High Resolution X-ray Crystallography Shows That Ascorbate Is a Cofactor for Myrosinase and Substitutes for the Function of the Catalytic Base
2000; Elsevier BV; Volume: 275; Issue: 50 Linguagem: Inglês
10.1074/jbc.m006796200
ISSN1083-351X
AutoresW.P. Burmeister, Sylvain Cottaz, Patrick Rollin, A. VASELLA, Bernard Henrissat,
Tópico(s)Protein Hydrolysis and Bioactive Peptides
ResumoMyrosinase, an S-glycosidase, hydrolyzes plant anionic 1-thio-β-d-glucosides (glucosinolates) considered part of the plant defense system. AlthoughO-glycosidases are ubiquitous, myrosinase is the only knownS-glycosidase. Its active site is very similar to that of retaining O-glycosidases, but one of the catalytic residues in O-glycosidases, a carboxylate residue functioning as the general base, is replaced by a glutamine residue. Myrosinase is strongly activated by ascorbic acid. Several binary and ternary complexes of myrosinase with different transition state analogues and ascorbic acid have been analyzed at high resolution by x-ray crystallography along with a 2-deoxy-2-fluoro-glucosyl enzyme intermediate. One of the inhibitors, d-gluconhydroximo-1,5-lactam, binds simultaneously with a sulfate ion to form a mimic of the enzyme-substrate complex. Ascorbate binds to a site distinct from the glucose binding site but overlapping with the aglycon binding site, suggesting that activation occurs at the second step of catalysis,i.e. hydrolysis of the glycosyl enzyme. A water molecule is placed perfectly for activation by ascorbate and for nucleophilic attack on the covalently trapped 2-fluoro-glucosyl-moiety. Activation of the hydrolysis of the glucosyl enzyme intermediate is further evidenced by the observation that ascorbate enhances the rate of reactivation of the 2-fluoro-glycosyl enzyme, leading to the conclusion that ascorbic acid substitutes for the catalytic base in myrosinase. Myrosinase, an S-glycosidase, hydrolyzes plant anionic 1-thio-β-d-glucosides (glucosinolates) considered part of the plant defense system. AlthoughO-glycosidases are ubiquitous, myrosinase is the only knownS-glycosidase. Its active site is very similar to that of retaining O-glycosidases, but one of the catalytic residues in O-glycosidases, a carboxylate residue functioning as the general base, is replaced by a glutamine residue. Myrosinase is strongly activated by ascorbic acid. Several binary and ternary complexes of myrosinase with different transition state analogues and ascorbic acid have been analyzed at high resolution by x-ray crystallography along with a 2-deoxy-2-fluoro-glucosyl enzyme intermediate. One of the inhibitors, d-gluconhydroximo-1,5-lactam, binds simultaneously with a sulfate ion to form a mimic of the enzyme-substrate complex. Ascorbate binds to a site distinct from the glucose binding site but overlapping with the aglycon binding site, suggesting that activation occurs at the second step of catalysis,i.e. hydrolysis of the glycosyl enzyme. A water molecule is placed perfectly for activation by ascorbate and for nucleophilic attack on the covalently trapped 2-fluoro-glucosyl-moiety. Activation of the hydrolysis of the glucosyl enzyme intermediate is further evidenced by the observation that ascorbate enhances the rate of reactivation of the 2-fluoro-glycosyl enzyme, leading to the conclusion that ascorbic acid substitutes for the catalytic base in myrosinase. 2-F-glucotropaeolin 4-morpholineethanesulfonic acid Glucosinolates are anionic β-d-S-glucosides found prominently in plants of the genus Brassica (cabbage, mustard, rapeseed, and otherCruciferae). They constitute a large family ofS-glucosides that differ by their aglycon (Ref. 1Fenwick G.R. Heaney R.K. Mullin W.J CRC Crit. Rev. Food Sci. Nutr. 1983; 18: 123-201Crossref Scopus (1185) Google Scholar and Fig. 1 a). The same plants produce myrosinase (thioglucoside glucohydrolase, EC 3.2.3.1), an S-glucosidase hydrolyzing glucosinolates. Myrosinase and glucosinolates are stored in different compartments of the plant, especially in the seeds. Mixing of enzyme and substrate (for example through mastication) induces glucosinolate hydrolysis. The biological function of myrosinase and glucosinolates is only partly elucidated; it has been suggested that they represent a defense system of the plant. Glucosinolates may as well serve to store inactive precursors of hormones such as 3-indolylacetic acid. 3-Indolyl acetonitrile and related indoles (2Gmelin R. Virtanen A.I Ann. Acad. Sci. Fenn. A. 1961; 11: 3-25Google Scholar) are released from indolyl glucosinolates by myrosinase (3Bones A.M. Rossiter J Physiol. Plant. 1996; 97: 1194-1208Crossref Scopus (586) Google Scholar). Cleavage of indol-3-ylmethyl glucosinolate (glucobrassicin) by myrosinase in the presence of ascorbic acid produces ascorbigen, a condensation product of ascorbic acid with 3-hydroxymethylindole (4Kiss G. Neukom H Mitt. Geb. Lebensm. Hyg. 1966; 57: 443-448Google Scholar). Thus, the myrosinase system could be involved in the storage and in the inactivation of ascorbic acid. A detailed review on myrosinase is given by Bones and Rossiter (3Bones A.M. Rossiter J Physiol. Plant. 1996; 97: 1194-1208Crossref Scopus (586) Google Scholar).Figure 1a, structure of glucosinolates.r = allyl, sinigrin; r =p-hydroxybenzyl, sinalbin, the main glucosinolate inS. alba grains and thus the natural substrate of the myrosinase used in this study. b, 2-F-GTL, the substrate used to produce the glucosyl enzyme (f). c–e, transition state analogues. f, the 2-F-glucosyl group.g, the activator ascorbate. h, nonhydrolyzable substrate analogue.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Myrosinase hydrolyzes the S-glycosides with retention of the anomeric configuration (5Cottaz S. Henrissat B. Driguez H Biochemistry. 1996; 35: 15256-15259Crossref PubMed Scopus (39) Google Scholar). Retaining glycosidases operate by a double displacement at the anomeric center promoted by two carboxylic residues acting as acid/base and as nucleophile, respectively. In the first step (glycosylation), the glycosidic oxygen is protonated by the carboxyl group acting as general acid, whereas the second catalytic residue, a carboxylate group, performs a nucleophilic attack at the anomeric carbon. A glycosyl enzyme intermediate with inverted anomeric configuration is formed via a transition state featuring a (more or less) planar (trigonal) anomeric carbon. In the second step (deglycosylation), the carboxylate group corresponding to the catalytic acid residue activates a water molecule that attacks at C-1 of the glycosyl enzyme to yield the hemiacetal and the free enzyme, againvia a transition state with a trigonal anomeric carbon (6McCarter J.D. Withers S.G Curr. Opin. Struct. Biol. 1994; 4: 885-892Crossref PubMed Scopus (806) Google Scholar). Many carbohydrate derivatives with a planar anomeric carbon and a half-chair or a distorted half-chair conformation inhibit glycosidases. Three such inhibitors, d-glucono-1,5-lactone (Refs. 7Conchie J. Gelman A.L. Levvy G.A Biochem. J. 1967; 103: 609-615Crossref PubMed Scopus (138) Google Scholar and 8Levvy G.A. Snaith S.M Adv. Enzymol. Rel. Areas Mol. Biol. 1972; 36: 151-181PubMed Google Scholarand Fig. 1 c), nojiritetrazole (Refs. 9Ermert P. Vasella A Helv. Chim. Acta. 1991; 74: 2043-2053Crossref Scopus (120) Google Scholar and 10Ermert P. Vasella A. Weber M. Rupitz K. Withers S.G Carbohydr. Res. 1993; 250: 113-128Crossref Scopus (79) Google Scholar and Fig. 1 d), andd-gluconhydroximo-1,5-lactam (Ref. 11Hoos R. Naughton A.B. Thiel W. Vasella A. Weber W Helv. Chim. Acta. 1993; 76: 2666-2678Crossref Scopus (61) Google Scholar and Fig. 1 e), have been studied. A detailed review is given by Heightman and Vasella (12Heightman T.D. Vasella A.T Angew. Chem. Int. Ed. 1999; 38: 750-770Crossref PubMed Google Scholar). Myrosinase belongs to family 1 of the glycoside hydrolases (13Henrissat B. Bairoch A Biochem. J. 1993; 293: 781-788Crossref PubMed Scopus (1768) Google Scholar, 14Henrissat B. Bairoch A Biochem. J. 1996; 316: 695-696Crossref PubMed Scopus (1183) Google Scholar, 15Davies G.J. Henrissat B Structure. 1995; 3: 853-859Abstract Full Text Full Text PDF PubMed Scopus (1609) Google Scholar) but is an unusual member of this family in that it lacks the acid/base residue in its active site. Although classical glycosidases activate the glycosidic oxygen by the catalytic acid residue in the glycosylation step, no such activation appears necessary nor indeed possible for myrosinase. Once the glycosyl enzyme intermediate is formed, the glutamine residue that replaces the catalytic glutamate residue of the classical β-glucosidases ensures the correct positioning of a water molecule, without deprotonating it. This positioning is sufficient to allow hydrolysis of the glycosyl enzyme intermediate and the release of the products (16Burmeister W.P. Cottaz S. Driguez H. Iori R. Palmieri S. Henrissat B Structure. 1997; 5: 663-675Abstract Full Text Full Text PDF PubMed Google Scholar). The covalent glycosyl enzyme intermediate of β-glycosidases can be trapped using substrates that are fluorinated at C-2 (i.e.adjacent to the scissile glycosidic bond) and that carry an aglycon with good leaving group ability (17Withers S.G. Rupitz K. Street I.P J. Biol. Chem. 1988; 263: 7929-7932Abstract Full Text PDF PubMed Google Scholar, 18Withers S.G. Warren R.A.J. Street I.P. Rupitz K. Kempton J. Aebersold R J. Am. Chem. Soc. 1990; 112: 5887-5889Crossref Scopus (207) Google Scholar) (Fig. 1 b) This approach has made it possible to observe several relatively stable 2-fluoro-glycosyl enzyme intermediates by x-ray crystallography (16Burmeister W.P. Cottaz S. Driguez H. Iori R. Palmieri S. Henrissat B Structure. 1997; 5: 663-675Abstract Full Text Full Text PDF PubMed Google Scholar, 19Davies G.J. Mackenzie L. Varrot A. Dauter M. Brzozowski M.A. Schülein M. Withers S.G Biochemistry. 1998; 37: 11707-11713Crossref PubMed Scopus (238) Google Scholar, 20White A. Tull D. Johns K. Withers S.G. Rose D.R Nat. Struct. Biol. 1996; 3: 149-154Crossref PubMed Scopus (186) Google Scholar, 21Cutfield S.M. Davies G.J. Murshudov G. Anderson B.F. Moody P.C.E. Sullivan P.A. Cutfield J.F J. Mol. Biol. 1999; 294: 771-783Crossref PubMed Scopus (86) Google Scholar) (Fig. 1 f). Even though myrosinase shows considerable activity in absence ofl-ascorbic acid, its activity is enhanced by ascorbate, as first described by Nagashima and Uchiyama (22Nagashima Z. Uchiyama M J. Agric. Chem. Soc. Japan. 1959; 33: 980-984Google Scholar). The physiological significance of this activation stems from an optimal effect byl-ascorbate compared with its derivatives (23Ettlinger M.G. Dateo G.P. Harrison B.W. Mabry T.J. Thompson C.P Proc. Natl. Acad. Sci. U. S. A. 1961; 47: 1875-1880Crossref PubMed Scopus (85) Google Scholar), suggesting that the enzyme has evolved to work with the naturally occurringl-ascorbate. A physiological role of the activation by ascorbate is further evidenced by the ascorbate concentration needed for optimal activation of about 1.5 mm, in the range of the global ascorbate concentration in the concerned plant tissues,e.g. 2 mm in horseradish root (24Grob K. Matile P Z. Pflanzenphysiol. 1980; 98: 235-243Crossref Google Scholar). Ascorbic acid is stored in the vacuoles of plant cells (25Matile P Biochem. Physiol. Pflanzen. 1980; 175: 722-731Crossref Google Scholar) where the local concentration is much higher (24Grob K. Matile P Z. Pflanzenphysiol. 1980; 98: 235-243Crossref Google Scholar). A 25-fold increase of the enzymatic activity of Brassica juncea seed myrosinase on the natural substrate sinigrin upon addition of 1 mm ascorbic acid has been found (26Tsuruo I. Hata T Agric. Biol. Chem. 1967; 31: 27-32Crossref Scopus (23) Google Scholar, 27Ohtsuru M. Hata T Biochim. Biophys. Acta. 1979; 567: 384-391Crossref PubMed Scopus (40) Google Scholar). Ettlinger et al. (23Ettlinger M.G. Dateo G.P. Harrison B.W. Mabry T.J. Thompson C.P Proc. Natl. Acad. Sci. U. S. A. 1961; 47: 1875-1880Crossref PubMed Scopus (85) Google Scholar) observed a 400-fold acceleration of the reaction rate for a crude myrosinase preparation fromSinapis alba grains upon addition of ascorbate, using sinigrin as the substrate. Variable degrees of activation, ranging from 1.8- to 11-fold depending on the source of myrosinase (Brassica napus, Brassica campestris, andS. alba), the isoenzyme used, and the substrate have been reported by Björkman and Lönnerdal (28Björkman R. Lönnerdal B Biochim. Biophys. Acta. 1973; 327: 121-131Crossref PubMed Scopus (88) Google Scholar). These authors also found an increase in Km upon activation by ascorbate and concluded that the stability of the enzyme-substrate complex was reduced, presumably because of an increase in the rate constant for the formation of the product. More recently, Shikita et al. (29Shikita M. Fahey J.W. Golden T.R. Holtzclaw D. Talalay P Biochem. J. 1999; 341: 725-732Crossref PubMed Google Scholar) described a 140-fold increase of Vmax in the presence of 0.5 mm ascorbate for the cleavage of sinigrin by myrosinase from Raphanus sativus. This increase inVmax was paralleled by an increase ofKm, an observation that has been interpreted as the result of a noncompetitive activation, because of binding of ascorbate to the enzyme-substrate complex. All the authors quoted above noticed the dual behavior of ascorbate, which activates myrosinase at 0.1–1.0 mm and acts as a competitive inhibitor at higher concentrations, typically above 1.5 mm. Ettlinger et al. (23Ettlinger M.G. Dateo G.P. Harrison B.W. Mabry T.J. Thompson C.P Proc. Natl. Acad. Sci. U. S. A. 1961; 47: 1875-1880Crossref PubMed Scopus (85) Google Scholar) have studied the specificity of myrosinase activation by different derivatives ofl-ascorbate, concluding that an acidic group is essential for the activation and that activation is independent of the reducing property of ascorbate. These investigators suggested that ascorbate plays the role of the catalytic acid/base. We have initiated a program to analyze the details of the mechanism of action of myrosinase and the effect of l-ascorbate using a myrosinase from white mustard (S. alba) grains. This enzyme is a dimer with a mass of 130 kDa, of which 30 kDa are due to glycosylation (16Burmeister W.P. Cottaz S. Driguez H. Iori R. Palmieri S. Henrissat B Structure. 1997; 5: 663-675Abstract Full Text Full Text PDF PubMed Google Scholar). A detailed view on the enzymatic mechanism of myrosinase is derived from high resolution (1.2–1.6 Å) x-ray crystal structure analysis of binary and ternary complexes of myrosinase with inhibitors mimicking the transition state, with ascorbic acid, and with the stable 2-fluoro-glucosyl enzyme intermediate. 2-F-GTL1(Fig. 1 b) was synthesized as described previously (30Cottaz S. Rollin P. Driguez H Carbohydr. Res. 1997; 298: 127-130Crossref Scopus (22) Google Scholar). d-Glucono-1,5-lactone (Fig. 1 c) and ascorbic acid (Fig. 1 g) were purchased from Sigma. Gluco-tetrazole (Fig. 1 d) and gluco-hydroximolactam (Fig. 1 e) were synthesized as described (9Ermert P. Vasella A Helv. Chim. Acta. 1991; 74: 2043-2053Crossref Scopus (120) Google Scholar, 11Hoos R. Naughton A.B. Thiel W. Vasella A. Weber W Helv. Chim. Acta. 1993; 76: 2666-2678Crossref Scopus (61) Google Scholar). C-GTL, the C-glycosidic analogue of glucotropaeolin (Fig. 1 h), was synthesized as a mixture of Z- and E-stereoisomers as described earlier (31Aucagne V. Gueyrard D. Tatibouët A. Quinsac A. Rollin P Tetrahedron. 2000; 56: 2647-2654Crossref Scopus (18) Google Scholar). Myrosinase crystals were prepared as described (16Burmeister W.P. Cottaz S. Driguez H. Iori R. Palmieri S. Henrissat B Structure. 1997; 5: 663-675Abstract Full Text Full Text PDF PubMed Google Scholar) using ammonium sulfate as precipitant. The purified myrosinase remained suitable for crystallization after storage for 3 years at 4 °C in 20 mm HEPES buffer, pH 6.5. For cryoprotection, a crystal was transferred to a solution containing 66% (v/v) saturated ammonium sulfate, 100 mm HEPES, pH 6.5, and 10% (v/v) glycerol. Before freezing in a nitrogen stream at 100 K, the crystal was dipped for a few seconds into a solution containing 20% glycerol. For some trials glycerol was replaced by ethylene glycol. The inhibitors were bound by soaking crystals in artificial mother liquor containing the compound. To obtain the 2-fluoro-glucosyl enzyme, the native crystals were soaked overnight with 2.5 mm 2-F-GTL as the hydrolysis of this compound is slow. For the ternary complex with 2-F-GTL and ascorbate, the ascorbate was introduced subsequently in the cryoprotectant containing 10% glycerol. Data were collected at the European Synchrotron Radiation Facility (Grenoble, France) on experimental station ID14-3 using a 133-mm MarCCD detector or on ID14-1 using a Mar345 detector (gluconolactone data set). Data were processed with MOSFLM (32Leslie A.G.W. Wolf W.M. Wilson K.S. Joint CCP4 and ESF-EADBM Newsletter on Protein Crystallography , no. 26. SERC Daresbury Laboratory, Warrington, United Kingdom1992Google Scholar) and the CCP4 package (33Collaborative Computational Project, Number 4Acta Crystallogr. Sect. D Biol. Crystallogr. 1994; 50: 760-763Crossref PubMed Scopus (19748) Google Scholar). Statistics of data collection and refinements are given in Table I. The initial model was entry 1MYR deposited in the Protein Data Bank (16Burmeister W.P. Cottaz S. Driguez H. Iori R. Palmieri S. Henrissat B Structure. 1997; 5: 663-675Abstract Full Text Full Text PDF PubMed Google Scholar). The native structure has been refined using first X-plor 3.1 (34Brünger A.T. X-PLOR , version 3.1. Yale University, New Haven, CT1992Google Scholar) and then REFMAC (35Murshudov G.N. Lebedev A. Vagin A.A. Wilson K.S. Dodson E.J Acta Crystallogr. Sect. D Biol. Crystallogr. 1999; 55: 247-255Crossref PubMed Scopus (1009) Google Scholar) to a final resolution of 1.2 Å. During the refinement a number of changes in the sequence, which was based on an x-ray structure at 1.6 Å resolution, became apparent and have been corrected.Table IStatistics on the data sets and the refined structuresData setNativeGlucotetrazole (10 mm)Glucohydroximolactam (10 mm)Gluconolactone (20 mm)2-F-GTL (2.5 mm)Ascorbic acid (5 mm)Glucohydroximolactam (5 mm), ascorbate (10 mm)2-F-GTL (2.5 mm), ascorbic acid (10 mm)Protein Data Bank1c4m1c6q1c6s1c6x1c701c711c721c73Resolution (Å)1.20 (1.20–1.26)1.35 (1.35–1.42)1.35 (1.35–1.42)1.60 (1.60–1.66)1.65 (1.65–1.74)1.50 (1.50–1.58)1.60 (1.60–1.66)1.50 (1.50–1.58)Unique22380215109214130899783900171102398466298776Redundancy4.5 (4.0)3.1 (3.0)2.7 (2.5)3.3 (3.0)3.3 (2.4)3.7 (3.0)4.2 (3.3)4.2 (3.3)Complete (%)99.6 (99.6)95.8 (94.9)83.7 (73.6)97.1 (91.3)88.5 (76.2)95.6 (91.5)99.7 (99.7)82.7 (71.4)Rmerge(%)7.6 (38.6)7.2 (27.8)7.5 (40.0)8.9 (33.7)8.5 (35.0)6.5 (29.4)7.6 (25.6)6.3 (35.2)Rcryst(%)12.4 (19.5)11.9 (15.4)12.0 (18.6)13.8 (20.1)16.9 (29.3)12.3 (14.8)12.9 (16.3)13.4 (18.1)Rfree(%)14.2 (20.7)14.6 (19.6)15.2 (24.3)17.8 (28.6)19.5 (34.0)15.1 (19.1)15.7 (19.8)16.7 (24.0)Root mean square bond lengths (Å)0.0150.0170.0190.0160.0140.0140.0120.014Values for the highest resolution shell are given in parentheses. All structures with the exception of the soak with 2-F-GTL have been refined including constrained individual anisotropic B-factor refinement. The contribution of the hydrogen atoms of the protein has been included. Rfree is based on 5% of the reflections (44Brünger A.T. Nature. 1992; 355: 472-475Crossref PubMed Scopus (3860) Google Scholar). Open table in a new tab Values for the highest resolution shell are given in parentheses. All structures with the exception of the soak with 2-F-GTL have been refined including constrained individual anisotropic B-factor refinement. The contribution of the hydrogen atoms of the protein has been included. Rfree is based on 5% of the reflections (44Brünger A.T. Nature. 1992; 355: 472-475Crossref PubMed Scopus (3860) Google Scholar). The complexes have been analyzed with SIGMAA weightedFobs − Fcalc maps calculated with model phases. The model is based on the native high resolution structure from which the water and glycerol molecules in the active site have been removed. A direct interpretation of aFderiv − Fnat Fourier synthesis is hampered by the presence of a glycerol molecule and several well ordered water molecules in the active site. The structure of the complexes have been refined as described for the native structure. Coordinates and structure factors have been deposited in the Protein Data Bank (see Table I for accession numbers). Time-dependent reactivation of the 2-fluoro-glucosyl enzyme covalent intermediate in absence or in presence of ascorbic acid was determined essentially as described previously (5Cottaz S. Henrissat B. Driguez H Biochemistry. 1996; 35: 15256-15259Crossref PubMed Scopus (39) Google Scholar). Myrosinase was inactivated with 2.5 mm2-F-GTL (30Cottaz S. Rollin P. Driguez H Carbohydr. Res. 1997; 298: 127-130Crossref Scopus (22) Google Scholar) for 18 h at 40 °C, followed by four successive ultrafiltrations on Nanosep 30 kDa (Pall Filtron Corp.) to remove the excess of inactivator. The inactivated enzyme was incubated at 25 °C without or with ascorbic acid (at concentrations of 0.1 and 1 mm), and reactivation was monitored using aliquots (20 μl) from the solutions of inactivated enzyme at appropriate time intervals. The samples were assayed by usingp-nitrophenyl-β-d-glucopyranoside (36Botti M.G. Taylor M.G. Botting N.P J. Biol. Chem. 1995; 270: 20530-20535Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar) as the substrate, following the variation of absorbance at 430 nm (λ = 7002 m−1·cm−1 for 4-nitrophenol). First-order rate constants (kreact) were determined by fitting the recovered activity as a function of time. The inhibition constants for gluco-tetrazole and gluco-hydroximolactam have been determined with sinigrin as substrate. The hexokinase/glucose-6-phosphate dehydrogenase coupled enzyme system was used to assay myrosinase activity by measuring glucose release, following the variation of absorbance at 340 nm (37Wilkinson A.P. Rhodes M.J.C. Fenwick G.R Anal. Biochem. 1984; 139: 284-291Crossref PubMed Scopus (39) Google Scholar). Myrosinase was preincubated at 34 °C (10 μl at appropriate dilution) in the absence or presence of inhibitor (38 μl at final concentrations of 0.4, 0.8, and 2 mm for the gluco-tetrazole and 0.125, 0.5, and 1 mm for the gluco-hydroximolactam). Reactions were initiated by addition of substrate (332 μl at final concentration of 0.17, 0.24, 0.35, and 0.7 mm in 50 mm Mes buffer, pH 6.5, containing 3 mm MgCl2, 0.55 mm ATP, 0.72 mm NADP, 0.56 unit/ml hexokinase, and 0.35 unit/ml glucose-6-phosphate dehydrogenase). Kinetic data were fitted to a competitive inhibition model (38Dixon M. Webb E.C Enzymes. 2nd Ed. Longmans, London1964: 92-100Google Scholar). All reagents were purchased from Sigma. Three established or putative transition state analogues, gluco-tetrazole, d-glucono-1,5-lactone, and gluco-hydroximolactam were soaked into the crystals. The resulting electron densities (Fig. 2,a–c) show that the three inhibitors bind similarly, with all hydroxyl groups involved in identical hydrogen bonds. This recognition is similar to that observed for the 2-fluoro-glucosyl enzyme intermediate (Fig. 2 d), except that the three inhibitors display a somewhat distorted half-chair conformation, whereas the glucose ring in the 2-fluoro-glycosyl enzyme has a clear4 C1 chair conformation (Fig. 3). d-Glucono-1,5-lactone binds in a distorted half-chair conformation (Fig. 3) with O-1 of the lactone in van der Waals' contact with Oε1 of Gln 187 (Fig. 2 a). The binding ofd-glucono-1,5-lactone with full occupancy at a concentration of 20 mm in the active site at the exact position of the glucose moiety of the substrate is in contradiction with the experiments by Botti et al. (36Botti M.G. Taylor M.G. Botting N.P J. Biol. Chem. 1995; 270: 20530-20535Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar), who concluded that the d-glucono-1,5-lactone is a noncompetitive inhibitor with a Ki of 5 mm. The inhibition constants for gluco-tetrazole (Ki = 0.7 mm) and gluco-hydroximolactam (Ki = 0.6 mm) were determined using sinigrin as substrate (data not shown). The gluco-tetrazole binds in a pure half-chair conformation (Figs. 2 b and 3). There is a hydrogen bond from Gln187 Oε1 to N1 of the tetrazole for which there is no equivalent for the gluconolactone inhibitor. This hydrogen bond is in the plane of the tetrazole moiety and may explain why the gluco-tetrazole inhibitor is bound at a slightly higher position in the active site than the other inhibitors. It is noteworthy that the residue equivalent to myrosinase Gln187 in relatedO-glucosidases, such as the cyanogenic β-glucosidase from white clover (39Barrett T. Suresh C.G. Tolley S.P. Dodson E.J. Hughes M.A Structure. 1995; 3: 951-960Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar), is the catalytic acid/base glutamate that would be in an appropriate position for an “in plane” protonation of the substrate as predicted by Heightman and Vasella (12Heightman T.D. Vasella A.T Angew. Chem. Int. Ed. 1999; 38: 750-770Crossref PubMed Google Scholar). The structure of the complex with gluco-hydroximolactam shows a slightly distorted half-chair conformation for the inhibitor (Fig. 3). In addition to the inhibitor, a sulfate ion was found bound in the active site. This sulfate ion forms salt bridges and hydrogen bonds to Arg259 Nη2, Gln187 Nε2, and the hydroxyl group of the hydroximo group (Fig. 2 c). The N1 nitrogen atom of this group is in van der Waals' contact with the oxygen Oε1 of the carbonyl group of Gln187. Several, albeit unsuccessful, experiments have been undertaken to observe substrate binding directly. The diastereomeric mixture of C-GTL (Fig. 1 h) did not inhibit at concentrations up to 20 mm (enzyme assays; data not shown), nor did it bind in the crystal at concentrations of up to 100 mm. We also attempted to take advantage of the slow hydrolysis of 2-F-GTL by myrosinase and to trap the enzyme-substrate complex using flash freezing of the crystals. Cottaz et al. (5Cottaz S. Henrissat B. Driguez H Biochemistry. 1996; 35: 15256-15259Crossref PubMed Scopus (39) Google Scholar) determined inactivation parameters Ki of 0.9 mm andki of 0.083 min−1 for 2-F-GTL. These data suggest that it should be possible to observe the enzyme-substrate complex. A number of experiments using different soaking times (5–20 min) and different pH values of the buffer (pH 4.2–6.5) did not lead to the observation of any electron density corresponding to the substrate analogue. The nonglucosylation of Glu409 confirms that myrosinase is only very slowly inactivated by 2-F-GTL. To study the intriguing activation of myrosinase by ascorbate, myrosinase crystals were soaked with ascorbate alone. Omit maps showed clearly the presence of ascorbate together with a glycerol molecule in the active site (Fig. 4 a). Ascorbate is recognized by a salt bridge between Arg259Nε and the O-1 oxygen and by a hydrogen bond between the hydroxyl group at position 2 and Arg259 Nη2. O-3 forms a hydrogen bond with Nε2 of Gln187 (Fig. 4 a). A portion of the ascorbic acid molecule is located in the hydrophobic pocket, which binds the hydrophobic part of the glucosinolates. This pocket is formed by the residues Ile257, Phe331, Tyr330, Phe371, and Phe473, which are invariant in the nine known myrosinase sequences with exception of Phe331. Another conserved residue, Arg194 (Lys in one sequence), is not directly in contact but may play an electrostatic role. The binding of ascorbate is remarkably similar to the binding of the sulfate ion in the gluco-hydroximolactam inhibitor structure. A soak with a mixture of gluco-hydroximolactam and ascorbic acid showed that there is indeed competition between the binding of sulfate and ascorbate. The structure shows a partial occupancy for ascorbate (q = 0.6) and for sulfate (q= 0.4) (Fig. 4 b). Two of the oxygen atoms of the sulfate ion are in positions similar to O-2 and O-3 of ascorbate, interacting with Nε2 of Gln187 and Arg259 Nη2. From their common position, it is obvious that ascorbate and the intact substrate cannot bind together in the active site. However, the presence of both ascorbate and gluco-hydroximolactam (Fig. 4 b) or of ascorbate in the 2-fluoro-glucosyl enzyme (Fig. 4 c) shows that ascorbate can bind once the aglycon of the substrate has diffused away. In the presence of the 2-fluoro-glucosyl group or of the gluco-hydroximolactam, a hydrogen bonds is formed between the O-6 hydroxyl group of glucose and the O-6 hydroxyl group of ascorbic acid (Fig. 4, b and c). Ascorbate is placed ideally to act as a catalytic base and to activate a water molecule, substituting for the general acid/base in the canonical mechanism of retaining glycoside hydrolases. Such a water molecule is visible in Fig. 4 c. It is bound at a distance of 2.56 Å to O-3 of ascorbate, whereas the distance to the C-1 atom of the 2-fluoro-glucosyl enzyme is 3.22 Å. The water molecule is placed 3.70 and 3.99 Å away from Gln187 Nε2 and Oε1, respectively, making a contribution of this residue unlikely. In the 2-fluoro-glucosyl enzyme and in the absence of ascorbate, the water molecule is closer to Gln187 (3.27 and 2.79 Å away from Nε2 and Oε1, respectively) and 3.40 Å from C-1 of the glucose ring. The activation of the hydrolysis of the glucosyl enzyme by ascorbate has been verified using the 2-F-GTL-inactivated enzyme. In the absence of ascorbate, half-reactivation required 53 h. Addition of 1 mm ascorbate resulted in a 14-fold activation, shortening the half-reactivation time to 3.6 h (Fig. 5) and demonstrating that activation concerns hydrolysis of the glucosyl enzyme. The good affinity displayed by the transition state analogous inhibitors (gluco-tetrazole and gluco-hydroximolactam) is in agreement with the hypothesis that the reaction mecha
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