Proc. Natl. Acad. Sci. USA Vol. 90, pp. 8459-8463, September 1993 Biochemistry The role of active-site aromatic and polar residues in catalysis and substrate discrimination by xylose isomerase (glucose isomerase/site-directed mutagenesis/enzyme-active site/protein engineering/catalytic effciency) MENGHSIAO MENG*t, MICHAEL BAGDASARIAN*, AND J. GREGORY ZEIKUS*t§¶ Departments of *Microbiology and *Biochemistry, Michigan State University, East Lansing, MI 48824; and Michigan Biotechnology Institute, Lansing, MI 48909 Communicated by T. Kent Kirk, June 11, 1993 (receivedfor review March 18, 1993)

ABSTRACT The functions of individual amino acid resi- hydride shift between Cl and C2, assisted by a divalent metal dues in the active site of Thernoanaerobacterium thermosulfu- at position [II], (v) conformational rearrangement, ring- rigenes D-xylose ketol-isomerase (EC 5.3.1.5) were studied by closure, and release of product. Our previous work on the site-directed substitution. The role of aromatic residues in the isotope effect ofD-[2-2H]glucose on the reaction velocity (3), active-site pocket was not limited to the creation of a hydro- as well as the work of other groups (10), indicated that the phobic environment. For example, Trp-188 provided for sub- transfer of hydrogen between Cl and C2 is the rate-limiting strate binding and Trp-139 aflowed for the discrimination step of the isomerization pathway. Indications were also between D-xylose and D-glucose. Substrate discrimination was found that Trp-139 residue (corresponding to Met-87 in accomplished by steric hindrance caused by the side chain of Arthrobacter) may be a steric hindrance for accommodation Trp-139 toward the larger glucose molecule. Preference of the of glucose in the active-site pocket of the enzyme (11). enzyme for the a-anomer of glucose depended on the His- In the present work we provide further support for the 101/Asp-104 pair. Wide differences observed in the catalytic hydride-shift hypothesis formulated by Collyer et aL (6) and constant (kt) for a- versus i-glucose in the wild-type enzyme extend it by proposing that the hydride shift occurs in the and the fact that only the kt for a-glucose was changed in the cyclic form ofthe substrate rather than in the extended form. His-101-+ Asn mutants strongly suggest that the substrate We also examine the roles ofaromatic amino acid residues in molecule entering the hydride-shift step is stiUl in the cydic the active site of Thermoanaerobacterium thermosulfuri- form. On the basis of these results a revised hypothesis for the genes, including the residue that contributes to the discrim- catalytic mechanism of D-xylose isomerase has been proposed ination between D-xylose and D-glucose. that involves His-101, Asp-104, and Asp-339 functioning as a catalytic triad. MATERIALS AND METHODS D-Xylose ketol-isomerase (EC 5.3.1.5) catalyzes the revers- Strains, Plasmids, and Chemicals. E. coli strain HB101 [F- ible isomerization of D-xylose to D-xylulose as part of the hsdS20 ara-1 recA13 proA12 lacYI galK2 rpsL20 mtl-l xyl-SJ xylose metabolic pathway in microorganisms (1). Due to its (12) was used for expression of the T. thermosulfurigenes ability to use D-glucose as substrate and convert it to D-fruc- xylose isomerase gene as described (3). E. coli strain TG1 [thi tose this enzyme is widely used in industry for production of supE hisD A(lac-proAB)/F' traD36 proA+B+ lacIq sweeteners. Comparison of the primary structures of xylose lacZAM15] and bacteriophage M13mp19 (13) were used for isomerases, deduced from the nucleotide sequences of site-directed mutagenesis and nucleotide-sequence determi- cloned genes reveal that amino acid residues, considered to nation. a-Glucose and p-glucose were from Sigma. play important roles in the active site of the enzyme are DNA Manipulation. Site-directed mutagenesis was per- conserved among all species studied to date (2-5). It is formed by the method of Sayers et al. (14) using the kit from believed, therefore, that all known xylose isomerases use Amersham. Nucleotide sequences of the mutant genes were essentially the same mechanism of catalysis. Two classes of be Class confirmed by the dideoxynucleotide chain-termination xylose isomerases, however, may distinguished. I, method (15). His-101 -- Asn (3), Trp-139 -* Tyr, and Trp-139 represented by the enzymes from Arthrobacter, Streptomy- -- Phe (11) mutant enzymes were created previously. ces rubiginosus, Streptomyces olivochromogenes, and Acti- Protein Purification and Steady-State Kinetics. The previ- noplanes missouriensis, has the N-terminal portion shorter in ous protocol (3) was modified to purify xylose isomerase to comparison with the class II enzymes, represented by the homogeneity (on SDS/PAGE). Briefly, crude cell extract isomerases from Escherichia coli, Bacillus, and Thermoa- was incubated at 75°C for 15 min, precipitate was removed by naerobacterium [formerly classified as Clostridium (3)]. and was fractionated by DEAE- Crystal structures of the class I enzymes have been deter- centrifugation, supernatant mined but no three-dimensional structures ofthe class Sepharose followed by Sephacryl-300 chromatography (3, (4-9), 11). Phe-145 -- Lys and Trp-188 -* His mutant enzymes were II isomerases have been determined to date. heated at 60°C for 20 min, and Trp-139-* Lys mutant enzyme On the basis of the crystal structure of the Arthrobacter was heated at 65°C for 30 min instead of 75°C. The enzyme isomerase and its complexes with different substrates and reactions in 1 ml contained 20 mM Mops (pH 7.0), 1.0 mM inhibitors a reaction mechanism for aldose isomerization has CoCl2, substrate at concentrations of 0.3-2.5 times the KM, been proposed (6, 10). This mechanism included the follow- and enzyme at 10-1500 pg. Temperature and reaction con- ing steps: (i) binding of a-D-pyranose substrate to the en- in table. Reaction zyme, (ii) ring-opening, presumed to be catalyzed by the ditions are listed the footnote to each His-53 residue, (iii) conformational rearrangement of sub- products were determined by the cysteine/carbazole/ strate from pseudocyclic to an extended open-chain form, (iv) Abbreviations: kt, catalytic rate constant; KM(agjucosc)AFPP apparent KM for a-glucose; KM(pgIucose)AP, apparent KM for p-glucose. The publication costs ofthis article were defrayed in part by page charge tPresent address: Department of Biochemistry, The University of payment. This article must therefore be hereby marked "advertisement" Texas, Southwestern Medical Center, Dallas, TX 75235. in accordance with 18 U.S.C. §1734 solely to indicate this fact. 1To whom reprint requests should be addressed. 8459 Downloaded by guest on October 2, 2021 8460 Biochemistry: Meng et al. Proc. Natl. Acad. Sci. USA 90 (1993) Table 1. Experimental and corrected values for a-glucose The reaction constant k, calculated from the measurement of isomerization reaction velocity mutarotation and expressed in decimal logarithms and min-', Substate concentraion, mM Velocity, janol/min per mg was 0.0073 ± 0.0009. Because the spontaneous mutarotation rate is faster than the enzyme-catalyzed rate of glucose P-Glucose a-Glucose* Vtowt V.* VP§ isomerization, we assumed that 300 6.6 0.56 0.33 0.23 100 2.2 0.25 0.13 0.12 d[ja-glucose] d[a-glucose] 60 1.3 0.17 0.08 0.09 40 0.9 0.12 0.05 0.07 dt (Enzyme) = dt (buffer) = 143glucose]. [2] *Values were estimated from determination of mutarotation. tApparent values were determined experimentally. The content of p-glucose after 1.5 min of incubation under tVelocity was calculated from Eq. 4. conditions used for enzymatic reaction was 96.6% of total. Velocity was calculated from Yt.tj - V. The content at 0 time, obtained by extrapolation of the was 99.1%. As an approximation, there- sulfric acid method (16). Kinetic constants were determined mutarotation curve, from both Lineweaver-Burk and Eadie-Hofstee plots (17). fore, we can consider that during the first 1.5 min [p-glucose] We defined the catalytic constant (kc) as the turnover = constant and number per active site of enzyme at saturating substrate d[a-glucose] concentration and determined it from the equation kat[E]o = - ~~=constant. [31 V., where [EJ. = total active-site concentration. dt Correction for Spontaneous Mutarotation. In the determi- nation of V. and KM for p-glucose, the interference from It is reasonable, therefore, to use the average content of a-glucose, present as impurity and formed by spontaneous a-glucose = 2.2%, present in the solution of p-glucose during mutarotation, was considered because KM(algucose)App < < the initial 1.5 min ofincubation with the enzyme, to calculate KM(p.glucose)App, where KM values represent apparent values the apparent initial velocity offructose formation from a-glu- for a-glucose and p-glucose, respectively. If both anomers cose, Va, from the equation: are present, fructose may arise from two different reactions: [a-glucose] V - Vmax(a-glucose) [4] V [a-glucose] + p-glucose J fructose Km(,glucose) where we assumed Vn(aglucose) Vmax(cvgjucose)App and mutarotation ] | KM(a.glucose) " KM(a-gucose)App. The velocity of fructose formation from p-glucose at a given concentration of p-glucose, Vq, could thus be calcu- a-glucose Va fructose. lated and used to calculate Vma(frglucose) and KM(pgluwse). To show the corrections obtained by this method one set ofdata for the wild-type enzyme is presented in Table 1. The initial velocity of fructose formation from p-glucose The same method was used to correct for mutarotation in (VP) may be calculated by subtracting the initial velocity of the determination ofthe V (aguwse) and KM(a-glucose). It was fructose formation from a-glucose (that exists as impurity or that these were small. is formed from p-glucose by mutarotation), (Va), from the found corrections very apparent velocity offructose formation (Vs + Va) determined experimentally. To account for the spontaneous mutarota- RESULTS AND DISCUSSION tion we have followed the change in optical rotation of The Role of Aromatic Residues in the Active Site of Xylose p-glucose under the same conditions as were used to deter- Isomerase. In the active site of xylose isomerase from Ar- mine enzymatic glucose isomerization (20 mM Mops, pH throbacter (Fig. 1), the substrate analogue (a-5-thio-D- 7.0/1.0 mM CoCk2; 35°C; 1.5 min) with the Autopol II glucose) is sandwiched between Trp-15 and Trp-136. In the T. automatic polarimeter (Rudolph Research, Flanders, NJ). thermosulfurigenes enzyme these residues correspond to Because mutarotation is reversible and its rate constant is Trp-49 and Trp-188. In addition, the residue Met-87 in the independent of the concentration of sugar over a wide range Arthrobacter enzyme is conserved as tryptophan in all class of concentrations, at initial reaction conditions we have II enzymes [Trp-139 in the enzyme from T. thermosulfuri- d[a-glucose] - d[p-glucose] genes (3)]. This residue is very close to the C6-OH group of = k[p-glucose]. [1] glucose and may constitute a steric hindrance in the discrim- dt dt ination between D-xylose and D-glucose as substrate.

I K182 Wl36 K182 :3 -:W136 18 3j 1~18j8

D254

FIG. 1. Stereostructure ofthe active site of D-xylose isomerase from Arthrobacter containing the substrate analog a-5-thio-D-glucose [from the coordinates of Collyer et al. (6)]. The metal ion sites are represented by crosses. Amino acids are indicated in one-letter code. Downloaded by guest on October 2, 2021 Biochemistry: Meng et aL Proc. Natl. Acad. Sci. USA 90 (1993) 8461 Table 2. Kinetic constants for wild-type and mutant D-xylose isomerases substituted in the active-site aromatic amino acids Glucose Xylose Enzyme kcat, S-1 KM, mM kcat/KM kcat, S-1 KM, mM kcat/KM Wild type 11 ± 2.0 110 ± 8 0.10 23 ± 2.0 9.3 ± 1.8 2.5 Trp-139 Tyr 9 ± 0.4 91 ± 12 0.10 10 ± 1.0 14 ± 1.0 0.7 Trp-139 Phe 16 ± 1.0 65 ± 7 0.25 24 ± 2.0 16 ± 2.0 1.5 Trp-139 Met 11 ± 1.0 55 ± 2 0.20 10 ± 1.0 9.2 ± 0.2 1.1 Trp-139 Leu 8 ± 0.4 50 ± 4 0.16 14 ± 1.0 11 ± 1.0 1.3 Trp-139 Val 5 ± 0.2 35 ± 1 0.14 8 ± 0.1 6.4 ± 0.1 1.3 Trp-139 Ala 8 ± 0.2 31 ± 2 0.26 8 ± 0.2 5.9 ± 0.4 1.4 Trp-139 Lys 3 ± 0.2 21 ± 1 0.14 4 ± 0.1 5.4 ± 0.7 0.7 Trp-49 Phe 10 ± 0.3 330 ± 14 0.03 ND ND ND Trp-49 Ala 7 ± 0.3 710 ± 48 0.01 ND ND ND Trp-49 Arg 10 ± 1.0 110 ± 3 0.09 ND ND ND Wild type (37°C)* ND ND ND 1 ± 0.1 1.1 ± 0.1 0.9 Trp-188 His (3rC) ND ND ND 0.5 ± 0.02 820 ± 30 6 x 10-4 Phe-145 Lys (37°C) ND ND ND 1 ± 0.1 53 ± 6 2 x 10-2 ND, not determined. *Reactions were performed at 37°C, as indicated in parentheses; all other reactions were run at 65°C. ND, not determined. As shown in Table 2, substitution of Trp-139 by residues KM@UCOSC), respectively, with no appreciable change in k,.t with smaller side chains resulted in lower KMeUCOSC). Most Surprisingly, the substitution Trp-49 -) Arg did not change mutant enzymes thus obtained exhibited higher catalytic appreciably either KM or k,at (Table 2). According to the efficiency (kct/Km) for glucose and lower catalytic efficiency structure of the Streptomyces enzyme the NE1 of this indole for xylose than the wild-type enzyme. The correlation be- residue contributes to the construction of the active-site tween the water-accessible surface of the hydrophobic resi- hydrogen-bonding network (8). The loss of one hydrogen due in position 139 and the KMIUCOSc) (Fig. 2) suggests that, bond in the network is expected to affect indirectly the at least, part of the side chain of Trp-139 protrudes into the binding affinity for the substrate. It is possible that the Ng of cavity of the active-site pocket and constitutes a steric arginine may take a position in which it can perform the same hindrance against the binding of glucose. Reduction of this function as the Nel of Trp-49. steric hindrance creates mutant enzymes that accommodate Mutant enzymes obtained by substitution of Trp-188 (Ar- glucose better than the wild-type enzyme. The Trp-139 -* throbacter equivalent, Trp-136) with Lys, Asp, or Glu ex- Lys mutant fails out ofthe proportionality rule. The polar and hibited no detectable activity with either D-glucose or D-Xy- positively charged side chain may cause a local structure lose, although the proteins remained soluble under the con- change and thus affect the affinity of substrate indirectly or ditions of assay (65°C). The mutant Trp-188 -- His showed a increase the affimity directly by hydrogen-bonding to the low activity only with D-xylose. The KM(xyIose) ofthis mutant substrate. A methionine residue is present in the correspond- enzyme at 65°C was >2 M, and thus the kinetic constants ing position of the enzymes from Arthrobacter (5, 6), Strep- could not be measured precisely. They could, however, be tomyces (4, 8), and Actinoplanes (9). It is possible that the determined at 37°C. Under these conditions the KM(xy1ose) was KM@UCOSC) may be reduced for these enzymes if this methi- 800-fold higher than that of the wild-type enzyme, whereas onine is replaced with a smaller hydrophobic residue. the k,at was only lower by afactor of2 (Table 2). These results Substitution ofTrp-49 in the T. thermosulfurigenes enzyme are consistent with the prediction, deduced from the crystal (Trp-15 in the Arthrobacter enzyme) with phenylalanine or structure of xylose isomerase, that the hydrophobic interac- alanine residues resulted in a 3- and 6.5-fold increase in tions between the indole ofTrp-188 and the carbon backbone 4 on. of pyranose contribute strongly to substrate binding in the lcu I active site. The role of tryptophan residues in binding sub- Trp strates has been demonstrated in maltose-binding protein (19), arabinose-binding protein, and galactose-binding pro- 100- tein (20). Substitution of Phe-145 (Arthrobacter equivalent, Tyr Phe-93) with lysine resulted in a 50-fold increase ofKM(XYlOSe) 0 and an insignificant change in kcat (Table 2). This result 80- suggests that Phe-145 also plays an important role in substrate binding. Phe-145 may orient the indole group ofTrp-188 in an Phe 60- optimal position for substrate interaction. 2t Met Anomeric Specificity of Xylose Isomerase. The models of Leu . catalytic isomerization suggested by the crystallographic data include aring-opening step, presumed to be catalyzed by 40 - His-53 residue acting as a base (6, 8). We have, therefore, tested, by mutagenesis, the role ofthe corresponding residue, Ala 1 His-101, and Asp-104, a residue that may assist the function 20 . I I of His-101, in T. thermosulfurigenes xylose isomerase. As 100 150 200 250 300 was shown in the previous work (3) and here in Table 3, Water-accessible surface area (A substitution ofthe His-101 by residues incapable ofacting as FIG. 2. Correlation between the KMW1UCOSC) and the water- a base, but capable ofaccepting hydrogen bonds, resulted in accessible surface area ofthe side chain ofthe residue in position 139 mutant enzymes exhibiting considerable residual activity in T. thermosulfurigenes xylose isomerase. Values for water- with insignificant change of KM. Similar results have been accessible surface areas are from ref. 18. reported for the A. missouriensis enzyme (21). Downloaded by guest on October 2, 2021 8-162 Biochemistry: Meng et al. Proc. Natl. Acad. Sci. USA 90 (1993) Table 3. Comparison of a-D-glucose and 3-D-glucose kinetic constants for variant D-xylose isomerases a-n-Glucose P-D-Glucose kot/KM(a) Enzyme k,,, s 1 KM, mM kcats -1 KM, mM kw/KM(pO Wildtype 1.30 ± 0.03 24 ± 1 0.25 ± 0.03 136 ± 12 27 His-101 Asn 0.15 ± 0.01 30 ± 2 0.26 ± 0.03 130 ± 16 2.5 Asp-104 Asn 0.65 ± 0.01 33 ± 1 0.14 ± 0.02 204 ± 18 29 Asp-104 Ala 0.08 ± 0.01 45 ± 2 0.27 ± 0.01 275 ± 20 1.8 Reactions were started with freshly prepared substrate and run at 350C for 1.5 min. The figures have been corrected as described in Materials and Methods.

The activity ofthe wild-type and mutant enzymes was also In the second mechanism, a base attracts the proton from the determined for the a- and ,B-anomers of D-glucose. In the C2-OH, and this is followed by a hydride shift and ring wild-type enzyme kcat( glucose) was 5-fold lower than the opening. kCat(<,lU5C.). The substitution His-101 Asn reduced the Two arguments can be raised against the cis-enediol inter- kc.t(,,g..) to 12% of the wild-type value, whereas kw(p mediate mechanism: (i) no residue capable of acting as a lucose) did not change. The KM for either anomer remained general base has been seen near the C2 hydrogen in the essentially unchanged (Table 3). If the hydrogen transfer, available crystal structures; (ii) no exchange of proton with shown previously to be the rate-limiting step (3), occurred the medium occurs during the isomerization reaction (23). after the opening of the pyranose ring, it would be expected Therefore, we propose the second mechanism, involving the that kctwglucose) and kcat(a.g1ucosc) would be of the same order hydride shift particularly because the crystal structure ofthe ofmagnitude because anomers do not exist in the open-chain enzyme from Streptomyces (8) indicated that Asp-287 residue form. Moreover, it would be expected that both constants (corresponding to Asp-339 of T. thermosulfurigenes enzyme) would be affected to the same extent by the His-101 Asn is close enough to C2-OH of the substrate to attract its mutation. The results presented in Table 3 suggest that the proton. hydrogen transfer occurs when the structure of the substrate If the position of His-101 in the T. thermosulfurigenes molecule is cyclic rather than linear. This conclusion is isomerase is indeed equivalent to the position ofHis-53 ofthe consistent with the results of crystallographic studies done Arthrobacter enzyme, its major role would be as hydrogen- under steady-state conditions in a flow-cell by Farber et al. bond acceptor to stabilize the transition state of the rate- (4). The electron densities observed by these authors indi- limiting step (3). The Asp-104 residue could assist this cated that the rate-limiting step was preceded by a cyclic form function by stabilizing the His-101 residue. As shown in of the substrate. We hypothesize that hydrogen transfer and Table 3, substitution Asp-104 -o Asn resulted in a drop ofkcat ring opening occur as a concerted single-step reaction. Two by 501O. The mutant obtained by the Asp-104 -* Ala possible mechanisms for this step may be considered. In one substitution, exhibited a kCt(,g1uwse) of only 6% of the wild ofthem, a base attracts the proton from the C2 carbon ofthe type, whereas the kcat(*glucose) remained unchanged. This pyranose; this results in the formation of a cis-enediol inter- result suggested that anomeric specificity of xylose mediate and ring opening during the transfer of proton (22). isomerase depends not only on the presence of His-101 but also depends on the position ofthis residue. With Asp-104 in His-1011 place, either oxygen of its carboxyl group can function as hydrogen-bond acceptor. By providing a hydrogen bond to His-101, Asp-104 locks His-101 at one of the two possible tautomeric forms, thus ensuring that N82 ofHis-101 is always .-1NH N ------HO- Transition state a hydrogen-bond acceptor. In the Asp-104 -. Asn mutant lAsn-1041 only the oxygen ofthe amide group can provide this function (Fig. 3). Without the hydrogen bond provided by Asp-104 (e.g., in Asp-104 -- Ala mutant) the imidazole of His-101 \e<°" could rotate or take up the tautomeric form that is unfavor- NH2 able for the formation of the hydrogen bond to the transition state. In the previously proposed models, the extended-chain molecules ofthe substrate, identified in the crystal structures of the enzyme, were interpreted as being intermediates that IHis-10l precede the hydride shift (6, 8). It is possible, however, that these extended-chain sugar molecules were not intermediates that preceded the rate-limiting step. They could be, for example, the free ketose of xylulose because it is present as N N-H HO- Transition state lAsn-104I a 20.2% fraction in the aqueous solutions of xylulose at Table 4. Kinetic constants of variant D-xylose isomerases w .NNH2 toward xylose o k:at/KM(mutant) FIG. 3. Schematic representation of the interaction between Enzyme kw, s-1 KM, mM kcat/KM(wild type) His-101, Asn-104, and the Cl-OH of the transition state. Note that Wildtype 23 1.0 9.3 ±2.0 1.0 Asp-104, which occupies this position in the wild-type enzyme, can Asp-309 Asn 2.7 ± 0.1 5.8 ± 1.0 0.2 lock His-101 in a particular tautomeric form, regardless of the Asp-296 Asn 1.0 ± 0.1 140 ± 20 28 x 10-4 rotational position of its amide group, whereas Asn-104 can perform Asp-339 - Asn 0.08 ± 0.01 70 ± 10 4 x 10-4 this function only when its carboxyl group is in the position repre- sented at the top of the figure. Reactions were performed at 65°C for 30 min. Downloaded by guest on October 2, 2021 Biochemistry: Meng et aL Proc. Natl. Acad. Sci. USA 90 (1993) 8463

MM[I]II M[I] As p-339 | M[IJ |AsP-3391 O1.0 OH . --. MOH OH" Asp-339 - -

I His-1011 [Ap104 | IHis-1011 Asp-104 I His.1071I | sp14 FIG. 4. Proposed catalytic mechanism for D-xylose isomerase involving the cyclic substrate, amino acid catalytic triad, and divalent metal in position [I]. equilibrium (24). Whitlow et al. (8) actually suggested that the shift from Cl to C2 and simultaneously induces the ring extended species in the enzyme-xylose-MnCI2 structures opening. Metal [I] stabilizes the substrate and the transition observed in their diffraction pictures may be better described state by coordination and, perhaps, provides the electrostatic as xylulose. force to stabilize the developing negative charge at the C5-O. The Role of Metal-Coordinating Residues. To test the func- The involvement of three different amino acid residues in a tion ofeach ofthe two metal ions in the active-site pocket, we catalytic triad is another hypothesis for the xylose isomerase have substituted Asp-309, -296, and -339 residues (Arthro- mechanism. Nonetheless, the involvement of a specific cat- bacter equivalents -256, -244, and 292, respectively; Fig. 1). alytic triad has been reported for different hydrolytic sac- Asp-309 corresponds to the residue binding the metal at charidases, such as a-glucanases (28). position [II], whereas Asp-296 and Asp-339 correspond to We thank Paul Johnson for advice on mathematical correction for residues that bind the metal in position [I]. In addition to its mutarotation of anomeric substrate and David Blow for the discus- metal-coordinating function, Asp-257 in the Streptomyces sions on the structure ofxylose isomerase. This work was supported by a gant from the U.S. Department of Agriculture (90-34189-5014 and Actinoplanes isomerases (corresponding to Asp-256 in to Michigan Biotechnology Institute) and Research Excellence Fund the Arthrobacter and Asp-309 in Thermoanaerobacterium) from the State of Michigan. was proposed to act as a general base that initiated the 1. Chen, W. P. (1980) Process Biochem. 15, 36-41. hydride shift on the linear form of the substrate (8, 25). 2. Dekker, K., Yamagata, H., Sakaguchi, K. & Udaka, S. (1991) J. Substitution of each of these aspartate residues with as- Bacteriol. 173, 3078-3083. paragine resulted in enzymes that still required Co2+ for 3. Lee, C., Bagdasarian, M., Meng, M. & Zeikus, J. G. (1990) J. Biol. Chem. 265, 19082-19090. maximal activity and thermostability suggesting that the 4. Farber, G. K., Glasfeld, A., Tiraby, G., Ringe, D. &Petsko, G. A. (1989) metal-binding site still exists in these mutants. The Asp-309 Biochemistry 28, 7289-7297. -- Asn mutant enzyme exhibited =w20%o of the wild-type 5. Henrick, K., Coliyer, C. A. & Blow, D. M. (1989)J. Mol. Bio. 208,129-157. 6. Collyer,C. A., Hemick, K. &Blow, D. M. (L990)J. Mol.Biol. 212,211-235. catalytic efficiency (kvat/KM; Table 4). This result argues 7. Carrell, H. L., Glusker, J. P., Burger, V., Manfre, F., Tritsch, D. & against the hypothesis that Asp-309 might be the essential Biellmann, J. F. (1989) Proc. Nad. Acad. Sci. USA 86, 4440 -dd4. catalytic base that initiates the hydride shift. 8. Wbitlow, M., Howard, A. J., Finzel, B. C., Poulos, T. L., Winborne, E. -. -. & GilCiland, G. L. (1991) Proteins 9, 153-173. Substitutions Asp-296 Asn or Asp-339 Asn caused 9. Jenkins, J., Janin, J., Rey, F., Chiadmi, M., van Tilbeurgh, H., Lasters, drastic decreases in catalytic efficiency resulting from both I., De Maeyer, M., Van Belle, D., Wodak, S. J., Lauwereys, M., the increase in KM and decrease in ka (Table 4). Thus, these Stanssens, P., Mrabet, N. T., Snawaert, J., Matthyssens, G. & Lam- beir, A.-M. (1992) Biochemistry 31, 5449-5458. two residues, or the metal[I], seem to play an important role 10. Smart, 0. S., Akins, J. & Blow, D. M. (1992) Proteins 13, 100-111. in the stabilization of the substrate and the transition state. 11. Meng, M., Lee, C., Badasarian, M. & Zeikus, J. G. (1991) Proc. Nati. The four order-of-magnitude decrease in kat/KM due to the Acad. Sci. USA 88, 4015-4019. Asp-339 -- Asn substitution supports the hypothesis that 12. Boyer, H. W. & Roulland-Dussoix, D. (1969) J. Mol. Biol. 41,459-472. 13. Yanish-Perron, C., Vieira, J. & Messing, J. (1985) Gene 33, 103-119. Asp-339 is the essential base in the catalytic mechanisms of 14. Sayers, J. R., Schmidt, W. & Eckstein, F. (1988) Nucleic Acids Res. 16, xylose isomerase. 791-802. The Proposed Catalytic Mechanism of Xylose Isomerase. In 15. Sanger, F., Nicklen, S. & Coulson, A. R. (1977) Proc. Natd. Acad. Sci. the previously proposed models it was suggested that the USA 74, 5463-5467. 16. Dische, Z. & Borenfreund, E. (1951) J. Biol. Chem. 192, 583-587. hydride shift is catalyzed by the metal in site [I1 (6). Results 17. Fersht, A. R. (1985) Enzyme Structure and Mechanisms (Freeman, San ofthis work indicate that substitution ofamino acid residues Francisco), 2nd Ed. coordinating to metal site [I] has a much more drastic effect 18. Chothia, C. (1976) J. Mol. Biol. 105, 1-14. on the activity than the substitution of the residues coordi- 19. Martineau, P., Szmelcman, S., Spurlino, J. C., Quiocho, F. A. & Hof- nung, M. (1990) J. Mol. Biol. 214, 337-352. nating to metal site [1]. It is possible, therefore, that metal [I] 20. Quiocho, F. A. (1989) Pure Appl. Chem. 61, 1293-1306. stabilizes the substrate and the transition state by coordina- 21. Lanbeir, A. M., Lauwrreyes, M., Stanssens, P., Marabet, N. T., Snau- tion as well as by electrostatic interaction with the developing waert, J., van Tilbeurgh, H., Matthyssens, G., Lasters, I., De Macyer, negative charge of the transition state. Studies of the coor- M., Wodak, S. J., Jenkins, J., Chiadami, M. & Janin, J. (1992) Biochem- istry 31, 5459-5466. dination sphere of the two metal-binding sites by spectro- 22. Makkee, M., Kleboom, A. P. G. & van Bekkum, H. (1984) Recl. Trav. scopic methods have also suggested that metal site A, cor- Chim. Pays-Bas 103, 361-364. responding to the metal site [IE identified in the x-ray studies, 23. Bock, K., Meldal, M., Meyer, B. & Wiebe, L. (1983)Acta Chem. Scand. is the site responsible for catalysis (26, 27). Ser. B 37, 101-108. 24. Wu, J. & Serianni, A. S. (1990) Carbohydr. Res. 2/6, 1-12. We would like, therefore, to propose, in Fig. 4, another 25. van Tilbeurgh, H., Jenkdns, J., Chiadmi, M., Janin, J., Wodak, S., model for the reaction catalyzed by xylose isomerase. In this Mrabet, N. T. & Lambeir, A.-M. (1992) Biochemistry 31, 5467-5471. model, His-101 is locked in one tautomeric form by interac- 26. Sudfeldt, C., Schiffer, A., Kigi, J. H. R., Bogumil, R., Schulz, H.-P., tion with Asp-104, and it acts as a hydrogen-bond acceptor to Wulif, S. & Witzel, H. (1990) Eur. J. Biochem. 193, 863-871. 27. Bogumil, R., Hfttermann, J., Kappl, R., Stabler, R., Sudfeld, C. & stabilize the substrate and the transition state. Asp-339, Witzel, H. (1991) Eur. J. Biochem. 196, 305-312. acting as a base, attracts the proton from C2-OH of the 28. Podkovyrov, S. M., Burdette, D. & Zeikus, J. G. (1993) FEBS Lett. 317, substrate. This attraction facilitates the subsequent hydride 259-262. Downloaded by guest on October 2, 2021