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.