3057PubMed Google Scholar). This is similar to the of the inhibitor constant by Badet-Denisot (11Badet-Denisot M.-A. Leriche C. Massière F. Badet B. Bioorg. Med. Chem. Lett. 1995; 5: 815-820Crossref Scopus (35) Google 5-phosphate and 5-phosphate are as the forms to structural to the cis-enolamine The shown are the for the of in solution free and as outlined and The is shown to its structural to the cis-enolamine the shown is for the of constant at of glutamine from Badet B. Le Goffic F. Biochemistry. PubMed Scopus Google Scholar). a constant from (12Bearne S.L. J. Biol. Chem. 1996; 271: 3052-3057PubMed Google Scholar). This is similar to the of the inhibitor constant by Badet-Denisot (11Badet-Denisot M.-A. Leriche C. Massière F. Badet B. Bioorg. Med. Chem. Lett. 1995; 5: 815-820Crossref Scopus (35) Google Scholar). 5-phosphate and 5-phosphate are as the forms to structural to the cis-enolamine The shown are the for the of in solution free and as outlined and The is shown to its structural to the cis-enolamine the shown is for the of constant at of glutamine from Badet B. Le Goffic F. Biochemistry. PubMed Scopus Google Scholar). both arabinose 5-phosphate and 5-phosphate in free forms that structural to the cis-enolamine intermediate is in the forms of arabinose 5-phosphate and 5-phosphate approximately and of the of J. R. J. Am. Chem. Soc. Scopus Google Scholar). similar Fru-6-P in the and free forms J. R. J. Am. Chem. Soc. Scopus Google Scholar). the form of Fru-6-P is the for GlmS is Badet-Denisot (2Badet-Denisot M.-A. René L. Badet B. Bull. Soc. Chim. Fr. 1993; 130: 249-255Google Scholar) have that is for GlmS to the of of Fru-6-P J. R. J. Am. Chem. Soc. Scopus Google Scholar) is similar to the catalytic other that an such as isomerase E. A. J. Biol. Chem. Full Text PDF PubMed Google Scholar, J. Biol. Chem. Full Text PDF PubMed Google Scholar, J. Biol. Chem. 1973; Full Text PDF PubMed Google isomerase J. Biol. Chem. Full Text PDF PubMed Google and GlcN-6-P E. 1995; 3: Full Text Full Text PDF PubMed Scopus Google are to of the to form the corresponding to the of of the GlmS Fru-6-P binding M.-A. Le Goffic F. Badet B. Arch. Biochem. Biophys. 1991; 288: 225-230Crossref PubMed Scopus (42) Google Scholar) with ligands have been A. Badet-Denisot M.-A. Badet B. 1998; Full Text Full Text PDF PubMed Scopus Google GlcN-6-P A. Badet-Denisot M.-A. Badet B. 1998; Full Text Full Text PDF PubMed Scopus Google and GlcNol-6-P A. Badet-Denisot M.-A. Badet B. Sci. PubMed Scopus Google Scholar). on these and co-workers A. Badet-Denisot M.-A. Badet B. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar, A. Badet-Denisot M.-A. Badet B. Sci. PubMed Scopus Google Scholar) have that GlmS as a to of Fru-6-P. is that GlmS the of arabinose 5-phosphate and 5-phosphate in the and free forms in solution Biochemistry. PubMed Scopus Google Scholar). the free were the of the inhibition constant to the of free in solution yield an for the equal to to be to the inhibition constant in this oxime 5-phosphate is an analogue of thecis-enolamine that the and the function the 2-amino GlmS this analogue with an inhibition constant equal to 1.2 mm, approximately the for arabinose is important to that the inhibition was an containing of form and of the the to thecis-enolamine the to the of form in solution gives a equal to The affinity that GlmS displays for the oxime is the affinity with which GlmS this of binding affinity to the amino of the hydrogen on of the oxime with an amino group yield a that from thecis-enolamine intermediate by the of a at Because the amino function contributes approximately kcal/mol to the binding affinity the for such a be approximately This is approximately for GlcNol-6-P and as an of the for the affinity for the (10Corizzi V. Badet B. Badet-Denisot M.-A. J. Chem. Soc. Chem. Commun. 1992; : 189-190Crossref Google Scholar) have reported that the analogue of Fru-6-P is a competitive inhibitor of GlmS with to Fru-6-P the oxime of this was reported to have a affinity for the enzyme with a equal to (10Corizzi V. Badet B. Badet-Denisot M.-A. J. Chem. Soc. Chem. Commun. 1992; : 189-190Crossref Google which is the inhibition constant for arabinose oxime 5-phosphate in the This is the arabinose the oxime of the is with the One for this in binding affinities be that the oxime of the more in the the relative of and were reported for the oxime of the the in binding the of the structural to the The between an enzyme and a a of The to the affinity has been to into the of group by the in affinity which of the groups of interest is removed Sci. S. A. PubMed Scopus Google Scholar, J. PubMed Scopus Google Scholar, A. and in W. H. Freeman and Co., Scholar). This of be by a group from the enzyme using site-directed mutagenesis from the J. PubMed Scopus Google Scholar, Biochemistry. 25: PubMed Scopus Google Scholar). the is the m with the and a be by the of the on the free energy of the relative to the analogue inhibitors be and the in binding affinity as the contribution of the removed to binding R. 1989; PubMed Scopus Google Scholar). This is used in the to the contribution that the 2-amino function to the binding of the product (GlcN-6-P) and the reactive intermediate analogue 6-phosphate, which from GlcN-6-P by the absence of an amino function at the is by the enzyme This to a free energy of binding equal to kcal/mol with a of kcal/mol for the binding free energy of GlcN-6-P B. Le Goffic F. Biochemistry. PubMed Scopus Google Scholar). free energy of binding is using the and is the The contribution of the amino function to binding is using the the competitive inhibition for ligands and containing the 2-amino respectively. the 2-amino function contributes −3.0 kcal/mol GlcN-6-P to the free energy of product binding. Similarly, which from GlcNol-6-P by the absence of an amino function at the is by the enzyme This to a free energy of binding equal to kcal/mol with a of kcal/mol for the binding free energy of GlcNol-6-P (12Bearne S.L. J. Biol. Chem. 1996; 271: 3052-3057PubMed Google Scholar). the 2-amino function contributes −4.1 kcal/mol GlcNol-6-P to the free energy of reactive intermediate analogue binding. are similar to the binding free reported for amino in other of kcal/mol and kcal/mol have been reported for the binding contribution of the amino function on ligands with the Biochemistry. PubMed Scopus Google Scholar, R. J. Mol. Biol. 1973; PubMed Scopus Google Scholar) and E. A. Biochemistry. 1973; PubMed Scopus Google respectively. the and as as to kcal/mol have been reported for the energetic contribution of the amino function to the of J. S. J. Am. Chem. Soc. 1995; Scopus Google Scholar). the amino function at the 2-position contributes approximately the of binding energy to the binding of the as to the binding of the reactive intermediate The amino contributes to binding Biochemistry. 1976; PubMed Scopus Google Scholar, Chem. Ed. PubMed Scopus Google Scholar) of both the product and thecis-enolamine of thecis-enolamine intermediate relative to the be by binding the function at The of to and reactive intermediate analogues are in the role of in E. Annu. Rev. Biochem. 1990; PubMed Scopus Google Scholar). The of of the Fru-6-P binding of GlmS M.-A. Le Goffic F. Badet B. Arch. Biochem. Biophys. 1991; 288: 225-230Crossref PubMed Scopus (42) Google Scholar) with GlcN-6-P A. Badet-Denisot M.-A. Badet B. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar) and GlcNol-6-P A. Badet-Denisot M.-A. Badet B. Sci. PubMed Scopus Google Scholar) the between the 2-amino group and the isomerase which rise to the uniform binding The amino group of GlcN-6-P is hydrogen to the of and and to a water The water is hydrogen to the of A. Badet-Denisot M.-A. Badet B. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). of the 2-amino function is expected to these hydrogen in the of kcal/mol of binding free energy dGlc-6-P is the The amino group of GlcNol-6-P is hydrogen to the of and a water The water is hydrogen to the group of A. Badet-Denisot M.-A. Badet B. Sci. PubMed Scopus Google Scholar). of the amino function from GlcNol-6-P is expected to these two hydrogen in the of kcal/mol of binding energy is the the hydrogen between the amino function and the to from thecis-enolamine to the with the of a hydrogen in the the binding affinity for the amino function the of the enzyme amidotransferase and isomerase is be to the glutamine amidotransferase with the amino the between GlmS and the 2-amino function of its ligands are for the uniform binding of the product and thecis-enolamine intermediate as evidenced by the similar contribution of the amino group to the free energy of binding of GlcN-6-P and respectively. The amino function contributes to the free energy of binding both the product and the reactive intermediate that the 2-amino function is an important to be in the of carbohydrate-based GlmS

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