High Resolution Reaction Intermediates of Rabbit Muscle Fructose-1,6-bisphosphate Aldolase
2005; Elsevier BV; Volume: 280; Issue: 29 Linguagem: Inglês
10.1074/jbc.m502413200
ISSN1083-351X
AutoresM. St-Jean, Julien Lafrance‐Vanasse, B. Liotard, J. Sygusch,
Tópico(s)Diet, Metabolism, and Disease
ResumoCrystal structures were determined to 1.8 Å resolution of the glycolytic enzyme fructose-1,6-bis(phosphate) aldolase trapped in complex with its substrate and a competitive inhibitor, mannitol-1,6-bis(phosphate). The enzyme substrate complex corresponded to the postulated Schiff base intermediate and has reaction geometry consistent with incipient C3-C4 bond cleavage catalyzed Glu-187, which is adjacent by to the Schiff base forming Lys-229. Atom arrangement about the cleaved bond in the reaction intermediate mimics a pericyclic transition state occurring in nonenzymatic aldol condensations. Lys-146 hydrogen-bonds the substrate C4 hydroxyl and assists substrate cleavage by stabilizing the developing negative charge on the C4 hydroxyl during proton abstraction. Mannitol-1,6-bis(phosphate) forms a noncovalent complex in the active site whose binding geometry mimics the covalent carbinolamine precursor. Glu-187 hydrogen-bonds the C2 hydroxyl of the inhibitor in the enzyme complex, substantiating a proton transfer role by Glu-187 in catalyzing the conversion of the carbinolamine intermediate to Schiff base. Modeling of the acyclic substrate configuration into the active site shows Glu-187, in acid form, hydrogen-bonding both substrate C2 carbonyl and C4 hydroxyl, thereby aligning the substrate ketose for nucleophilic attack by Lys-229. The multifunctional role of Glu-187 epitomizes a canonical mechanistic feature conserved in Schiff base-forming aldolases catalyzing carbohydrate metabolism. Trapping of tagatose-1,6-bis(phosphate), a diastereoisomer of fructose 1,6-bis(phosphate), displayed stereospecific discrimination and reduced ketohexose binding specificity. Each ligand induces homologous conformational changes in two adjacent α-helical regions that promote phosphate binding in the active site. Crystal structures were determined to 1.8 Å resolution of the glycolytic enzyme fructose-1,6-bis(phosphate) aldolase trapped in complex with its substrate and a competitive inhibitor, mannitol-1,6-bis(phosphate). The enzyme substrate complex corresponded to the postulated Schiff base intermediate and has reaction geometry consistent with incipient C3-C4 bond cleavage catalyzed Glu-187, which is adjacent by to the Schiff base forming Lys-229. Atom arrangement about the cleaved bond in the reaction intermediate mimics a pericyclic transition state occurring in nonenzymatic aldol condensations. Lys-146 hydrogen-bonds the substrate C4 hydroxyl and assists substrate cleavage by stabilizing the developing negative charge on the C4 hydroxyl during proton abstraction. Mannitol-1,6-bis(phosphate) forms a noncovalent complex in the active site whose binding geometry mimics the covalent carbinolamine precursor. Glu-187 hydrogen-bonds the C2 hydroxyl of the inhibitor in the enzyme complex, substantiating a proton transfer role by Glu-187 in catalyzing the conversion of the carbinolamine intermediate to Schiff base. Modeling of the acyclic substrate configuration into the active site shows Glu-187, in acid form, hydrogen-bonding both substrate C2 carbonyl and C4 hydroxyl, thereby aligning the substrate ketose for nucleophilic attack by Lys-229. The multifunctional role of Glu-187 epitomizes a canonical mechanistic feature conserved in Schiff base-forming aldolases catalyzing carbohydrate metabolism. Trapping of tagatose-1,6-bis(phosphate), a diastereoisomer of fructose 1,6-bis(phosphate), displayed stereospecific discrimination and reduced ketohexose binding specificity. Each ligand induces homologous conformational changes in two adjacent α-helical regions that promote phosphate binding in the active site. Aldolases are ubiquitous enzymes and have been a subject of continuous interest because of their ability to catalyze carbon-carbon bond formation in living organisms. Their role is best known in glycolysis, where fructose 1,6-bis(phosphate) (FBP) 1The abbreviations used are: FBP, fructose-1,6-bis(phosphate); MBP, (2R)-mannitol-1,6-bis(phosphate); TBP, tagatose-1,6-bis(phosphate); DHAP, dihydroxyacetone phosphate; HBP, hexitol-1,6-bis(phosphate); GBP, (2S)-glucitol-1,6-bis(phosphate); DERA, d-2-deoxyribose-5-phosphate aldolase; KDPG, 2-keto-3-deoxy-6-phosphogluconate; r.m.s., root mean square. 1The abbreviations used are: FBP, fructose-1,6-bis(phosphate); MBP, (2R)-mannitol-1,6-bis(phosphate); TBP, tagatose-1,6-bis(phosphate); DHAP, dihydroxyacetone phosphate; HBP, hexitol-1,6-bis(phosphate); GBP, (2S)-glucitol-1,6-bis(phosphate); DERA, d-2-deoxyribose-5-phosphate aldolase; KDPG, 2-keto-3-deoxy-6-phosphogluconate; r.m.s., root mean square.aldolases (EC 4.1.2.13) promote the reversible cleavage of FBP to triose phosphates, d-glyceraldehyde 3-phosphate and dihydroxyacetone phosphate (DHAP). The class I enzyme uses covalent catalysis, implicating a Schiff base formed between a lysine residue on the enzyme and a ketose substrate. In vertebrates, there are three tissue-specific class I aldolases (aldolase A (found in skeletal muscle and red blood cells), aldolase B (found in liver, kidney, and small intestine), and aldolase C (found in neuronal tissues and smooth muscle)), and they are distinguishable on the basis of immunological and kinetic properties (1Penhoet E.E. Rutter W.J. J. Biol. Chem. 1971; 246: 318-323Abstract Full Text PDF PubMed Google Scholar). The catalytic mechanism has been extensively studied using class I aldolase A from rabbit muscle, and key intermediates are depicted in Scheme I. In the forward reaction, a reactive lysine residue in the active site attacks the ketose (2Grazi E. Rowley P.T. Chang T. Tchola O. Horecker B.L. Biochem. Biophys. Res. Commun. 1962; 9: 38-43Crossref PubMed Scopus (68) Google Scholar) of the acyclic FBP substrate (3Rose I.A. Warms J.V.B. Biochemistry. 1985; 24: 3952-3957Crossref PubMed Scopus (22) Google Scholar, 4Ray B.D. Harper E.T. Fife W.K. J. Am. Chem. Soc. 1983; 105: 3731-3732Crossref Scopus (15) Google Scholar). Transient formation of a dipolar tetrahedral carbinolamine with the keto function yields a neutral carbinolamine species 1, which is then dehydrated to the protonated imine form of the trigonal Schiff base 2 (5Jencks W.P. Catalysis in Chemistry and Enzymology. McGraw-Hill, New York1969Google Scholar, 6Avigad G. Englard S. Arch. Biochem. Biophys. 1972; 158: 337-346Crossref Scopus (13) Google Scholar). Proton abstraction of the C4 hydroxyl initiates a rearrangement resulting in cleavage of the substrate C3-C4 bond and enamine formation, shown as species 3, in the active site (7Kuo D.J. Rose I.A. Biochemistry. 1985; 24: 3947-3952Crossref PubMed Scopus (25) Google Scholar). Following d-glyceraldehyde 3-phosphate release, the enamine upon stereospecific protonation (8Rose I.A. Rieder S.V. J. Biol. Chem. 1958; 231: 315-329Abstract Full Text PDF PubMed Google Scholar) forms a Schiff base and is released as DHAP by the inverse reaction sequence shown in Scheme I. From crystallographic structure determination (9Sygusch J. Beaudry D. Allaire M. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 7846-7850Crossref PubMed Scopus (213) Google Scholar), the active site in rabbit muscle aldolase, shown in Fig. 1, contains a number of charged residues, vicinal to the Schiff base-forming Lys-229 (10Lai C.Y. Tchola O. Cheng T. Horecker B.L. J. Biol. Chem. 1965; 240: 1347-1350Abstract Full Text PDF PubMed Google Scholar), that can potentially participate in catalysis. These residues can mediate proton transfers as general acid/base catalysts and stabilize or destabilize charges, and because of their proximity to each other, they are susceptible to electrostatic modification of their pKa values, making role assignment of active site residues exceedingly complex. Residues such as Glu-187, adjacent to Lys-229, have, on the basis of mutagenic, kinetic, and structural data, more than one mechanistic role that includes substrate cleavage, charge stabilization, and mediating proton transfers at the level of the ketimine intermediate (11Maurady A. Zdanov A. de Moissac D. Beaudry D. Sygusch J. J. Biol. Chem. 2002; 277: 9474-9483Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). Other residues, such as Asp-33 and Lys-146, also have consequential roles in catalysis, since their catalytic activity is significantly compromised upon mutagenesis (12Morris A.J. Tolan D.R. J. Biol. Chem. 1993; 268: 1095-1100Abstract Full Text PDF PubMed Google Scholar, 13Morris A.J. Tolan D.R. Biochemistry. 1994; 33: 12291-12297Crossref PubMed Scopus (67) Google Scholar, 14Blonski C. de Moissac D. Perie J. Sygusch J. Biochem. J. 1997; 323: 71-77Crossref PubMed Scopus (41) Google Scholar); the mutation Lys-146 → Arg was shown to perturb substrate cleavage and Schiff base formation (13Morris A.J. Tolan D.R. Biochemistry. 1994; 33: 12291-12297Crossref PubMed Scopus (67) Google Scholar, 15Morris A.J. Davenport R.C. Tolan D.R. Protein Eng. 1996; 9: 61-67Crossref PubMed Scopus (22) Google Scholar). Asp-33 and Glu-187 are within hydrogen bonding distance of Lys-146 in the native structure, and these three residues, from their spatial disposition in the active site relative to Lys-229, must make separate and distinct interactions with bound substrate. Insight into how these and other active site residues participate in catalysis and, in particular, substrate cleavage has been hampered by an absence of structures of reaction intermediates shown in Scheme I. To examine the reaction mechanism in class I aldolases and the role of active site residues, a crystallographic study was undertaken of rabbit muscle class I FBP aldolase in complex with its substrate. Acid quenching experiments using excess rabbit muscle aldolase had previously identified a covalent complex formed at room temperature in the presence of FBP that at pH 7.5 represented ∼50% of substrate bound at equilibrium (3Rose I.A. Warms J.V.B. Biochemistry. 1985; 24: 3952-3957Crossref PubMed Scopus (22) Google Scholar). The nature of the covalent linkage was not resolved. In the same study, total FBP bound to enzyme returned to free solution ∼9 times for each net cleavage reaction, suggesting that steps after formation of the Schiff base 2 were limiting and that reaction intermediates 1 and/or 2 are most likely the preponderant equilibrium populations. However, naturally occurring covalent intermediates with FBP have not been observed to date in crystal structure determinations of class I FBP aldolases, even in the presence of excess FBP (16Dalby A. Dauter Z. Littlechild J.A. Protein Sci. 1999; 8: 291-297Crossref PubMed Scopus (90) Google Scholar, 17Choi K.H. Mazurkie A.S. Morris A.J. Utheza D. Tolan D.R. Allen K.N. Biochemistry. 1999; 38: 12655-12664Crossref PubMed Scopus (51) Google Scholar) or by sodium borohydride reduction (18Choi K.H. Shi J. Hopkins C.E. Tolan D.R. Allen K.N. Biochemistry. 2001; 40: 13868-13875Crossref PubMed Scopus (75) Google Scholar). In bacterial class I aldolases of different substrate specificities, flash freezing of recombinant native aldolase crystals to ∼100 K trapped at acid pH carbinolamine reaction intermediates (19Allard J. Grochulski P. Sygusch J. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 3679-3684Crossref PubMed Scopus (52) Google Scholar, 20Heine A. DeSantis G. Luz J.G. Mitchell M. Wong C.H. Wilson I.A. Science. 2001; 294: 369-374Crossref PubMed Scopus (259) Google Scholar) as well as a Schiff base intermediate in a mutant enzyme form (20Heine A. DeSantis G. Luz J.G. Mitchell M. Wong C.H. Wilson I.A. Science. 2001; 294: 369-374Crossref PubMed Scopus (259) Google Scholar). To investigate the nature of the covalent equilibrium complex in native rabbit muscle aldolase, aldolase crystals were incubated in the presence of saturating FBP concentrations in nonacidic buffer (pH 7.5), similar to those used in the kinetic studies. Flash freezing of rabbit muscle crystals briefly soaked in a saturating FBP solution trapped authentic Schiff base intermediate. Two stereoisomer analogues of FBP, (2R)-mannitol-1,6-bis(phosphate) (MBP) and (4S)-tagatose-1,6-bis(phosphate) (TBP), which form noncovalent adducts, were also trapped in the aldolase active site and provided further insight into the reaction pathway and substrate recognition. The structural analysis indicates a canonical reaction mechanism by which class I aldolases cleave a C-C bond and form Schiff base intermediates and that in the case of mammalian FBP aldolase involves a conformational change induced by active site binding. TBP cyclohexylammonium salt was a gift of Dr W. D. Fessner (University of Darmstadt). Prior to use, the cyclohexylammonium ion was exchanged for the sodium ion using a strong cationic exchanger and neutralized with NaOH. TBP sodium salt was used in all experiments described. Hexitol-1,6-bis(phosphate) (HBP) was prepared by NaBH4 reduction of FBP as described previously and yields a mixture of two diastereoisomers: (2R)-MBP and (2S)-glucitol-1,6-bis(phosphate) (GBP) (21Ginsburg A. Mehler A.H. Biochemistry. 1966; 5: 2623-2634Crossref PubMed Scopus (83) Google Scholar). The amount of Pi in TBP using 31P NMR was estimated at 9% and was negligible in the case of FBP and HBP. All figures in the present paper were prepared using the program PyMOL (22DeLano W.L. The PyMOL Molecular Graphics System. DeLano Scientific LLC, San Carlos, CA2004Google Scholar). Purification and Crystallization—Plasmid pPB14 coding for rabbit muscle aldolase (12Morris A.J. Tolan D.R. J. Biol. Chem. 1993; 268: 1095-1100Abstract Full Text PDF PubMed Google Scholar) was transformed and overexpressed in Escherichia coli strain BL21-SI (Invitrogen). Recombinant rabbit muscle aldolase was purified by a combination of anion and cation exchange chromatography and size exclusion chromatography. Aldolase concentration was determined by BCA protein assay reagent (Pierce) with bovine serum albumin serving as a standard. Enzymatic activity was monitored by spectrophotometry using a coupled assay and following NADH oxidation at 340 nm (23Racker E. J. Biol. Chem. 1947; 167: 843-854Abstract Full Text PDF PubMed Google Scholar). Aldolase crystals were grown by vapor diffusion from a 1:1 mixture of protein solution (10 mg/ml initial protein concentration made up in 20 mm Tris-HCl, pH 7.0) and precipitant buffer (17.5% polyethylene glycol 4000 in 0.1 m Na-HEPES, pH 7.5) that was equilibrated against a reservoir of precipitant. Data Collection and Processing—Aldolase crystals were soaked for 3 min in FBP buffer (mother liquor plus 10 mm FBP) or for 10 min in HBP buffer (mother liquor plus 1 mm HBP) or for 7 min in TBP buffer (mother liquor plus 2 mm TBP). Prior to data collection, crystals were cryoprotected by transfer through a cryobuffer solution (FBP or HBP or TBP buffer plus 20% glycerol) and immediately flash frozen in a stream of gaseous N2 cooled to 100 K. Diffraction data were collected from single crystals at beamline X8-C of the National Synchrotron Light Source (Brookhaven National Laboratory, Upton, NY) using a Quantum 4 charge-coupled device (Area Detector Systems, Poway, CA). As control, a native data set was also collected. All data sets were processed with HKL2000 (24Otwinowski Z. Minor W. Methods Enzymol. 1997; 276: 307-326Crossref PubMed Scopus (38527) Google Scholar), and the results are summarized in Table I.Table IData collection and refinement statisticsNativeFBPMBPTBPData collectionResolution (Å)50-1.8050-1.7620-1.8950.0-1.89(1.86-1.80)aAll values in parentheses are given for the highest resolution shell.(1.84-1.76)aAll values in parentheses are given for the highest resolution shell.(1.97-1.89)aAll values in parentheses are given for the highest resolution shell.(1.99-1.89)aAll values in parentheses are given for the highest resolution shell.Wavelength (Å)0.97950.97950.97951.0000ReflectionsObserved1,681,473955,9322,002,0151,461,622Unique127,047134,970104,042110,492Completeness (%)97.0 (87.1)96.7 (78.9)96.9 (77.2)97.8 (85.8)Average I/σ(I)22.5 (2.8)17.9 (2.0)12.6 (2.5)15.0 (2.1)RsymbRsym=∑hkl∑iIi(hkl)−Ii(hkl)/∑hkl∑iIi(hkl) with i running over the number of independent observations of reflection hkl.0.049 (0.39)0.068 (0.41)0.172 (0.78)0.070 (0.44)Space groupP21P21P21P21Unit cell parametersa (Å), b (Å), c (Å), β (°)83.1, 102.9, 84.7, 98.483.0, 103.2, 84.3, 98.883.0, 103.3, 84.4, 98.983.1, 103.2, 84.5, 98.6RefinementNo. of atomsProtein11,03211,03211,03211,032Water2427240826332215Hetero0768040σ cut-off; I / σ(I) >1111Rcryst (%)cRcryst=∑hkl||Io(hkl)|−|Ic(hkl)||/∑hkl|Io(hkl)|.16.715.516.716.7Rfree (%)dRfree=∑hkl∈T||Io(hkl)|−|Ic(hkl)||/∑hkl∈T|Io(hkl)|, where T is a test data set randomly selected from the observed reflections prior to refinement. The test data set was not used throughout refinement and contained 5, 7, 4, and 3% of the total unique reflections for native, FBP, MBP, and TBP, respectively.20.519.020.421.0r.m.s. deviationBond length (Å)0.0050.0050.0050.005Bond angle (degrees)1.2671.2651.2531.405〈B〉 (Å2)27.826.424.830.9Ramachandran analysiseAnalyzed by PROCHECK (29). (%)Most favorable89.290.189.789.8Allowed10.59.79.49.6Generously allowed0.30.20.90.6Luzzati plot (Å)0.180.160.190.19a All values in parentheses are given for the highest resolution shell.b Rsym=∑hkl∑iIi(hkl)−Ii(hkl)/∑hkl∑iIi(hkl) with i running over the number of independent observations of reflection hkl.c Rcryst=∑hkl||Io(hkl)|−|Ic(hkl)||/∑hkl|Io(hkl)|.d Rfree=∑hkl∈T||Io(hkl)|−|Ic(hkl)||/∑hkl∈T|Io(hkl)|, where T is a test data set randomly selected from the observed reflections prior to refinement. The test data set was not used throughout refinement and contained 5, 7, 4, and 3% of the total unique reflections for native, FBP, MBP, and TBP, respectively.e Analyzed by PROCHECK (29Laskowski R.A. MacArthur M.W. Moss D.S. Thornton J.M. J. Appl. Crystallogr. 1993; 26: 283-291Crossref Google Scholar). Open table in a new tab Structure Solution and Refinement—Initial phases used for model building of native and liganded structures were obtained by molecular replacement using a previously determined structure for the rabbit muscle aldolase tetramer (25Blom N. Sygusch J. Nat. Struct. Biol. 1997; 4: 36-39Crossref PubMed Scopus (110) Google Scholar) (Protein Data Bank entry 1ADO). All crystal structures belong to the monoclinic space group P21 and have one aldolase homotetramer in the asymmetric unit. All reflections having I/σ(I) > 1 were used in refinement; however, electron density maps were calculated to the resolution shown in Table I and corresponded to completeness of ∼80% in the highest resolution shell. The structures were subjected to iterative rounds of refinement (simulated annealing and minimization) with CNS (26Brunger A.T. Adams P.D. Clore G.M. DeLano W.L. Gros P. Grosse-Kunstleve R.W. Jiang J.-S. Kuszewski J. Nilges N. Pannu N.S. Read R.J. Rice L.M. Simonson T. Warren G.L. Acta Crystallogr. Sect. D Biol. Crystallogr. 1998; 54: 905-921Crossref PubMed Scopus (16957) Google Scholar) and model building using O (27Jones T.A. Zou J.Y. Cowan S.W. Kjeldgaard Acta Crystallogr. Sect. A. 1991; 47: 110-119Crossref PubMed Scopus (13009) Google Scholar). Water molecules were automatically added by CNS in initial rounds and manually near the end of refinement. C-terminal regions (residues 344-363) were traced in all subunits with the exception of residues 352-357 in three subunits and the C-terminal residue Tyr-363 in the remaining subunit that were associated with regions of weak electron density. Ligand modeling was based on interpretation of electron density shapes of 2Fo - Fc and Fo - Fc annealed omit maps and using PRODRG for topology and parameter generation (28Schuttelkopf A.W. van Aalten D.M. Acta Crystallogr. Sect. D. Biol. Crystallogr. 2004; 60: 1355-1363Crossref PubMed Scopus (4255) Google Scholar). Binding by FBP and HBP, as the MBP stereoisomer, were readily discernable and were associated with clearly defined electron densities in the active site. Difference electron density (Fo - Fc) annealed omit maps calculated in the final round of refinement confirmed identical binding of ligands in all four subunits. Electron density associated with TBP was not clearly defined, and only phosphate groups that were visible in the active sites were refined. Final model statistics, calculated with CNS and PROCHECK (29Laskowski R.A. MacArthur M.W. Moss D.S. Thornton J.M. J. Appl. Crystallogr. 1993; 26: 283-291Crossref Google Scholar), are shown in Table I. The coordinates and structure factors of native aldolase, covalently bound FBP, and MBP and TBP as noncovalent complexes have been deposited with the Protein Data Bank (entry codes 1ZAH, 1ZAI, 1ZAJ, and 1ZAL, respectively). The final structure models of native aldolase and covalently bound FBP, MBP, and TBP noncovalent complexes have an Rcryst (Rfree) of 0.167 (0.205), 0.155 (0.190), 0.167 (0.204), and 0.167 (0.210), respectively. The corresponding Luzzati atomic coordinate error was estimated at 0.18, 0.16, 0.19, and 0.19 Å, respectively. Ramachandran analysis with PROCHECK placed at least 89% of nonglycine and nonproline residues of the four structures in the most favorable region and with the remainder found in allowed and generously allowed regions, attesting to good model geometry in the structures. Errors in hydrogen bond distances, positional differences, and B-factors are reported as S.D. values and were estimated based on their value in each aldolase subunit. Structure Comparisons—Superpositions were performed with the program PyMOL (22DeLano W.L. The PyMOL Molecular Graphics System. DeLano Scientific LLC, San Carlos, CA2004Google Scholar) using Cα atom coordinates of identical blocks of amino acid sequences and comparing native with liganded aldolase tetramers. Due to conformational heterogeneity among subunits in N-terminal and C-terminal regions, comparisons were performed using residues 10-343. Root mean square (r.m.s.) deviations based on superposition of equivalent Cα atoms are reported in Table II. Repeated superpositions were performed using stretches of 50 amino acids to detect secondary structure elements that were conformationally invariant to binding events. Lowest r.m.s. deviation corresponded to residues 150-250. This stretch of residues (positions 150-250) represented secondary structure elements: β-strands 5 and 6 as well as α-helices 4-6 of the β-barrel structure. Residues 158-259 encompassing these structural elements were then used in all subsequent structure superpositions to discover regions in the liganded structures that underwent conformational changes upon ligand binding. Intersubunit variability within a tetramer was analyzed by the program Polypose (31Diamond R. Protein Sci. 1992; 1: 1279-1287Crossref PubMed Scopus (106) Google Scholar) and yielded r.m.s. differences, based on Cα atom coordinates, that were less than the error in the atomic coordinates for each structure.Table IIConformational changes induced by ligand bindingResidues selectedFBPMBPTBP10-3430.440.460.28158-2590.120.140.1334-65aCalculation of r.m.s. differences for these residues was based on superpositions of liganded complexes onto native enzyme that used exclusively residues 158-259, as described under "Materials and Methods."1.010.950.61302-329aCalculation of r.m.s. differences for these residues was based on superpositions of liganded complexes onto native enzyme that used exclusively residues 158-259, as described under "Materials and Methods."0.800.910.4935aCalculation of r.m.s. differences for these residues was based on superpositions of liganded complexes onto native enzyme that used exclusively residues 158-259, as described under "Materials and Methods."1.250.970.7238aCalculation of r.m.s. differences for these residues was based on superpositions of liganded complexes onto native enzyme that used exclusively residues 158-259, as described under "Materials and Methods."0.640.500.21303aCalculation of r.m.s. differences for these residues was based on superpositions of liganded complexes onto native enzyme that used exclusively residues 158-259, as described under "Materials and Methods."0.750.790.47a Calculation of r.m.s. differences for these residues was based on superpositions of liganded complexes onto native enzyme that used exclusively residues 158-259, as described under "Materials and Methods." Open table in a new tab Comparison among structures of Schiff base intermediates in class I aldolases was made at the level of the covalent intermediate by superposing the Schiff base structure with that of the Schiff base intermediate from rabbit muscle aldolase. Superposition consisted of matching the atomic positions of Lys-229 Nz and FBP carbon atoms C1, C2, and C3 in the Schiff base structure of mammalian FBP aldolase against equivalent atoms in the target aldolase structures. In E. coli d-2-deoxyribose-5-phosphate aldolase (DERA) mutant structure (Protein Data Bank entry 1JCJ) (20Heine A. DeSantis G. Luz J.G. Mitchell M. Wong C.H. Wilson I.A. Science. 2001; 294: 369-374Crossref PubMed Scopus (259) Google Scholar), Lys-167 Nz atom and DERA substrate C1 and C2 carbon atoms were formally equivalent to Lys-229 Nz, FBP C2, and FBP C3 atoms, respectively. In E. coli transaldolase B, equivalent atoms in the reduced Schiff base dihydroxyacetone analogue (Protein Data Bank entry 1UCW) (32Jia J. Schorken U. Lindqvist Y. Sprenger G.A. Schneider G. Protein Sci. 1997; 6: 119-124Crossref PubMed Scopus (63) Google Scholar) were Lys-132 Nz and carbon atoms C1, CH, and C2 of the dihydroxyacetone ligand, respectively. For the carbinolamine precursor formed with pyruvate in E. coli KDPG aldolase (Protein Data Bank entry 1EUA) (19Allard J. Grochulski P. Sygusch J. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 3679-3684Crossref PubMed Scopus (52) Google Scholar), atoms equivalent to Lys-229 Nz, FBP C1, and FBP C2 were Lys-133 Nz and pyruvate carbon atoms C1 and C2. In the structure of the covalent complex formed by archaeal Thermoproteus tenax FBP class I aldolase with DHAP (Protein Data Bank entry 1OK4) (33Lorentzen E. Pohl E. Zwart P. Stark A. Russell R.B. Knura T. Hensel R. Siebers B. J. Biol. Chem. 2003; 278: 47253-47260Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar), equivalent atoms were Lys-177 Nz and carbon atoms C1, C2, and C3 of the DHAP ligand. Superposition of the structure of S. pyogenes TBP class I aldolase 2B. Liotard and J. Sygusch, unpublished data. used 166 residues homologous with rabbit muscle FBP aldolase and yielded an r.m.s. deviation of 1.64 Å based on Cα atoms, as determined by the program DeepView/Swiss-PdbViewer (34Guex N. Peitsch M.C. Electrophoresis. 1997; 18: 2714-2723Crossref PubMed Scopus (9561) Google Scholar). Moreover, the carbinolamine intermediate of the native DERA (PDB entry 1JCL) (20Heine A. DeSantis G. Luz J.G. Mitchell M. Wong C.H. Wilson I.A. Science. 2001; 294: 369-374Crossref PubMed Scopus (259) Google Scholar) was structurally identical to the Schiff base intermediate in the DERA mutant structure, (r.m.s. deviation = 0.14 Å using all Cα atoms), allowing comparison to be made solely among native liganded structures. Modeling and Energy Minimization—The acyclic form of FBP was built using topology parameters from PRODRG and manually docked into the active site of rabbit muscle aldolase as a noncovalent complex. The rabbit muscle aldolase-MBP complex served as template and in the initial docking was used to align the noncovalent FBP complex. The FBP complex was then energy-minimized by 2000 steps of conjugated gradient minimization in CNS. Schiff Base—Flash freezing of rabbit muscle aldolase crystals in the presence of the substrate trapped a covalent complex in the active site under equilibrium conditions. Continuous electron density, extending beyond Lys-229 Nz in each subunit, shown in Fig. 2A, indicates formation of a stable covalent adduct with FBP. The planar shape of the electron density observed about the FBP C2 carbon indicates trigonal hybridization and is consistent with trapping of a Schiff base intermediate in each aldolase subunit. Comparison of average B-factors between bound FBP and interacting side chains, 25.0 ± 2.8 and 20.0 ± 6.0 Å2, respectively, suggests nearly full active site occupancy by FBP. Furthermore, the conformation of the crystallized enzyme is not inconsistent with that of a catalytically active conformer. Within measurement errors, kinetic parameters of soluble rabbit muscle aldolase (not shown) were unaffected by crystallization buffer, precipitant concentration used for crystallization, or glycerol cryoprotectant, and full activity was recovered upon dissolution of the crystalline enzyme. The Schiff base intermediate, shown in Fig. 2B, engages in numerous hydrogen bonding and electrostatic interactions with active site residues. The binding mode by the P1 phosphate was isomorphous with that reported for the NaBH4 reduced covalent complex with DHAP (Protein Data Bank entry 1J4E) (18Choi K.H. Shi J. Hopkins C.E. Tolan D.R. Allen K.N. Biochemistry. 2001; 40: 13868-13875Crossref PubMed Scopus (75) Google Scholar) (r.m.s. deviation = 0.14 Å based on equivalent Cα atoms) wherein Arg-303 curls around and interacts electrostatically with the oxyanion, creating a phosphate oxyanion binding pocket. In addition to the electrostatic interactions, five hydrogen bonding interactions were made with the P1 phosphate oxyanion in the binding pocket including an unusually short hydrogen bond between Ser-271 side chain and the oxyanion (2.45 ± 0.02 Å) and indicating strong active site attachment by the FBP P1 phosphate oxyanion. The P6 phosphate binding site includes active site residue Lys-107 whose participation in P6 phosphate binding is corroborated from affinity labeling of Lys-107 by pyridoxal-P that abrogated FBP binding but not DHAP (35Anai M. Lai C.Y. Horecker B.L. Arch. Biochem
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