A metal-mediated hydride shift mechanism for xylose isomerase based on the 1.6 A Streptomyces rubiginosus structures with xylitol and D-xylose

The crystal structure of recombinant Streptomyces rubiginosus D-xylose isomerase (D-xylose keto-isomerase, EC 5.3.1.5) solved by the multiple isomorphous replacement technique has been refined to R = 0.16 at 1.64 A resolution. As observed in an earlier study at 4.0 A (Carrell et al., J. Biol. Chem. 259: 3230-3236, 1984), xylose isomerase is a tetramer composed of four identical subunits. The monomer consists of an eight-stranded parallel beta-barrel surrounded by eight helices with an extended C-terminal tail that provides extensive contacts with a neighboring monomer. The active site pocket is defined by an opening in the barrel whose entrance is lined with hydrophobic residues while the bottom of the pocket consists mainly of glutamate, aspartate, and histidine residues coordinated to two manganese ions. The structures of the enzyme in the presence of MnCl2, the inhibitor xylitol, and the substrate D-xylose in the presence and absence of MnCl2 have also been refined to R = 0.14 at 1.60 A, R = 0.15 at 1.71 A, R = 0.15 at 1.60 A, and R = 0.14 at 1.60 A, respectively. Both the ring oxygen of the cyclic alpha-D-xylose and its C1 hydroxyl are within hydrogen bonding distance of NE2 of His-54 in the structure crystallized in the presence of D-xylose. Both the inhibitor, xylitol, and the extended form of the substrate, D-xylose, bind such that the C2 and C4 OH groups interact with one of the two divalent cations found in the active site and the C1 OH with the other cation. The remainder of the OH groups hydrogen bond with neighboring amino acid side chains. A detailed mechanism for D-xylose isomerase is proposed. Upon binding of cyclic alpha-D-xylose to xylose isomerase, His-54 acts as the catalytic base in a ring opening reaction. The ring opening step is followed by binding of D-xylose, involving two divalent cations, in an extended conformation. The isomerization of D-xylose to D-xylulose involves a metal-mediated 1,2-hydride shift. The final step in the mechanism is a ring closure to produce alpha-D-xylulose. The ring closing is the reverse of the ring opening step. This mechanism accounts for the majority of xylose isomerase's biochemical properties, including (1) the lack of solvent exchange between the 2-position of D-xylose and the 1-pro-R position of D-xylulose, (2) the chemical modification of histidine and lysine, (3) the pH vs. activity profile, and (4) the requirement for two divalent cations in the mechanism.     

Xylose isomerase in substrate and inhibitor michaelis states: atomic resolution studies of a metal-mediated hydride shift

Xylose isomerase (E.C. 5.3.1.5) catalyzes the interconversion of aldose and ketose sugars and has an absolute requirement for two divalent cations at its active site to drive the hydride transfer rates of sugar isomerization. Evidence suggests some degree of metal movement at the second metal site, although how this movement may affect catalysis is unknown. The 0.95 A resolution structure of the xylitol-inhibited enzyme presented here suggests three alternative positions for the second metal ion, only one of which appears positioned in a catalytically competent manner. To complete the reaction, an active site hydroxyl species appears appropriately positioned for hydrogen transfer, as evidenced by precise bonding distances. Conversely, the 0.98 A resolution structure of the enzyme with glucose bound in the alpha-pyranose state only shows one of the metal ion conformations at the second metal ion binding site, suggesting that the linear form of the sugar is required to promote the second and third metal ion conformations. The two structures suggest a strong degree of conformational flexibility at the active site, which seems required for catalysis and may explain the poor rate of turnover for this enzyme. Further, the pyranose structure implies that His53 may act as the initial acid responsible for ring opening of the sugar to the aldose form, an observation that has been difficult to establish in previous studies. The glucose ring also appears to display significant segmented disorder in a manner suggestive of ring opening, perhaps lending insight into means of enzyme destabilization of the ground state to promote catalysis. On the basis of these results, we propose a modified version of the bridged bimetallic mechanism for hydride transfer in the case of Streptomyces olivochromogenes xylose isomerase.     

Crystal structure of the yeast His6 enzyme suggests a reaction mechanism

The Saccharomyces cerevisiae His6 gene codes for the enzyme phosphoribosyl-5-amino-1-phosphoribosyl-4-imidazolecarboxamide isomerase, catalyzing the fourth step in histidine biosynthesis. To get an insight into the structure and function of this enzyme, we determined its X-ray structure at a resolution of 1.30 A using the anomalous diffraction signal of the protein's sulphur atoms at 1.77 A wavelength. His6 folds in an (alpha/beta)8 barrel similar to HisA, which performs the same function in bacteria and archaea. We found a citrate molecule from the buffer bound in a pocket near the expected position of the active site and used it to model the open form of the substrate (phosphoribulosyl moiety), which is a reaction intermediate. This model enables us to identify catalytic residues and to propose a reaction mechanism where two aspartates act as acid/base catalysts: Asp134 as a proton donor for ring opening, and Asp9 as a proton acceptor and donor during enolization of the aminoaldose. Asp9 is conserved in yeast His6 and bacterial or archaeal HisA sequences, and Asp134 has equivalents in both HisA and TrpF, but they occur at a different position in the protein sequence.     

Crystal structure of rabbit phosphoglucose isomerase complexed with its substrate D-fructose 6-phosphate.

Phosphoglucose isomerase (PGI, EC 5.3.1.9) catalyzes the interconversion of D-glucose 6-phosphate (G6P) and D-fructose 6-phosphate (F6P) and plays important roles in glycolysis and gluconeogenesis. Biochemical characterization of the enzyme has led to a proposed multistep catalytic mechanism. First, the enzyme catalyzes ring opening to yield the open chain form of the substrate. Then isomerization proceeds via proton transfer between C2 and C1 of a cis-enediol(ate) intermediate to yield the open chain form of the product. Catalysis proceeds in both the G6P to F6P and F6P to G6P directions, so both G6P and F6P are substrates. X-ray crystal structure analysis of rabbit and bacterial PGI has previously identified the location of the enzyme active site, and a recent crystal structure of rabbit PGI identified Glu357 as a candidate functional group for transferring the proton. However, it was not clear which active site amino acid residues catalyze the ring opening step. In this paper, we report the X-ray crystal structure of rabbit PGI complexed with the cyclic form of its substrate, D-fructose 6-phosphate, at 2.1 A resolution. The location of the substrate relative to the side chains of His388 suggest that His388 promotes ring opening by protonating the ring oxygen. Glu216 helps to position His388, and a water molecule that is held in position by Lys518 and Thr214 accepts a proton from the hydroxyl group at C2. Comparison to a structure of rabbit PGI with 5PAA bound indicates that ring opening is followed by loss of the protonated water molecule and conformational changes in the substrate and the protein so that a helix containing amino acids 513-520 moves in toward the substrate to form additional hydrogen bonds with the substrate.     

Hydrogen location in stages of an enzyme-catalyzed reaction: time-of-flight neutron structure of D-xylose isomerase with bound D-xylulose

The time-of-flight neutron Laue technique has been used to determine the location of hydrogen atoms in the enzyme d-xylose isomerase (XI). The neutron structure of crystalline XI with bound product, d-xylulose, shows, unexpectedly, that O5 of d-xylulose is not protonated but is hydrogen-bonded to doubly protonated His54. Also, Lys289, which is neutral in native XI, is protonated (positively charged), while the catalytic water in native XI has become activated to a hydroxyl anion which is in the proximity of C1 and C2, the molecular site of isomerization of xylose. These findings impact our understanding of the reaction mechanism.     

The reaction pathway of the isomerization of d-xylose catalyzed by the enzyme d-xylose isomerase: A theoretical study

Different pathways of the metal-induced isomerization of D-xylose to D-xylulose are investigated and compared in detail using energy minimization and molecular dynamics simulation. Two theoretical models are constructed for the reaction: in vacuum and in the enzyme D-xylose isomerase. The vacuum model is constructed based on the X-ray structure of the active site of D-xylose isomerase. It contains the atoms directly involved in the reaction and is studied using a semi-empirical molecular orbital method (PM3). The model in the enzyme includes the effects of the enzyme environment on the reaction using a combined quantum mechanical and molecular mechanical potential. For both models, the structures of the reactants, products, and intermediate complexes along the isomerization pathway are optimized. The effects of the position of the “catalytic Mg²⁺ ion” on the energies of the reactions are studied. The results indicate: 1) in vacuum, the isomerization reaction is favored when the catalytic metal cation is at site A, which is remote from the substrate; 2) in the enzyme, the catalytic metal cation, starting from site A, moves and stays at site B, which is close to the substrate; analysis of the charge redistribution of the active site during the catalytic process shows that the metal ion acts as a Lewis acid to polarize the substrate and catalyze the hydride shift; these results are consistent with previous experimental observations; and 3) Lys183 plays an important role in the isomerization reaction. The ϵ-NH₃⁺ group of its side chain can provide a proton to the carboxide ion of the substrate to form a hydroxyl group after the hydride shift step. This role of Lys183 has not been suggested before. Based on our calculations, we believe that this is a reasonable mechanism and consistent with site-directed mutation experiments. © 1997 Wiley-Liss Inc.     

The catalytic mechanism of galactose mutarotase

Galactose mutarotase catalyzes the first step in normal galactose metabolism by catalyzing the conversion of beta-D-galactose to alpha-D-galactose. The structure of the enzyme from Lactococcus lactis was recently solved in this laboratory and shown to be topologically similar to domain 5 of beta-galactosidase. From this initial X-ray analysis, four amino acid residues were demonstrated to be intimately involved in sugar binding to the protein: His 96, His 170, Asp 243, and Glu 304. Here we present a combined X-ray crystallographic and kinetic analysis designed to examine the role of these residues in the reaction mechanism of the enzyme. For this investigation, the following site-directed mutant proteins were prepared: H96N, H170N, D243N, D243A, E304Q, and E304A. All of the structures of these proteins, complexed with either glucose or galactose, were solved to a nominal resolution of 1.95 A or better, and their kinetic parameters were measured against D-galactose, D-glucose, L-arabinose, or D-xylose. From these studies, it can be concluded that Glu 304 and His 170 are critical for catalysis and that His 96 and Asp 243 are important for proper substrate positioning within the active site. Specifically, Glu 304 serves as the active site base to initiate the reaction by removing the proton from the C-1 hydroxyl group of the sugar substrate and His 170 functions as the active site acid to protonate the C-5 ring oxygen.     

Crystal structure of phenylacetic acid degradation protein PaaG from Thermus thermophilus HB8

Microbial degradation of phenylacetic acid proceeds via the hybrid pathway that includes formation of a coenzyme A thioester, ring hydroxylation, non‐oxygenolytic ring opening, and β‐oxidation‐like reactions. A phenylacetic acid degradation protein PaaG is a member of the crotonase superfamily, and is a candidate non‐oxygenolytic ring‐opening enzyme. The crystal structure of PaaG from Thermus thermophilus HB8 was determined at a resolution of 1.85 Å. PaaG consists of three identical subunits related by local three‐fold symmetry. The monomer is comprised of a spiral and a helical domain with a fold characteristic of the crotonase superfamily. A putative active site residue, Asp136, is situated in an active site cavity and surrounded by several hydrophobic and hydrophilic residues. The active site cavity is sufficiently large to accommodate a ring substrate. Two conformations are observed for helix H2 located adjacent to the active site. Helix H2 is kinked at Asn81 in two subunits, whereas it is kinked at Leu77 in the other subunit, and the side chain of Tyr80 is closer to Asp136. This indicates that catalytic reaction of PaaG may proceed with large conformational changes at the active site. Asp136 is the only conserved polar residue in the active site. It is located at the same position as those of 4‐chlorobenzoyl‐CoA dehalogenase and peroxisomal Δ³,Δ²‐enoyl‐CoA isomerase, indicating that PaaG may undergo isomerization or a ring‐opening reaction via a Δ³,Δ²‐enoyl‐CoA isomerase‐like mechanism. Proteins 2009. © 2009 Wiley‐Liss, Inc.     

Structure of the complex between trypanosomal triosephosphate isomerase and N-hydroxy-4-phosphono-butanamide: binding at the active site despite an "open" flexible loop conformation.

The structure of triosephosphate isomerase from Trypanosoma brucei complexed with the competitive inhibitor N-hydroxy-4-phosphono-butanamide was determined by X-ray crystallography to a resolution of 2.84 A. Full occupancy binding of the inhibitor is observed only at one of the active sites of the homodimeric enzyme where the flexible loop is locked in a completely open conformation by crystal contacts. There is evidence that the inhibitor also binds to the second active site of the enzyme, but with low occupancy. The hydroxamyl group of the inhibitor forms hydrogen bonds to the side chains of Asn 11, Lys 13, and His 95, whereas each of its three methylene units is involved in nonpolar interactions with the side chain of the flexible loop residue Ile 172. Interactions between the hydroxamyl and the catalytic base Glu 167 are absent. The binding of this phosphonate inhibitor exhibits three unusual features: (1) the flexible loop is open, in contrast with the binding mode observed in eight other complexes between triosephosphate isomerase and various phosphate and phosphonate compounds; (2) compared with these complexes the present structure reveals a 1.5-A shift of the anion-binding site; (3) this is the first phosphonate inhibitor that is not forced by the enzyme into an eclipsed conformation about the P-CH2 bond. The results are discussed with respect to an ongoing drug design project aimed at the selective inhibition of glycolytic enzymes of T. brucei.     

Comparative kinetics of D-xylose and D-glucose isomerase activities of the D-xylose isomerase from Thermus aquaticus HB8

The D-xylose isomerase from T. aquaticus accepts, besides D-xylose, also D-glucose, and, with lower efficiency, D-ribose, and D-arabinose as alternative substrates. The activity of the enzyme is strictly dependent on divalent cations. Mn2+ is most effective in the D-xylose isomerase reaction and Co2+ in the D-glucose isomerization. Mg2+ is active in both reactions, Zn2+ only in the further one. The enzyme is strongly inhibited by Cu2+, and weakly by Ni2+, Fe2+, and Ca2+. A hyperbolic dependence of the reaction velocity of the D-xylose isomerase on the concentration of D-xylose xylose and of D-glucose was found, while biphasic saturation curves were obtained by variation of the metal ion concentrations. The D-glucose isomerization reaction shows normal behaviour with respect to the metal ions. A kinetic model was derived on the basis of the assumption of two binding sites for divalent cations, one cofactor site with higher affinity and a second, low affinity site, which modulates the activity of the enzyme.     

Atomic resolution crystallography reveals how changes in pH shape the protein microenvironment

Hydrogen atoms are a vital component of enzyme structure and function. In recent years, atomic resolution crystallography (>or=1.2 A) has been successfully used to investigate the role of the hydrogen atom in enzymatic catalysis. Here, atomic resolution crystallography was used to study the effect of pH on cholesterol oxidase from Streptomyces sp., a flavoenzyme oxidoreductase. Crystallographic observations of the anionic oxidized flavin cofactor at basic pH are consistent with the UV-visible absorption profile of the enzyme and readily explain the reversible pH-dependent loss of oxidation activity. Furthermore, a hydrogen atom, positioned at an unusually short distance from the main chain carbonyl oxygen of Met122 at high pH, was observed, suggesting a previously unknown mechanism of cofactor stabilization. This study shows how a redox active site responds to changes in the enzyme's environment and how these changes are able to influence the mechanism of enzymatic catalysis.     

Anomeric specificity of D-xylose isomerase.

Crystal structures of complexes of D-xylose isomerase with deoxysugars have been determined. Deoxynojirimycin is a structural analogue of alpha-pyranose and mimics the binding of these aldose substrates. The structure of this complex supports the hypothesis that an imidazole group catalyzes ring opening of the pyranose. The steric restrictions in the active site of the enzyme prevent a beta-pyranose from binding in the same way. For the reverse reaction with ketoses, the anomeric specificity is less certain. Dideoxyimino-D-glucitol is a structural analogue of the ketose alpha-D-furanose. The binding of the inhibitor dideoxyimino-D-glucitol to the crystals of the enzyme does not mimic the binding of the reactive alpha-D-fructofuranose. Superposition of the nonphysiological substrate alpha-D-fructofuranose onto the atomic positions of dideoxyimino-D-glucitol is not possible due to the steric restrictions of the active site. However, by utilizing the approximate 2-fold symmetry of the sugar, a stereochemically sensible model is produced which is consistent with other data. In addition to reaction with alpha-D-furanose, the enzyme probably reacts with open ring keto sugars which are present at significant concentrations. Other sugars which resemble furanoses either do not inhibit significantly or are not observed in the crystals bound in a single conformation.     

Crystal structure of a zinc-activated variant of human carbonic anhydrase I,CA I Michigan 1: evidence for a second zinc binding site involving arginine coordination

The human genetic variant carbonic anhydrase I (CA I) Michigan 1 results from a single point mutation that changes His 67 to Arg in a critical region of the active site. This variant of the zinc metalloenzyme appears to be unique in that it possesses an esterase activity that is specifically enhanced by added free zinc ions. We have determined the three-dimensional structure of human CA I Michigan 1 by X-ray crystallography to a resolution of 2.6 A. In the absence of added zinc ions, the mutated residue, Arg 67, points out of the active site, hydrogen bonding with the carboxylate of Asn 69. This contrasts with the orientation of His 67, in the native isozyme, which points into the active site. The orientations of His 94, His 96, and His 119, that coordinate the catalytic zinc ion, and of the catalytically critical Thr 199-Glu 106 hydrogen bonding system, are largely unchanged in the mutant. The structure of an enzyme adduct with a second zinc bound was determined to a resolution of 2.0 A. The second zinc ion is coordinated to His 64, His 200, and Arg 67. This arginine residue reverses its orientation on zinc binding and turns into the active site. The residues at these three positions have been implicated in determining the specific kinetic properties of native CA I. This is, to our knowledge, the first example of a zinc ion coordinating with an arginine residue in a Zn(II) enzyme.     

Heteronuclear NMR and crystallographic studies of wild-type and H187Q Escherichia coli uracil DNA glycosylase: electrophilic catalysis of uracil expulsion by a neutral histidine 187.

The nature of the putative general acid His187 in the reaction catalyzed by Escherichia coli uracil DNA glycosylase (UDG) was investigated using X-ray crystallography and NMR spectroscopy. The crystal structures of H187Q UDG, and its complex with uracil, have been solved at 1.40 and 1.60 A resolution, respectively. The structures are essentially identical to those of the wild-type enzyme, except that the side chain of Gln187 is turned away from the uracil base and cannot interact with uracil O2. This result provides a structural basis for the similar kinetic properties of the H187Q and H187A enzymes. The ionization state of His187 was directly addressed with (1)H-(15)N NMR experiments optimized for histidine ring spin systems, which established that His187 is neutral in the catalytically active state of the enzyme (pK(a) <5.5). These NMR experiments also show that His187 is held in the N(epsilon)()2-H tautomeric form, consistent with the crystallographic observation of a 2.9 A hydrogen bond from the backbone nitrogen of Ser189 to the ring N(delta)()1 of His187. The energetic cost of breaking this hydrogen bond may contribute significantly to the low pK(a) of His187. Thus, the traditional view that a cationic His187 donates a proton to uracil O2 is incorrect. Rather, we propose a concerted mechanism involving general base catalysis by Asp64 and electrophilic stabilization of the developing enolate on uracil O2 by a neutral His187.     

Hydration Structures of the Human Protein Kinase CK2α Clarified by Joint Neutron and X-ray Crystallography

Casein kinase 2 (CK2) has broad phosphorylation activity against various regulatory proteins, which are important survival factors in eukaryotic cells. To clarify the hydration structure and catalytic mechanism of CK2, we determined the crystal structure of the alpha subunit of human CK2 containing hydrogen and deuterium atoms using joint neutron (1.9 Å resolution) and X-ray (1.1 Å resolution) crystallography. The analysis revealed the structure of conserved water molecules at the active site and a long potential hydrogen bonding network originating from the catalytic Asp156 that is well known to enhance the nucleophilicity of the substrate OH group to the γ-phospho group of ATP by proton elimination. His148 and Asp214 conserved in the protein kinase family are located in the middle of the network. The water molecule forming a hydrogen bond with Asp214 appears to be deformed. In addition, mutational analysis of His148 in CK2 showed significant reductions by 40%–75% in the catalytic efficiency with similar affinity for ATP. Likewise, remarkable reductions to less than 5% were shown by corresponding mutations on His131 in death-associated protein kinase 1, which belongs to a group different from that of CK2. These findings shed new light on the catalytic mechanism of protein kinases in which the hydrogen bond network through the C-terminal domain may assist the general base catalyst to extract a proton with a link to the bulk solvent via intermediates of a pair of residues.     

Crystal structure of thiamin phosphate synthase from Bacillus subtilis at 1.25 A resolution.

The crystal structure of Bacillus subtilis thiamin phosphate synthase complexed with the reaction products thiamin phosphate and pyrophosphate has been determined by multiwavelength anomalous diffraction phasing techniques and refined to 1.25 A resolution. Thiamin phosphate synthase is an alpha/beta protein with a triosephosphate isomerase fold. The active site is in a pocket formed primarily by the loop regions, residues 59-67 (A loop, joining alpha3 and beta2), residues 109-114 (B loop, joining alpha5 and beta4), and residues 151-168 (C loop, joining alpha7 and beta6). The high-resolution structure of thiamin phosphate synthase complexed with its reaction products described here provides a detailed picture of the catalytically important interactions between the enzyme and the substrates. The structure and other mechanistic studies are consistent with a reaction mechanism involving the ionization of 4-amino-2-methyl-5-hydroxymethylpyrimidine pyrophosphate at the active site to give the pyrimidine carbocation. Trapping of the carbocation by the thiazole followed by product dissociation completes the reaction. The ionization step is catalyzed by orienting the C-O bond perpendicular to the plane of the pyrimidine, by hydrogen bonding between the C4' amino group and one of the terminal oxygen atoms of the pyrophosphate, and by extensive hydrogen bonding and electrostatic interactions between the pyrophosphate and the enzyme.     

Structural studies on 3-hydroxyanthranilate-3,4-dioxygenase: the catalytic mechanism of a complex oxidation involved in NAD biosynthesis

3-Hydroxyanthranilate-3,4-dioxygenase (HAD) catalyzes the oxidative ring opening of 3-hydroxyanthranilate in the final enzymatic step of the biosynthetic pathway from tryptophan to quinolinate, the universal de novo precursor to the pyridine ring of nicotinamide adenine dinucleotide. The enzyme requires Fe2+ as a cofactor and is inactivated by 4-chloro-3-hydroxyanthranilate. HAD from Ralstonia metallidurans was crystallized, and the structure was determined at 1.9 A resolution. The structures of HAD complexed with the inhibitor 4-chloro-3-hydroxyanthranilic acid and either molecular oxygen or nitric oxide were determined at 2.0 A resolution, and the structure of HAD complexed with 3-hydroxyanthranilate was determined at 3.2 A resolution. HAD is a homodimer with a subunit topology that is characteristic of the cupin barrel fold. Each monomer contains two iron binding sites. The catalytic iron is buried deep inside the beta-barrel with His51, Glu57, and His95 serving as ligands. The other iron site forms an FeS4 center close to the solvent surface in which the sulfur atoms are provided by Cys125, Cys128, Cys162, and Cys165. The two iron sites are separated by 24 A. On the basis of the crystal structures of HAD, mutagenesis studies were carried out in order to elucidate the enzyme mechanism. In addition, a new mechanism for the enzyme inactivation by 4-chloro-3-hydroxyanthranilate is proposed.     

Catalytic mechanism of xylose (glucose) isomerase from Clostridium thermosulfurogenes. Characterization of the structural gene and function of active site histidine

The gene coding for thermophilic xylose (glucose) isomerase of Clostridium thermosulfurogenes was isolated and its complete nucleotide sequence was determined. The structural gene (xylA) for xylose isomerase encodes a polypeptide of 439 amino acids with an estimated molecular weight of 50,474. The deduced amino acid sequence of thermophilic C. thermosulfurogenes xylose isomerase displayed higher homology with those of thermolabile xylose isomerases from Bacillus subtilis (70%) and Escherichia coli (50%) than with those of thermostable xylose isomerases from Ampullariella (22%), Arthrobacter (23%), and Streptomyces violaceoniger (24%). Several discrete regions were highly conserved throughout the amino acid sequences of all these enzymes. To identify the histidine residue of the active site and to elucidate its function during enzymatic xylose or glucose isomerization, histidine residues at four different positions in the C. thermosulfurogenes enzyme were individually modified by site-directed mutagenesis. Substitution of His101 by phenylalanine completely abolished enzyme activity whereas substitution of other histidine residues by phenylalanine had no effect on enzyme activity. When His101 was changed to glutamine, glutamic acid, asparagine, or aspartic acid, approximately 10-16% of wild-type enzyme activity was retained by the mutant enzymes. The Gln101 mutant enzyme was resistant to diethylpyrocarbonate inhibition which completely inactivated the wild-type enzyme, indicating that His101 is the only essential histidine residue involved directly in enzyme catalysis. The constant Vmax values of the Gln101, Glu101, Asn101, and Asp101 mutant enzymes over the pH range of 5.0-8.5 indicate that protonation of His101 is responsible for the reduced Vmax values of the wild-type enzyme at pH below 6.5. Deuterium isotope effects by D-[2-2H]glucose on the rate of glucose isomerization indicated that hydrogen transfer and not substrate ring opening is the rate-determining step for both the wild-type and Gln101 mutant enzymes. These results suggest that the enzymatic sugar isomerization does not involve a histidine-catalyzed proton transfer mechanism. Rather, essential histidine functions to stabilize the transition state by hydrogen bonding to the C5 hydroxyl group of the substrate and this enables a metal-catalyzed hydride shift from C2 to C1.     

Fluorescent properties of the Escherichia coli D-xylose isomerase active site.

The consequences of active site mutations of the Escherichia coli D-xylose isomerase (E.C. 5.3.1.5) on substrate binding were examined by fluorescence spectroscopy. Site-directed mutagenesis of conserved tryptophan residues in the E. coli enzyme (Trp49 and Trp188) reveals that fluorescence quenching of these residues occurs during the binding of xylose by the wild-type enzyme. The fluorescent properties of additional active site substitutions at His101 were also examined. Substitutions of His101 which inactivate the enzyme were shown to have altered spectral characteristics, which preclude detection of substrate binding. In the case of H101S, a mutant protein with measurable isomerizing activity, substrate binding with novel fluorescent properties was observed, possibly the bound pyranose form of xylose under steady-state conditions.