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W professor Date /'?///’/7//§?Z MSU is an Affirmative Action/Equal Opportunity Institution 0-12771 LIERARY Michigan State Unlversity “n“ —‘q F.“ PLACE IN RETURN BOX to remove this checkout from your record. TO AVOID FINES return on or before date due. DATE DUE DATE DUE DATE DUE tom 2 3 1995 ’h 3% fir u MSU Is An Aflirmdive Action/Equal'Opportunity Institution chS-DJ THE MECHANISM OF ACTION AND STRUCTURE/FUNCTION RELATIONSHIP OF D—XYLOSE ISOMERASE FROM THERMOANAEROBACTERIUM THERMOS ULFURIGENES By Meng-Hsiao Meng A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Microbiology and Public Health 1992 ABSTRACT THE MECHANISM OF ACTION AND STRUCTURE/FUNCTION RELATIONSHIP OF D-XYLOSE ISOMERASE FROM THERMOANAEROBACTERIUM THERMOSULFUROGENES By Meng-Hsiao Meng The mechanism of catalytic reaction and the structure/function relationship of Thermoanaerobacterium D-xylose isomerase (EC. 3.5.1.5) was studied by site- directed mutagenesis based on the available information of X-ray crystallographic structure of Arthrobacter and Streptomyces enzymes and on the comparison of amino acid sequence of D-xylose isomerases from different sources. Kinetic data indicated that isomerization and ring-opening occur at a concerted step and isomerization happens via hydride shift mechanism. His—101 acts as a hydrogen- bond acceptor to stabilize the transition state of the rate-limiting step. Asp-104 assists this function of His-101 by locking the imidazole ring in a tautomeric form convenient for acceptance of a hydrogen bond. Steric hindrance by Trp-139 against the accommodation of glucose is the major mechanism for Thermoanaerobacterium D-xylose isomerase to discriminate between xylose and glucose. Sequential decrease in K, for glucose was observed when Trp-l39 was substituted by Tyr, Phe, Met, Leu, Val and Ala, in the order shown. Hydrophobic interactions between the hydrocarbon backbone of sugar and Trp-188 and Phe-l45 provide strong binding energy for substrate binding. If the architecture of the active site pocket was kept intact, reduction of the area of water- accessible hydrophobic surface enhanced the thermostability of enzyme. Rate-determining step during the process of irreversible thennoinactivation of Thermoanaerobacterium D—xylose isomerase is the formation of incorrectly folded protein which is a monomolecular event. Besides the well known divalent cations such as Mg+2 and Co”, we found that monovalent cations, particularly K+ also enhance the thermostability of the enzyme. To My parents, wife and daughters with love ACKNOWLEDGMENT I would like to express my sincere appreciation to Professor Michael Bagdasarian, who is my scientific advisor and philosophical mentor, for his guidance, encouragement and patience through my graduate school years. I would also like to thank all of the members of my guidance committee, Drs. J. Gregory Zeikus, Larry Snyder, Wendy Champness and Rawle Hollingsworth, for their advice and invaluable time. I would like to acknowledge the help from Dr. Paul Johnson for the advice in mathematic treatment of mutarotation data for correction of the kinetic constants of anomeric glucose, and the assistant from Dr. K. Padmanabhan for computer- aided molecular modeling in Silicon Graphic Computer System. I also should thank Drs. Robert Hausinger and Thomas Deits for helpful discussion and Dr. John Wilson for providing facilities and advice for the DSC measurement. Finally I am most grateful to my wife Pai-Ying for her dedication and encouragement. I could not have undertaken this endeavor without the understanding of my wife Pai-Ying. iv TABLE OF CONTENTS LIST OF TABLES LIST OF FIGURES ABBREVIATIONS CHAPTER I. General Introduction - References ---- CHAPTER H. Mechanism of Reaction Catalyzed by D-Xylose Isomerase Abstract ---- Introduction Materials and Methods Results Discussion References CHAPTER HI. Mechanism for Discrimination between Xylose and Glucose and the Role of Active Site Aromatic Amino Acids ------------------ Abstract -—-- Introduction Materials and Methods Results page vii viii 22 25 26 27 31 39 63 69 71 72 74 78 81 CHAPTER IV. CHAPTER V. Discussion References ---- Effect of Salt on Thermostability: the Dominant Factor Governing the Irreversible Thermoinactivation of Thermoanaerobacterium Xylose Isomerase ---- Abstract ---- Introduction Materials and Methods Results Discussion References -_-_ Conclusion References vi 96 102 104 105 106 107 110 130 132 134 138 LIST OF TABLES Chapter H 1. Sequences of oligonucleotides used for site-directed mutagenesis 2. Kinetic constants of wild-type and His-101 substitution mutant D-xylose isomerase 3. Deuterium isotope effect on catalytic constant, kcat, of wild-type and His- 101 —) Gln mutant enzyme 4. Comparison of kinetic constants of variant D-xylose isomerases between Ot-D-glucose and B-D-glucose 5. Kinetic constants of variant D-xylose isomerase towards xylose Chapter [H 1. Sequences of oligonucleotides used for site-directed mutagenesis 2. Comparison of kinetic constants of variant D-xylose isomerases for glucose and xylose 3. Kinetic constants of wild-type and active site aromatic amino acids substituted mutant D-xylose isomerases (I) 4. Kinetic constants of wild-type and active site aromatic amino acids substituted mutant D-xylose isomerases (H) Chapter IV 1. Effect of CoCl2 on the half-life of D-xylose isomerase at 85°C 2. Effect of various salts on thermostability of wild-type D-xylose isomerase vii LIST OF FIGURES Chapter I 1. Schematic illustration of the reactions catalyzed by D-xylose isomerase 2. Stereo structure of the active site of Arthrobacter D-xylose isomerase 3. Comparison of amino acid sequence of D-xylose isomerase from different microorganisms 4. Schematic illustration of cis-enediol mechanism 5. Schematic diagrams of the active site structure containing cyclic form and that containing linear form of the substrate Chapter H 1. Schematic illustration of two possible mechanisms in which ring opening and isomerization could occur as a concerted step 2. Schematic illustration of the possible interaction between His-101, amino acid residue at position 104 and Cl-OH of the transition state 3. Plot of pH dependence of kcat 4. Structure of ester derivative of D-mannitol 5. Mass spectrum of ester derivative of D-mannitol 6. The proposed mechanism for the isomerization reaction catalyzed by D- xylose isomerase Chapter III 1. Stereo analysis of the interaction between hydrophobic amino acids and a—thio-D-glucose in the active site pocket of Arthrobacter D-xylose viii isomerase 2. The correlation between Km(glucose) and the water-accessible surface area of the side chain of amino acid at position 139 of the Thermoanaerobacterium xylose isomerase 3. Time course of irreversible thermoinactivation of factitious variants of Thermoanaerobacterium xylose isomerase 4. Schematic illustration of the reduction of hydrophobic surface area as Trp-139 changed to Phe or Ala Chapter IV 1. Effect of initial protein concentration on the inactivation process of D- xylose isomerase at 85°C 2. Arrhenius plot of thermoinactivation of D-xylose isomerase 3. Effect of KCl on the half-life of D—xylose isomerase at 88°C 4. Differential scanning rnicrocalorimetric plots of D-xylose isomerase 5. SDS-PAGE of wild-type and Phe-60 —) His mutant xylose isomerase 6. Time course of irreversible thermoinactivation of wild-type and Phe—60 -—>His mutant enzymes ix DEAE DSC EDTA EPR GC HFCS MES MOPS PAGE SDS ENDOR ABBREVIATIONS diethylaminoethyl differential scanning calorimetry ethylenediamine tetraacetic acid electron paramagnetic resonance gas chromatography high fructose corn syrup 2-(N-morpholino)propanesulfonic acid 3-[N-morpholino]propanesulfonic acid nuclear magnetic resonance polyacrylarnide gel electrophoresis sodium dodecyl sulfate electron nucleic double resonance CHAPTER 1 GENERAL INTRODUCTION D-xylose isomerase (EC. 5.3.1.5), often referred to as D-glucose isomerase, catalyzes the reversible isomerization of D—xylose to D-xylulose and D-glucose to D-fructose (Figure 1). The ability of catalyzing the latter reaction makes D-xylose isomerase an important industrial enzyme for the production of high fructose corn syrup (HFCS). In the United States alone, immobilized xylose isomerase is used to produce over 4 million tons of HFCS annually (Layman, 1986). The second major commercial interest in the enzyme is in the production of ethanol from xylose which is the predominant sugar in hemicellulose present in waste material of plant origin. D-xylose isomerases have been isolated from many bacterial species, and the properties of these enzymes such as substrate specificity, divalent metal cation activation, optimum pH etc. have been intensely investigated, especially in the enzymes from Streptomyces, Lactobacillus, and Bacillus (Antrim et al., 1979). D- xylose isomerases require the presence of a divalent cation such as Mn”, Co+2 or Mg+2 for catalytic activity and thermostability. However, a specific cation that activates xylose isomerase from one organism may have no effect on xylose isomerase from a different organism. Moreover, a specific cation will often enhance the activity of xylose isomerase more toward one substrate than another. In all D—xylose isomerases studied to date D-xylose is a more favorable substrate than D-glucose, mainly due to the much lower KM of D-xylose. The enzyme also Figure 1: Schematic illustration of the reactions catalyzed by D-xylose isomerase. H ' o H OH H H OH xylopyranose CH2OH L o H OH H H OH glucopyranose OH I 11 H H O H- C-OH H HO H OH OH H xylulofuranose H I cum 0 H- C-OH H HO H OH OH H fructofu ranose 5 shows anomeric specificity in that it prefers to catalyze the isomerization of the Ot- anomer of the substrate. The enzymatic isomerization of D-glucose to D-fructose monitored by 13C NMR spectroscopy showed that the Ot-D-glucopyranose and the Ot-D-fructofuranose are the reactive species for the enzyme (Makkee et al., 1984). During isomerization the proton at C2 position of the aldose is transferred to the l—pro-R position of ketose without exchange with solvent (Bock et al., 1983). Crystal structures of D—xylose isomerase, bound to divalent metal cations, substrate or inhibitors, from Arthrobacter species (Henrick et al., 1989; Collyer et al., 1990), Streptomyces rubiginosus (Carrell et al., 1989; Whitlow et al., 1991), Streptomyces olivochromogenes (Farber et al., 1989) and Actinoplanes missouriensis (Jenkins et al., 1992) have been solved. The enzyme is a homotetramer, each subunit consisting of a parallel Ot/B barrel domain and an extended C-terminal tail interacting with neighboring subunit. The active site pocket is located near the C-terminal ends of B strands of the barrel domain and includes residues from a second subunit. Two adjacent divalent metal ions are coordinated by the amino acid residues of the active site. The active site can be described as an amphipathic pocket with hydrophobic residues lining one side and hydrophilic residues the other (Figure 2). Genes encoding D-xylose isomerase have been isolated from at least twelve different microorganisms. The alignment of amino acid sequences from five of them is shown in Figure 3. The one from Thermoanaerobacterium Figure 2: Stereo structure of active site Arthrobacter D-xylose isomerase . Substrate analog, a—S-thio-glucose (white), positioned in the middle of active site pocket, is in contact with His-53 (pink) through the axial Cl-OH. Active site surface can be described as a amphipathic pocket with hydrophilic amino acids lining one side (yellow) and hydrophobic amino acids the other (green). T wo metal cations (red cross) are coordinating to hydrophilic amino acid residues and substrate analog. Figure 3: Comparison of amino acid sequences of D—xylose isomerases from different microorganisms. Boldfaced letters indicate residues changed in this work. S.r., Streptomyces rubiginosus; S.v., Streptomyces violaceoniger; A.m., Ampullariella strain 3876; Art., Arthrobacter strain B3726; T.t., T hermoanaerobacterium thermosulfurigenes; E.c., E. coli. {11:33me Oflfig<fi attrzvmm ” .. 0""?4'1 3??? P Flt-3 Oflfl5<fi 53??? nflflBGlu 5'-CCGTATTTCTGCTTCQ A QGATAGAGATATTGCC-3' His-101—)Asp 5'-CCGTAT'ITCTGCTTCQAIGATAGAGATATTGCC-3' Asp-104—>Asn 5'-GATAGAA_ATATTGCCCCTGAA-3' Asp-104—>Ala 5'-GATAGAG_(;TATTGCCCCTGAA-3' Asp-339—)Asn 5'-CTCAACTTC_AATGCGAAAGT—3' Asp-296—Msn 5'-GGATCGATTAACGCAAATAC-3' Asp-309%Asn 5'-TGGGATACA_A_ATCAGTTCCC-3' Glu-232—9Asp 5'-CAGTTCT'I‘GATTGAICCGAAGCCAAAGGAG-3' New triplets are shown in bold face. Underlined nucleotides indicate the introduced mismatches. 33 termination method (Sanger et al., 1977). The 1.4-kilobase EcoRI/BamHI fragments containing the mutant genes were excised from the M13mp19 replicative form DNA, inserted into the vector pMMB67EH (Fiirste et al., 1986), and introduced into E. coli strain HBlOl. Protein Purification. Wild type and mutant xylose isomerases, expressed by E. coli HBlOl, were purified through a heat step of 75°C, DEAE-Sepharose and Sephacryl-300 chromatography as described previously (Lee et al., 1990), which gave enzymes homogeneous on SDS/PAGE. Protein concentration was determined by the method of Lowry et al. (1951) with bovine serum albumin as standard. Steady-State Kinetics. One ml of reaction mixture contained 20 mM MOPS (pH 7.0), 1.0 mM CoClz, substrate at concentrations of 0.3-2.5 K"I and enzyme 10-1500 ug. Reactions were run at 65°C for 30 min and product formed was determined by cysteine/carbazole/sulfuric acid method (Dishe & Borenfreund, 1951). Enzyme concentrations were adjusted such that less than 5% of the original substrate was converted in 30 min which allowed the determination of initial reaction velocities. Kinetic constants for a- and B- anomer of glucose were determined with 0.3 - 1.5 mg of enzyme in 1.0 ml. Reactions were started by the addition of freshly dissolved substrate and run at 35°C for 1.5 min. Kinetic constants were determined from both Lineweaver-Burk and Eadie-Hofstee plots (Fersht, 1985). Each figure 34 of the kinetic constants shown in this study is represented as an average value from at least two independent experiments. We defined km, as turnover number per active site of enzyme at saturating substrate concentration, and determined it from the equation kca,[E]o=Vmax, where [E]°=total active site concentration. For monitoring the dependence of activity on pH, reactions were run in 1 ml MES buffers (45 mM) containing 1.0 mM Co”. In the case of Asp-104 -) Asn mutant, preincubation at 65°C was found necessary to attain maximal activity. For preincubation, this enzyme in MOPS buffer (pH 7.0) was kept at 65°C for 1 hour; then 200 pg of it was added to the incubation mixture containing MES buffer at the desired pH to start the reaction. Correction for spontaneous mutarotation. In the determination of Vm and K M for B-glucose, the interference from a—glucose, formed by spontaneous mutarotation was considered since K magma App << K “(Hm App. If both of the anomers are initially present, fructose will arise from two different reactions: Va B-glucose ——-—> fructose mutarotation H Va a—glucose ———>fructose 35 The initial velocity of fructose formation from B-glucose (VB) may be calculated by subtracting the initial velocity of fructose formation from (It-glucose (that exists as impurity or is formed from B-glucose by mutarotation), (Va), from the apparent velocity of fructose formation (VB + Va) determined experimentally. To account for the spontaneous mutarotation we have followed the change in optical rotation of B-glucose under the same conditions as were used to determine enzymatic glucose isomerization (20 mM Mops, pH 7.0, 1.0 mM CoClz; 35°C; 1.5 min) with the Autopol II automatic polarimeter (Rudolph Research, Flanders, NJ). Since mutarotation is reversible and its rate constant is independent of the concentration of sugar over a wide range of concentrations at initial reaction conditions we have: d[a-g1uc] _ -d[B-gluc] dt dt -k [B-gl uc] (1) The reaction constant k calculated from the measurement of mutarotation and expressed in decimal logarithms and min‘1 was 0.00727 $00009. Since the spontaneous mutarotation rate is faster than the enzyme-catalyzed rate of glucose isomerization rate, we assumed that: d[a—gluc] d[a-—gluc] 1([8 - - -gl no] (It (B'nz) dt (buffer) (2) 36 The content of B-glucose after 1.5 min of incubation under conditions used for enzymatic reaction was 96.6% of total. The content at 0 time, obtained by extrapolation of the mutarotation curve was 99.1%. As an approximation, therefore, we can consider that during the first 1.5 min [B-gluc] = constant and d[a-gluc] -const dt: (3) It is reasonable, therefore, to use the average content of Ot-glucose = 2.2%, present in the solution of B-glucose during the initial 1.5 min of incubation with the enzyme, to calculate the apparent initial velocity of fructose formation from 0:- glucose, Va, from the equation: V - VIII-Xfl-gluc) “-91 UC] ‘ [a-gluc] +KMI-g1uc) (4) in which we assume Vmu(a,8.uc) = memwgluc) and KM(a,8,uc) = KMAMMM. The velocity of fructose formation from B-glucose at a given concentration of B- glucose, V.,, could thus be calculated and used to calculate memc, and K WWW. To determine the catalytic constants, Vm and KM, for (It-glucose, the correction for 37 the interference from B-glucose must also be introduced. Using the method described above, we have determined the average content of B-glucose, present in the solution of tat-glucose during the initial 1.5 min of reaction, to be 5.6%. The corrected mem and meuc) were used in the Michaelis-Menten equation to calculate the formation rate of fructose from B-glucose present aS an impurity in the various concentrations of (It-glucose. By subtracting VB from total fructose formation rate, Va at various concentrations of (It-glucose could be obtained and thus be used to calculate the corrected Vm(a,8.uc) and KMmgm). It was found that the corrections of catalytic constants for or-glucose are very small. Conservation of deuterium between the 2-position of glucose and the 1-position of fructose. D—[2-2H]-glucose was incubated overnight with wild-type enzyme at 60°C under the following condition: 20 mM MOPS buffer (pH 7.0), 1 mM Co“, 750 mM substrate and 0.45mg/ml enzyme. The enzyme was removed by passing the reaction mixture through a Centricon 10 membrane (10,000 mw cutoff, Amicon). In a glass vial a small amount of sodium borohydride, freshly dissolved in water, was added dropwise to 0.1 ml of the filtrate. After l-hour at room temperature, the mixture was dried in a stream of nitrogen. A few ml of methanol/acetic acid (50:1) was then added to the residue and the mixture was vortexed and dried again. The addition of methanol/acetic acid and the drying was repeated several times. To the final dry residue 75 ul of acetyl anhydride and 75 38 III of pyridine were added. Reaction was run at 85°C for 1.5 hours with vortexing every 15 min. The reaction mixture was dried and 100 [.11 of water was added to the residue. The product was then extracted with 200 til-chloroform . The dry residue, after removal of chloroform, was redissolved in small amount of chloroform and injected into GC and GC-Mass spectrometer (Jeol JMS-AX505H). The column for GC separation was DB225. Temperature range was 200°C - 230°C and the temperature-rising speed 2°C/min. RESULTS Effect of mutation at His-101. His-101 was substituted with glutamine, asparagine, glutamate and aspartate. The kinetic constants of variant D-xylose isomerase are shown in Table 2. Such substitutions caused the decrease in km, but no significant change in KM. At neutral pH the mutant enzymes exhibited 5-15% of activity of a wild type enzyme (expressed as kcat). Since glutamine and asparagine can not function as a base catalyst to open the ring of substrate, the observed properties of the mutant enzyme raise several questions: (i) What is the base that opens the ring in His-101 —> Gln and His-101 —> Asn mutant enzymes ? (ii) Is it possible that the activity observed in these mutant enzymes results from the reaction with the free aldehyde glucose present in the solution? At equilibrium solution, the free aldehyde only represents 0.002% of total sugar. A big increase in Km”, should be expected if this were the case. The insignificant change in KM observed suggests that these two mutant enzymes use the same form of substrate as does the wild type enzyme. In other words, this data do not support the idea that His-101 acts as a base. The change in energy of interaction, AAG‘, between the enzyme and transition state due to the mutation can be calculated from the equation given by the transition state theory (Fersht 1985): 39 40 Table 2: Kinetic constants of wild-type and His-101 substitution mutant D-xylose isomerases pH7.0 pH5.5 Enzyme kcat(s") KM(mM) kcat(s") KM(mM) WT 11.4 120 5.7 150 H101—)Q 0.6 140 0.6 180 H101-9N 1.9 170 1.9 250 H101—)E 0.6 250 0.5 280 H101-)D 0.9 200 0.8 370 Reaction was performed at 65°C. 4 l AAG'E-RTTn(kw/KM)mMi/(kca/Ku)wud type This calculation suggests that a hydrogen bond between enzyme and transition state was lost in the mutant enzymes. When activity was measured at pH 5.5 the km, of the wild type enzyme dropped by approximately 50%, whereas no significant change was observed in mutant enzymes. At lower pH the imidazole group of His- 101 becomes more protonated. This data suggest that the deprotonated form of imidazole group is required to provide a hydrogen bond to transition state. Therefore, His-101 might act as a hydrogen-bond acceptor to stabilize transition State. Side chains of Gln, Glu, Asp, and Asn may act as a hydrogen-bond acceptor. A difference in position of these side chains in the active center, as compared with the position of the His-101 imidazole, may be the reason for the lower kw, observed in the mutant enzymes. Deuterium isotope effect on catalytic constants. In order to identify the rate- limiting step during the isomerization reaction, deuterium isotope effects on the catalytic constants of both wild type and His-101 —> Gln mutant enzyme were tested. The results are shown in Table 3. D—[2-2H] glucose slowed the reaction rate of both wild type and mutant enzymes by a factor of approximately four. The results of this experiment suggest that the breakage of C2-H bond is involved in the rate-limiting step. They also indicate that His-101 —> Gln mutant uses the same 42 Table 3: Deuterium isotope effect on catalytic constant, kcat, of wild-type and His- 101 —) Gln mutant enzyme kcat(s“) kcat(glucose) Enzyme D-glucose D-[2-2H]-glucose kcat([2-2H]-glucose) WT 13 3.5 3.7 H101—)Q 0.5 0.13 3.8 Reaction was performed at 65°C. 43 mechanism of catalysis as the wild type enzyme. Effect of mutations on the anomer-specificity. The suggestion, based on crystallographic data, that His-54 (His- 101 in the Thermoanaerobacterium enzyme), may be the base responsible for the Opening of the ring (Collyer et al., 1990; Whitlow et al., 1991), was tested by site-directed substitution of this residue and determination of the kinetic constants with both anomeric forms of glucose for the mutant enzymes. The results are shown on Table 4. In the wild type enzyme both the catalytic constant, km, and the substrate affinity (reflected by KM) for [3- glucose are approximately fivefold lower than those for (Jr-glucose. When His-101 was substituted by Asn (which can not act as a base), kcaumw dropped to 12% of its wild type value whereas the kmmm did not change significantly. K M remained essentially unchanged for either of the anomers. Thus, the substitution His-104 —> Asn has substantially decreased the preference of the mutant enzyme for the Ot- anomer. The ratio of catalytic efficiency between or and B anomer was reduced more than ten-fold, from 27 to 2.5. This indicated that His-101 does not act as a base; however, it still confers the anomeric specificity on xylose isomerase by increasing the kagM. As indicated by the determination of the primary isotope effect observed with D-[2-2H]glucose as substrate, the constant contributing most to the km, would be the rate constant of the transfer of hydrogen atom between Cl and C2 of substrate. The hydride shift was assumed to take place on the open- Table 4: Comparison of kinetic constants of variant D-xylose isomerase between a-D-glucose and B-D-glucose or-D-glucose B-D—glucose kcat/KMW) Enzyme kcar(s“) KM(mM) kcar(s") KM(mM) [teat/KM(B) WT 1.30:|:0.03 24:1:1 0.25i0.03 136:12 27 D104—>N 0.65:1:0.01 33:1 0.14i0.02 2041-18 29 H101—9N 0.15i0.01 30:1:2 0.26:0.03 130:16 2.5 D104—)A 0.08i0.01 45:1:2 0.27:1:0.01 275120 1 .8 Reactions were started with freshly preparated substrate and run at 35°C for 1.5 nun The figures have been corrected as described in Materials and Methods. 45 chain form of the substrate (Collyer et al., 1990; Whitlow et al., 1991; Jenkins et al., 1992). If this were the case, kmmmc) should have been of the same order of magnitude as the kcamgm, since stereo-anomers cease to exist when substrate is in the open-chained form. Moreover, these two catalytic constants should have been affected to the same extent by the substitution of Asn for His-101. Since there is no other amino acid residue with basic side chain close to equatorial Cl-OH (B- conformation) or axial Cl-OH (or-conformation) position in the active pocket of xylose isomerase (Collyer et al., 1990; Whitlow et al., 1991), the pyranose ring could either be opened by a water molecule present in the active site of the His- 101 —> Asn mutant, or, more likely, the mechanism of ring-opening is different from the one that has been proposed. The last conclusion is based on the following arguments: (l) the observation of primary isotope effect in both wild-type and His- 101 -—> Gln mutant enzymes with D—[2-2H]-glucose suggested that the transfer of C2- hydrogen is the rate-limiting step (Lee et al. 1990; Smart et al., 1992). (2) mutant xylose isomerases in which His-101 has been substituted by residues unable to function as a base still exhibit catalytic activity equal to 10 - 14% of the wild type enzyme. (3) significant difference between kcwmm) and km,(‘,_g.uc) suggests that the structure of the substrate immediately prior to the rate-limiting step is a cyclic structure rather than an extended open-chained structure. We would like to suggest, therefore, that isomerization and ring opening occur as a concerted step. Two concerted mechanisms may be proposed as shown in Figure 1. In one of 46 them, a base attracts the proton from the C2 carbon of the pyranose. This results in the formation of a cis-enediol intermediate and ring opening during the transfer of hydrogen. Two arguments may be raised against this mechanism: (1) no residue capable of acting as a general base has been observed near the C2 hydrogen in the available crystal structures; (2) no exchange of proton with the medium occurs during the isomerization reaction (Bock et al., 1983). In the second mechanism, a base attracts the proton from the C2—OH. This is followed by a hydride shift and ring opening. From the crystal structure of Streptomyces enzyme with 1.6 A resolution (Whitlow et al., 1991), it follows that Asp-287 (corresponding to Asp-339 of Thermoanaerobacterium enzyme) is close to C2-OH. It is possible that this residue acts as a base in this catalytic reaction. If the position of His-101 in the Thermoanaerobacterium enzyme is indeed equivalent to the position of His-53 of the Arthrobacter enzyme, its role would be hydrogen-bonding to the axial Cl-OH, of the substrate in the (Jr-pyranose form, and stabilization of the transition state. Asp-104 could assist this function by stabilization of the His residue. Substitution Asp-104 —> Ala resulted in a drop of k C ammo, to about 6% of the wild-type value and a two-fold increase of K M whereas the kmwuc, remained unchanged (Table 4). This suggested that anomeric specificity depends not only on the presence of His-101, but also on the correct position of this residue. Without the hydrogen-bonding provided by Asp-104 the imidazole group of His-101 could rotate and take up positions that are unfavorable 47 Figure 1: schematic illustration of two possible mechanisms in which ring opening and isomerization could occur as a concerted step. 48 \ \ \ 3‘ +8. B: H O 0' Hv— / HO HO HO’ R N CHon HO OH OH HO OH OH HO OH cis-ene—diol-intermediate ['1 0‘3 0- o Ho 0 OH OH OH HO k\ HO (:0 r’ H ° B-H B: /B' / / [”1 49 for the formation of hydrogen bond to the transition state. When, on the other hand, Asp-104 was substituted by Asn, km, dropped to approximately 50% of the wild type value for both anomers (Table 4). A hydrogen bond can be formed between Asn-104 and His-101, but only the oxygen of the amide group is a hydrogen-bond acceptor. In contrast to this, both oxygens of the carboxyl group in Asp-104 can be hydrogen-bond acceptors. Another function of Asp-104, therefore, seems to be the maintenance of His-104 imidazole group in a particular tautomeric form, most favorable for its function as a hydrogen-bond acceptor in the stabilization of the transition state (Figure 2). Substitution of the metal-coordinating amino acids. In order to test the function of each metal cation in the active site pocket, Asp-309, Asp-296, Asp-339 and Glu- 232 were substituted. Asp-309 is considered to bind the metal at position [11] whereas Asp-296, Asp-339 and Glu-232 bind the metal at position [I] (Figure 5 in Chapter I). Asp-257 in Streptomyces enzyme (corresponding to Asp-309 in Thermoanaerobacterium enzyme) also was proposed to be a base catalyst to initiate hydride shift occurring in linear form substrate molecule (Whitlow et al., 1991; van Tilbeurgh et al., 1992). Substitution of each of these aspartate residues with Asn resulted in mutant enzymes that still required Co2+ for maximal activity and for thermostability (measured as residual activity after 20 min at 75° C). This suggested that the metal binding site still exists in these mutants and that individual 50 Figure 2: Schematic illustration of the possible interaction between His-101, amino acid residue at position 104 and Cl-OH of transition state. Dashed lines indicate possible hydrogen bonds. The configuration of transition state is believed to be a cyclic form, and His-101 specifically interacts with its axial C1- OH. 51 His-101 C N N ------ HO—Transition State I \ <\ N N ------ Ho—Transition State Asn-104 .X' L -H HO-Transition State His-101 N\\ N—H HO—Transition State Ala-104 \\CH3 52 mutations have brought about only local alterations such as perhaps changes in geometry of coordination due to the loss of a negative charge, the inability of -NH2 in Asn to coordinate metal and a consequent change in the metal position. However, the exact structural changes in these mutants will have to be revealed by X-ray diffraction. Kinetic constants of the mutant enzymes are shown in Table 5. Asn-309 mutant enzyme exhibited approximately 20% of the wild type catalytic efficiency (km/KM). This result argues against the hypothesis that Asp-309 might act as a base to initiate the hydride shift on the open-chain form of the substrate. It is consistent, however, with the supposition that Asp-309 or the metal[II] stabilizes the transition state. The substitution of Asn for either Asp-296 or Asp- 339 caused drastic decrease in catalytic efficiency resulting from both the increase in KM and decrease in km. It seems, thus, that these two residues, or the metal[I], play an important role not only in the stabilization of the substrate but also in the stabilization of the transition state. The reduction of kcat/KM by four order of magnitude in Asp-339 -—) Asn mutant enzyme consists with the hypothesis that Asp- 339 may act as a base catalyst during catalysis of the enzyme. Glu-232 —> Asp mutant enzyme lost solubility at temperatures above 45°C, and no activity was detected in this mutant enzyme. The metal binding site [I] probably has been destroyed in this mutant. Dependence of the catalytic constant, k“, on pH. It has been suggested that in 53 Table 5: Kinetic constants of variant D-xylose isomerases towards xylose xylo se heat/KM(mM) Enzyme kcar(s") KM(mM) kcat/Kanmype) WT 23¢] 9.3i2.0 l D309—)N 2.7:t0.1 5.8i1.0 0.2 D296—>N l.0:l:0.1 140320 28x104 D339—>N 0.08:1:0.01 70i10 4x10'4 E232->D‘ no activity Reaction was performed at 65°C for 30 min. a: E232—>D mutant enzyme precipitated when temp. was above 45°C. 54 the xylose isomerase from Thermoanaerobacterium the protonation of His-101 is responsible for the decrease of Van“ with the decrease of pH below 7.0 (Lee et al., 1990). It was considered possible, therefore, that the pK, of enzyme-glucose complex, which can be estimated form the plot of km vs. pH, might be lowered by the removal of a negative charge from the neighborhood of His-101. The dependence of km, on the pH for the wild-type enzyme and the two mutants, His- 101 —-> Asn and Asp-104 —) Asn is shown in Figure 3. Because the free enzyme is unstable at pH lower than 5.4, the activities were assayed between 5.5 and 7.0. From the equation (kw)H=kca,-(kca,)H[H*]/K,, the pK, of the enzyme-glucose complex for wild-type enzyme was calculated to be 5.4. Although the pK, for the mutant enzymes could not be detemtined exactly from the data obtained in this experiment, it must be far below 5.5 for each of the mutant enzymes. The apparent KM did not change significantly for either the wild type or the mutant enzymes between pH 7.0 and pH 5.5. Thus, Asp-104 seems to contribute significantly to the overall negative-charge environment around His-101. If this charge is removed the apparent pK, of the enzyme-glucose complex is lowered considerably. Conservation of deuterium during the isomerization reaction. Isomerization of glucose performed in D20 indicated that practically no exchange of deuterium occurred between the solvent and the product (Bock etal., 1983). Crystal structure of the Arthrobacter enzyme with 5-thio-or-D-glucose (Collyer et a1, 1990) and of 55 Figure 3: Plot of pH dependence of kcat. ) Kcat(sec 56 10 3' \NT a 6-1 LAsn104 I—T-u—‘4-4—-L - t 4-1 2‘ Asn101 —H+'—I-——.~ I o r 1 ' I Ifi T I r ' I I 5A. 5L6 53 61) (12 6A. 63 71) pH 66 72 57 the Streptomyces enzyme with cyclic xylose (Whitlow et al., 1991) indicated that the hydrogen at C2 is positioned near a T rp side chain and is far from any residue with a potential base character. It seems to be very unlikely, therefore, that the isomerization proceeds by the mechanism [1] (Figure 2) involving an cis-enediol intermediate. To provide further insights into the reaction mechanism we have evaluated the possibility of free hydrogen radical generation upon the breakage of the C2-H bond. To do this, we have reduced the reaction mixture with sodium borohydride to convert D-glucose to D—sorbitol and D-fructose to D-mannitol and D-sorbitol. These were acetylated and subjected to GC-Mass analysis. If the isomerization takes place by a hydride shift, then deuterium from the C2 atom of glucose will be quantitatively transferred to C1 atom of fructose. Consequently, deuterium will be present at C1 of all D-mannitol molecules. If the transfer of hydrogen would take place via an cis-enediol intermediate or by generation of free hydrogen radical, the conversion of deuterium would be lower than 100%. The structure of the ester derivatives of D-mannitol, derived from the isomerization of 2-[D]-D-glucose, is shown in Figure 4 in which the number along the dash arrow line indicates the molecular weight of the fragment generated after bombardment. If deuterium is present at C1 of all such derivatives, the ratio of fragment with molecular weight 362 to that with 361 should be close to unity, so will be the ratio of 290/289 and so on. Tire Mass spectrum of the ester derivatives of D-mannitol (Figure 5) shows 58 Figure 4: Structure of ester derivative of D-mannitol Number along the dashed arrow indicates molecular weight of the fragment after bombardment. 59 DCHOAC i 74 3611 ACOCH 1146 289 a --------- I I ACOCIIH I 218 217 I HCOAC ‘ 290 145 i """"" I """"" HCOAC I 362 73 s --------- I I CHZOAC Ester-derivative Of D-mannitol Figure 5: Mass spectrum of ester derivatives of D—mannitol. The mass range was selected between 200 and 370 for clarity of presentation. 61 Relative Abundance So. em .3 no.” Mam Mao O . __-... ..__ MOO . _tq NM :1 .r q a — MAO DI D ’pr 1 .mmo Nam _. mm» . tr: .e. XS woo. 5N wwm . was. . wmo 1' I V 62 that the ratio of abundance of 361/362, 289/290 and 217/218 particles is very close to unity. This result strongly supports the conclusion that hydrogen transfer between C1 and C2 atoms of the substrate occurs by the hydride shift. DISCUSSION The models of xylose/glucose isomerization published to date postulated the opening of the Ot-pyranose ring as a necessary step prior the transfer of the C2 hydrogen. These models were supported by the finding of extended-chain species of the substrate in the crystal structures of the enzyme/xylose-xylulose complexes. These extended-chain substrates were interpreted as reaction intermediates (Carrell et al., 1989; Collyer et al., 1990). However, the extended-chain species of sugar, observed in the crystal structure, do not necessarily have to be reaction intermediates preceding the hydride shift. Since D-xylulose in aqueous solution contains 20.2% free ketose (Wu and Serianni, 1990), the extended species could be the free ketose of xylulose. In these models, the active site histidine (His-54 of S treptomyces enzyme or His-101 of T hermoanaerobacterium enzyme) was assigned as a base to either open the ring or attract the proton from C2 of the linear subsu'ate. Effects of substitution of His-101 by Asn and Gln in Thermoanaerobacterium enzyme do not support the idea that His-101 acts as a base. Recently, Lambier et al., (1992) challenged the hypothesis that assigns to His-54 of Actinoplanes missouriensis isomerase (His-53 of Arthrobacter, His-101 of the Thermoanaerobacterium enzyme) the role of ring opening. They found that variant enzymes, obtained by substitution of His-54 by different residues capable 63 64 of hydrogen-bonding to the substrate, retained approximately 10% of wild type activity and acted by the same mechanism as the wild-type enzyme similarly to the mutants of the Thermoanaerobacterium xylose isomerase described in this work and our previous work (Lee et al., 1990). However, these authors still postulated the ring opening as obligatory step preceding the hydride transfer. Kinetic data for the two anomers of glucose for the wild type and mutant enzymes, presented in this work, suggested that the substrate preceding the rate- lirniting step is in the cyclic form. This conclusion is consistent with the results of crystallographic studies performed under steady-state conditions in a flow-cell (Farber et al., 1989). The electron densities observed by these authors indicated that the rate-limiting step was preceded by a cyclic form of the substrate. Our results of the isotope effect on the isomerization kinetics have indicated that hydrogen transfer is the rate-limiting step. We propose, therefore, that hydrogen transfer and ring opening are performed as a concerted step. We also provide further support for hydride shift mechanism of the hydrogen transfer. This support is based on the following arguments: (1) no exchange of the proton between the substrate and the medium takes place during the reaction; (2) deuterium at C2 of glucose is transferred to C1 of fructose with the efficiency close to 100%; (3) there is no basic amino acid residue, capable of attracting the proton, in the vicinity of the C2-H of the substrate. Metal[I] coordinates to C3-OH and C4-OH of the Ot-pyranose (Collyer et al., 65 1990; Whitlow et al., 1991). Although metal[I] does not seem to interact directly with CS-O of (rt-pyranose, the distance between metal[I] and C5-O could become shorter if a distortion of the transition state occurred. It is possible, thus, that metal[I] provides the electrostatic interaction to stabilize the developing negative charge at C5-O in the transition state. Asp-339 of the Thermoanaerobacterium enzyme (Asp-287 of Streptomyces), coordinating to metal[I], also hydrogen-bonds to C2-OH of the Ot-pyranose (Whitlow et al., 1991). It could, therefore, attract the proton from the C2-OH to initiate the hydride shift. The drop of catalytic efficiency, by four orders of magnitude, upon substitution of this residue by Asn (Table 5) is consistent with this hypothesis. Based on all these data, we would like to propose a new model for the reaction catalyzed by xylose isomerase (Figure 6). In this model, His-101, locked at one tautomeric form by Asp-104, acts as a hydrogen-bond acceptor to stabilize the substrate as well as the transition state. Asp-339, acting as a base, attracts the proton from C2-OH. This facilitates the subsequent hydride shift from C1 to C2, and simultaneously induces the opening of the ring. Metal[I] stabilizes the substrate and transition state by coordination. It may also provide the electrostatic force to stabilize the developing negative charge at C5-oxygen. Metal[H] probably helps maintain the active site structure and affects the activity indirectly. The product is formed after the attack of the CS-oxygen on the C2 keto group and closing of the ring. 66 Figure 6: The proposed mechanism for the isomerization reaction catalyzed by D- xylose isomerase. 67 EELAE guru?»— /|Ao was A if m J .. .. ctr/o .o E . 1%... m0. 272$... d l Wso O EELQM gears..— EAEE 7532. 68 The exact three-dimensional structure of xylose isomerase from Thermoanaerobacterium thermosulfurigenes is not available at present. The inferences concerning the positions of amino acid residues, addressed in this dissertation are based on the known crystal structure of enzymes from Arthrobacter (Henrick, et al., 1989; Collyer and Blow, 1990; Collyer et al., 1990) and of Streptomyces (Farber et al., 1989; Whitlow et al., 1991) and on the conservation of several domains in the primary structure of xylose isomerases from many different sources, particularly in the region of the active site. Although the results presented in this study are consistent with the belief that the same mechanism of isomerization is functioning in the catalysis by several different xylose isomerases, it is possible that minor structural differences in the relative positions of different amino acid residues might lead to misinterpretation of the results provided by the kinetic data. It is important, therefore, that the conclusions presented here are confirmed by the determination of three-dimensional structure of appropriate mutant enzymes. REFERENCES Bock, K., Mcldal, M., Meyer, B., & Wiebe, L. (1983) Acta C hemica Scandinavica B 37, 101-108. Bogumil, R., Hiittermann, J., Kappl, R., Stabler, R., Sudfeld, C., & Witzel, H. (1991) Eur. J. Biochem. 196, 305-312. Boyer, H. W. & Roulland-Dussoix, D. (1969) J. Mol. Biol. 41, 459-472. Carrell, H. L., Glusker, J. P., Burger, V., Manfre, F., Tritsch. D., & Biellmann, J .- F. (1989) Proc. Natl. Aced. Sci. USA 86, 4440-4444. Collyer, C. A., Henrick, K. & Blow, D. M. (1990) J. Mol. Biol. 212, 211-235. Dische, Z. & Borenfreund, E. (1951) J. Biol. Chem. 192, 583-587. Farber, G. K., Glasfeld, A., Tiraby, G., Ringe, D. & Petsko, G. A. (1989) Biochemistry 28, 7289-7297. Fersht, A. R. (1985) Enzyme Structure and Mechanisms (Freeman, San Francisco, CA) 2nd Ed. Fiirste, J. P., Pansegrau, W., Frank, R., BlOcker, H., Scholz, P., Bagdasarian, M. & Lanka, E. (1986) Gene 48, 119-131. Jenkins, J., Janin, J., Rey, lF., Chiadmi, M., van Tilbeurgh, H., Lasters, 1., De Macyer, M., Van Belle, D., Wodak, S. J., Lauwereys, M., Stanssens, ,P., Mrabet, N. T., Snauwaert, J., Matthyssens, G. & Lambeir, A.-M. (1992) Biochemistry 31, 5449-5458. Henrick, K., Collyer, C. A. & Blow, D. M. (1989)J. Mol. Biol. 208, 129-157. Larnbair, A. M., Lauwrreyes, M., Stanssens, P., Marabet, N. T., Snauwaert, J ., van Tilbeurgh, H., Matthyssens, G., Lasters, 1., De Macyer, M., Wodak, S. J ., Jenkins, J ., Chiadami, M. and Janin, J. (1992) Biochemistry 31, 5459-5466. Lee, C., Bagdasarian, M., Meng, M. & Zeikus, J. G. (1990) J. Biol. Chem. 265, 19082—19090. 69 70 Lowry, O. H., Rosebrough, N. J., Farr, A. L., & Randall, R. J. (1951) J. Biol. Chem. 193, 265-275. Sanger, F., Nicklen, S. & Coulson, A. R. (1977) Proc. Natl. Aced. Sci USA 74, 5463-5467. Sayers, J. R., Schmidt, W. & Eckstein, F. (1988) Nucleic Acids Res. 16, 791-802. Smart, 0, S., Akins, J., & Blow, D. M. ( 1992) Proteins, Struct. F unct. Genet. 13, 100-1 11. Sudfeldt, C., Schaffer, A., Kiigi, J. H. R., Bogumil, R., Schulz, H.-P., Wulff, S., & Witzel, H. (1990) Eur. J. Biochem. 193, 863-871. van Tilbeurgh, H., Jenkins, J ., Chiadmi, M., Janin, J ., Wodak, S., Mrabet, N. T. & Lambeir, A.-M. (1992) Biochemistry 31, 5467-5471. Whitlow, M., Howard, A. J., Finzel. B. C., Poulos, T. L., Winbome, E., & Gilliland, G. L. (1991) Protein: Structure, F unction, and Genetics 9, 153- 173. Wu, J., & Serianni, A. S. (1990) Carbohydr. Res. 206, 1-12. Yanish-Perron, C., Vieira, J. & Messing, J. (1985) Gene 33, 103-119. CHAPTER III MECHANISM FOR DISCRIMINATION BETWEEN XYLOSE AND GLUCOSE AND THE ROLE OF ACTIVE SITE AROMATIC AMINO ACIDS 71 ABSTRACT The structural basis for substrate specificity of the thermophilic xylose isomerase from Thermoanaerobacterium thermosulfurigenes was examined by using predictions from the known crystal structure of the Arthrobacter enzyme and amino acid residue substitutions by site-directed mutagenesis of the xylA gene of T. thermosulfurigenes. The locations of Met-87, Thr-89 and Val-134, which contact the C6-OH group of D-5-thio-glucose in xylose isomerase from Arthrobacter (Collyer et al., 1990) are equivalent to those of Trp-139, Thr-141 and Val-186 in the Thermoanaerobacterium enzyme. The comparison of kinetic data between wild type and mutant enzymes suggests that the major mechanism for discrimination between D-xylose and D-glucose in T. thermosulfurigenes enzyme is the steric hindrance between the indole group of Trp-139 and the C6-methanolic group of D- glucose. Sequential decrease in KM toward glucose was observed when Trp-139 was substituted by Tyr, Phe, Met, Leu, Val or Ala, in the order shown. Although Thr-141 and Val-186 cause no steric hindrance against glucose binding, introduction of an additional hydrogen bond by replacing Val-186 with Thr also increased affinity toward glucose. Besides Trp-139, the functional roles of four other aromatic amino acids in the active site pocket have been examined. The indole group of Trp-188, which is presumed to be positioned parallel to the hydrophobic backbone of the sugar, and believed to be interacting with it, plays an 72 73 essential role in the binding of the sugar substrate. Trp-49, which presumably hydrogen bonds to Asp-339 coordinating to metal [I], helps in the binding of substrate to the enzyme by maintaining the structure of the active site. Reducing the area of water-accessible hydrophobic surface of the active Site pocket by replacing Trp-139 with smaller hydrophobic amino acids or replacing Trp-49 with Arg enhanced the thermostability of the enzyme. INTRODUCTION Specificity of enzymes towards their substrates is determined in part by molecular residues that provide for binding of the substrate and which maintain substrate steric configuration in the active site. A variety of factors influence enzyme-substrate complementarity and catalytic efficiency including steric fit, charge interactions, hydrogen bonding and hydrophobic interactions (Craik et al., 1985). Until recently, the main strategy to reveal and study the molecular basis of these factors was to determine the three-dimensional structure of the enzyme- substrate complexes by X-ray crystallography. Redesigning proteins by engineering of their genes is now a viable approach that complements structural studies and enables determination of the effect caused by amino acid substitution on the function of mutant enzyme. Thus, substrate specificity has been altered by redesigning the structural frame of an enzyme (Wilks et al., 1988; Bone et al., 1989; Scrutton et al., 1990), its electrostatic network (Wilkinson et al., 1984; Wells et al., 1987; Dean etal., 1990; & Evnin et al., 1990) or its hydrophobic interaction with the substrate (Estell et al., 1989). Catalytic function of an enzyme can also be changed and regulated by modifications of the physical microenvironment of its catalytic site (Hurley et al., 1990; Higaki et al., 1990). In all D-xylose isomerases studied to date D-xylose is a more favorable substrate than D-glucose, mainly due to the lower KM of D-xylose. D-xylose and 74 75 D—glucose have identical configuration, except for the presence of an additional - CHZOH group at the C6 position in the glucose molecule. This extra methanolic group must, therefore, be responsible for the differences in the K M exhibited by xylose isomerase towards glucose versus xylose. According to the alignment of amino acid sequences from different D-xylose isomerases and the active site structures of D—xylose isomerases from Streptomyces and Arthrobacter (Figure 1 see also Collyer et al., 1990; & Whitlow er al., 1991), Trp-139, Val-186 and T hr- 141 of Thermoanaerobacterium enzyme might cause such steric hindrance against glucose binding. Mutation at the codons specifying these amino acids have been performed to test this hypothesis. Besides Trp-139, there are four more aromatic amino acids constituting the hydrophobic surface of the active site pocket. The role of the aromatic amino acid residues for binding the sugar have been demonstrated in maltose-binding protein (Martineau et al., 1990), arabinose-binding protein and galactose-binding protein (Quiocho et al., 1989). In this dissertation, the role of these aromatic amino acids in substrate binding and on thermostability of the enzyme have been examined. 76 Figure 1: Stereo analysis of the interaction between hydrophobic amino acids and a-S-thio-D—glucose in the active site pocket of Arthrobacter D-xylose isomerase. Substrate analog is sandwiched between two indole groups (yellow). C6-OH of analog is close to residue of Val-134 (red), Thr-89 (green) and Met-87 (light blue). In Thermoanaerobacterium enzyme the corresponding amino acids are believed to be Val-186, Thr-141 and Trp-139, respectively. 77 MATERIALS AND METHODS Strains, Plasmids & Site-directed mutagenesis. Were as described in Chapter II. The oligonucleotides used for site-directed mutagenesis are shown in Table 1. Protein Purification. Wild type and most mutant xylose isomerases, expressed by E. coli H8101, were purified through a heat step of 75°C for 15 min, DEAE- Sepharose and Sephacryl-300 chromatography as described previously (Lee et al., 1990). Phe-145 -—> Lys, and Trp-188 —> His mutant enzymes were heated at 60°C for 20 min, and Trp-139 —-) Lys was heated at 65°C for 30 min instead of 75°C. After purification through DEAE-Sepharose and Sephacryl-300 all mutant enzymes were homogeneous on SDS/PAGE. Protein concentration was determined by the method of Lowry et al. (1951) with bovine serum albumin as standard (from Pierce). The apoenzyme was obtained by dialysis of the homogeneous enzyme against 100-time volume of MOPS buffer (10 mM pH 7.0) with EDTA (10 mM) for 36 hours and then against MOPS without EDTA for another 36 hours. Dialysis buffer solutions were changed every 12 hours. Steady-State Kinetics. Kinetic constants were determined as described in Chapter II. 78 79 Table 1: Sequences of oligonucleotides for site-directed mutagenesis Mutation sequence Trp139—>Tyr 5'-ACGAAAGTI'ITGT_A_T_GGTACTGCGAAT-3' Trp-139—9Phe 5'-ACGAAAGT'I"ITGT'_I"_LGGTACTGCGAAT-3' Trp-139—>Met 5'-AAAGTTI'I‘GflGGGTACTGCG-3' Trp139—9Leu 5'-CGAAAGT'I'I'I‘GC_'[GGGTACTGCGA-3' Trp-139—>Val 5'-CGAAAGTTI‘TGG_T_GGGTACTGCGA-3' Trp-139—9Ala 5'-CGAAAGTTTTG$GGGTACTGCGA-3' Trp-139—>Lys 5'-ACGAAAGT'ITTGAA_GGGTACTGCGAA-3' Trp-49—)Phe 5'-ATAGCT'I‘AT'I"I__‘_'_I‘CACACTTTT-3' Trp-49—)Ala 5'-ATAGCTTATQ_C_GCACACT'ITT-3' . Trp-49—Mrg 5'-CTATAGCTTAT&GCACACTTIT-3' Trp-l88—9Asp 5'-AACTACGTATTC_(_}_A_I_GGTGGAAGAGAA-3' Trp-188—9His 5'-AACTACGTATI‘C§A’[GGTGGAAGAGAA-3' Trp-l88—-)Lys 5'-ACTACGTATTC&GGGTGGAAG-3' Trp-188—>Glu 5'-ACTACGTATTCQ_AGGGTGGAAG-3' Phe- 145 ->Lys 5'-TACTGCGAATCTTiAATCCAATCCAAGAT—3' Phe-60—>His 5'-GAACAGATCAAQATGGCAAAGCTAS' New triplets are shown in bold face. introduced mismatches. Underlined nucleotides indicate the 80 Kinetics of Irreversible Thermoinactivation. The time course of irreversible thermoinactivation of D-xylose isomerase was measured by incubating 0.6 ml enzyme solution (0.1mg/ml) in 10 mM MOPS buffer (pH 7.0 at 85°C) containing 50 11M CoCl2 at 85°C for various periods of time and then detennining the residual activity at 65°C. First order rate constants of irreversible thermoinactivation were obtained by linear regression in semilogarithmic coordinates. “:2 ‘ikfifihrfi. I RESULTS The residues causing steric hindrance against glucose binding. In order to find out what is the mechanism for discrimination between the two substrates, Thr-141, Trp-139, and Val-186 were chosen as the target amino acids for substitution. We changed Thr-141 to Ser, Trp-139 to Phe and Tyr, and Val-186 to Thr, Ser, and Ala. The steady-state kinetic constants of mutant enzymes are shown in Table 2. The variant Thr-141 -) Ser exhibited a two-fold lower catalytic efficiency, keg/KM, toward glucose than wild type. It was considered, therefore, that Thr-141 does not cause steric hindrance against glucose binding in Thermoanaerobacterium xylose isomerase. In the Trp-139 —> Phe variant an increase of the catalytic efficiency toward glucose but decrease toward xylose was observed, implying that the Side chain of Trp-139 does cause a steric hindrance against the binding of glucose. The enlarged pocket in Trp-139 —> Phe mutant enzyme presumably accommodates glucose better, but may be too large for xylose. Catalytic efficiencies for both glucose and xylose in Val-186 —> Ala variant are practically the same as in the wild type enzyme. This indicates that the side chain of Val-186 does not cause steric hindrance against the binding of glucose. The increase in catalytic efficiency for glucose in the Val-186 —> Thr mutant variant should be attributed, therefore, to the introduction of a new hydrogen bond between the enzyme and the substrate since both side chains of Thr and Val are of approximately the same size. T rp-139 —> 81 82 Table 2: Comparison of kinetic constants of variant D—xylose isomerases for glucose and xylose Glucose Xylose Enzyme kcar(s“) KM(mM) kcar/KM kcar(s") KM(mM) kcat/K M WT 11:2 110:8 0.10 23:1 93:18 2.5 W139-—>F 16:1 65:8 0.25 24:2 16:2 1 .5 W139—-)Y 90:04 91:12 0.10 10:1 14:1 0.7 V186—>T 15:2 91:7 0.16 23:1 98:18 2.3 V186—>S 13:1 140:7 0.09 18:2 17:1 1.1 Vl86—>A 9.0:1 100:10 0.09 31:3 14:2 2.3 T141—)S 7.8:05 160:20 0.05 46:2 28:2 1.7 W139—> Vl86—>T 16:2 29:4 0.55 24:1 13:2 1.8 W139—> V186—)S 12:1 58:4 0.21 95:04 21:2 0.45 Reaction was performed at 65°C in 20 mM MOPS buffer (pH 7.0) containing 1 mM CoClz. 83 Tyr and Val-186 —> Ser mutant variants have same level of specificity constant for glucose as the wild type, but the specificity constant for xylose is three- and two- folds lower, respectively. Introduction of an inappropriate hydrogen bonding in an enlarged substrate-binding pocket may by responsible for this decrease. Finally, two double mutant enzymes, Trp-139 —-> Phe/Val-186 —) Thr and Trp-139 —) Phe/Val- 186 —) Ser, were constructed to see whether the effects on the increase of catalytic efficiency for glucose are additive. Catalytic efficiency toward glucose increased further in Trp-139 -) Phe/Val-186 —> Thr due to a further decrease of KM. The AAG‘t between Val-186 —) T hr and the wild type enzyme, and between Trp-139 —> Phe/Val-l86 —-) Thr and Trp-139 -) Phe were 1.5 and 2.2 KJmol", respectively. This values are consistent with the presumption that the hydroxyl group of Thr-186 participates in a new hydrogen bonding between the enzyme and C6—OH of glucose. To elucidate further the functional role of Trp-139 we have substituted this residue with a series of hydrophobic amino acids. As shown in Table 3, substitution of Trp-139 with residues having smaller side chains increased the catalytic efficiency for glucose while decreasing the efficiency for xylose. This confirms the conclusion that the bulky side chain of Trp-139 hinders the accommodation of glucose in the catalytic pocket of the Thermoanaerobacterium xylose isomerase. 84 Table 3: Kinetic constants of wild-type and active site aromatic amino acids substituted mutant D-xylose isomerases (I) glucose xylose Enzyme kcat(s'l) KM(mM) kcat/KM kcat(S'l) KM (mM) kcat/K M WT 11:2 110:8 0.10 23:2 93:18 2.5 W139—9Y 90:04 91:12 0.10 10:1 14:1 0.7 W139—>F 16:1 65:7 0.25 24:2 16:2 1 .5 W139—9M 11:1 55:2 0.20 10:1 92:02 1.1 W139—>L 7.7:0.4 50:4 0.15 14:1 11:1 1.3 W139—)V 45:02 35:1 0.13 7.7:0.l 64:01 1.2 W139—>A 82:02 31:2 0.26 84:02 59:04 1.4 W139->K 3.1:0.2 21:1 0.15 35:01 54:07 0.7 W49—>R 10:1.0 1 10:3 0.09 W49—9F 10:0.3 330:14 0.03 W49-)A 7 .2:0.3 710:48 0.01 F60—>H 2.1:0.1 140:4 0.02 Reaction was performed at 65°C. 85 Correlation between KM(mM) and the Water Accessible Surface Area of the Side Chain of the Residue at position 139. As shown in Table 3, a progressive decrease in KM for glucose was observed when Trp-139 was substituted by T yr, Phe, Met, Leu, Val or Ala, whereas the KM(xylose) was changed more or less randomly. Figure 2 shows that there is a correlation between the KM(glmw, and the water-accessible surface of the side chain of the residue in position 139. This suggests that this side chain protrudes into the cavity of the active site pocket and this protrusion is the reason for the steric hindrance against glucose binding. This suggestion is consistent with the predictions based on the crystal structure of the active site of Arthrobacter enzyme (Collyer et al., 1990) in which Met-87 (corresponding to Trp-139 of Thermoanaerobacterium enzyme) is indeed in the proximity of C6-methanolic group of or-thio-D-glucose (Figure l). Lys, whose side chain has the size comparable to that of Leu, falls out of the proportionality rule. Its placement in position 139 resulted in a mutant enzyme with the lowest KM and k C 0, toward both substrates. We believe that the NH; group of Lys side chain may be responsible for this anomaly. There are several carboxyl groups coordinating to metal ions and/or hydrogen bonding to substrate in the immediate vicinity of the residue 139. The positive charge of Lys may interact with them, causing local structural changes, and, consequently confer upon the Trp-139 —> Lys mutant enzyme properties different from those expected from a simple change in the hydrophobic side chain size. It is also possible that amino group of Lys forms 86 Figure 2: The correlation between KM(glucose) and the water-accessible surface area of the side chain of amino acid at position 139 of the Thermoanaerobacterium xylose isomerase The value of the accessible surface area of the side chain for each amino acid is from C. Chothia, J. Mol. Biol. 105, 1-14, 1975. glucose Km (mM) 87 120 100- Ala E‘ Val 20 100 I ‘ I ' I 150 200 250 . o 2 Water-accessrble surface area (A ) 300 88 a hydrogen bond to the substrate and increases the affinity for both substrates in a way similar to that which has been observed in the case of Val-186 -—> Thr substitution. The Role of Other Aromatic Amino Acids in the Active Site. Besides Trp-139, there are four other aromatic amino acid residues in the active site pocket. In Arthrobacter xylose isomerase the pyranose ring of the substrate is sandwiched between Trp-15 and Trp-136 (corresponding to Trp-49 and Trp-188 of the Thermoanaerobacterium enzyme, respectively). The indole ring of Trp-136, surrounded by Phe-93 and Phe-25 (from the neighboring subunit), interact hydrophobically with the carbon backbone of the substrate pyranose ring (Figure 1). These Phe residues correspond to Phe-145 and Phe-60 of the Thermoanaerobacterium enzyme, respectively. The N‘1 of the Trp-15 in the Arthrobacter enzyme hydrogen bonds to Asp-292 (corresponding to Asp-339 in Thermoanaerobacterium) which coordinates to metal ion[I] (Collyer et al., 1990). When Trp-49 of Thermoanaerobacterium enzyme was substituted by Phe or Ala, the KM(g,,,m, increased 3 and 6.5 folds, respectively, with slight decrease in the km, (Table 3). The loss of the hydrogen bond between Trp-49 and Asp-339 may be expected to affect the structure of the active site. A decrease in the binding affinity for the substrate is consistent with this expectation. Surprisingly, the substitution of Trp-49 with Arg did not result in appreciable change of either K M 89 or k 0,. Introduction of the positive charge of Arg seemed not to change the active site structure. It is possible that the Ne of Arg may superimpose on the NI of tryptophan and hydrogen bond to Asp-339 thus maintaining a structure of the active site close enough to the wild type to be functionally indistinguishable. When Trp-188 was substituted by Lys, Asp, Glu or His none of the four mutant proteins exhibited any detectable catalytic activity toward glucose. With D-xylose as substrate, Trp-188 -) Lys, Trp-188 —> Asp and Trp-188 -—> Glu still did not show any activity but in the Trp-188 —> His mutant a low activity could be detected. The protein solubility of Trp-188 —-> Glu and Trp-188 -> His after incubation at 65°C was checked by applying the supernatant, after centrifugation, onto SDS-PAGE. The results showed that the protein still remains soluble. The lack of activity in these mutants must, therefore, be attributed to the loss of catalytic ability rather than the instability of proteins at 65°C. Although Trp-188 —) His mutant showed activity towards xylose, the K mm, was very high so that the catalytic constants could not be measured precisely at 60°C. However, it was found that KM of this variant xylose isomerase is temperature dependent (Table 4). At 37°C KM(xylm) was 820 mM which is about 800 times higher than that of the wild-type enzyme whereas km, was lower only by a factor of 2 as compared with the wild-type xylose isomerase (Table 4). It is possible that the loss of catalytic activity in the Trp-188 -—) Lys, Trp-188 —> Asp or Trp-188 —> Glu mutant enzymes was also due to the inability of these proteins to bind the substrate. These data 90 Table 4: Kinetic constants of wild-type and active site aromatic amino acids substituted mutant D-xylose isomerases (H) xylose Enzyme kcar(s") KM(mM) kcat/KM WT(65°C)' 23:1 93:18 2.5 WT(37°C)° 1.0:0.1 1.1:0.1 0.9 F145—->K(37°C)b l.1:0.1 53:6 2x102 W188-—>H(37°C)b 0.50:0.02 820:30 6x10“4 W188—-)D no activity W188—)E no activity W188—>K no activity a: Reaction was performed at 65°C. b: Reaction was performed at 37°C. 91 indicate that the hydrophobic interaction between the indole ring of Trp-188 and the hydrophobic backbone of pyranose plays an important role in the binding of sugar substrates to the xylose isomerase catalytic site. Substitution of Phe-145 with Lys resulted in a fifty-fold increase of KM(xym, and an insignificant change in km, at 37°C (Table 4). This suggests that Phe-145 also plays an important role in substrate binding. Since the phenyl group of Phe-145 does not interact directly with the hydrophobic backbone of the substrate, the role of Phe-145 may be to maintain the indole group of Trp-188 in an optimal position for the interaction with the substrate. Substitution of Phe-60 with His did not change the K MWCOSC, significantly, but this mutant enzyme exhibited only 20% of km, in comparison with the wild type (Table 3). Phe-60 is involved in the association of monomers to form the active dimers. As is shown by SDS-PAGE, Phe-60 -) His has stronger interaction at subunit interface of active dimer (see Figure 6 in Chapter IV). This indicates that the active site structure was changed somehow in Phe-60 -> His enzyme, and this change must account for the change of kinetic constants. However, a crystal structure of the mutant enzyme is required to fully understand the mechanism of this defect. Effect of Mutations on the Thermostability of D-Xylose isomerase. D-xylose isomerase is an enzyme of considerable thermostability in aqueous solutions at neutral pH. This property has been of great advantage in the purification of this 92 enzyme when it was synthesized in E. coli. Incubation of crude cell extract at 75°C precipitated the majority of the host proteins while Thermoanaerobacterium xylose isomerase was left as soluble and active enzyme. Among the mutant proteins created in this study, Trp-139 -> Lys, Trp-188 —) His and Phe-145 —> Lys were no longer resistant to the heat treatment at 75°C. This indicates that a disruption of the hydrophobic interactions between aromatic residues in the active site, by substituting them with charged residues, destabilizes the enzyme. For comparison of the thermostability between wild type and mutant proteins we have determined the time course of inactivation at 85°C. The optimal concentration of CoCl2 for these experiments was found to be 50 mM (data not shown). D-xylose isomerase did not follow the reversible two-state process of thermal inactivation, Native .- Unfolded form. Upon prolonged incubation at 85°C enzymatic activity was lost progressively and was accompanied by precipitation of protein. This irreversible thermoinactivation process followed a first order kinetics (Fig. 3), and the reaction rate constant was independent of the initial protein concentration within the range 50-500 ug/ml (data not shown). From the reaction rate constant, the half-life for each variant xylose isomerase was determined. We tested the thermostability of Trp-139 —9 Phe, Trp-139 —-> Met, Trp— 139 -> Ala, Trp-49 —> Arg, Trp-49 —> Phe and Trp-49 —) Ala. The mutant protein Trp-49 —-> Ala lost activity and precipitated immediately after heating at 85°C. Trp-49 -—) Phe was also very unstable with half-life less than 1.5 min. The gain in thermostability of Trp- 93 Figure 3: Time course of irreversible thermoinactivation of factitious variants of Thermoanaerobacterium xylose isomerase. The half-life (t‘”) of enzyme was determined from the equation: tm=(ln 2)/k, in which k is the first order rate constant of thermoinactivation. Ln (residual activity) 94 O In , 1 \f‘: 1 (min) .‘ \ ' El WT 35 0 R49 55 - Ir F139 67 e A139 50 - I F49 o . a M139 58 -1 .. n o D u Ii ' u -2 r r ' I 0 50 100 Time (min) 150 95 49 —-> Arg mutant suggests again that N8 of Arg may take the position of NEl of Trp-49. The increments in thermostability of Trp-49 —> Arg were the same at pH 7.0 and pH 5.0 (data not shown), suggesting that the positive charge of Arg residue may not be the factor responsible for the increase in the thermostability of the mutant enzyme. The results of mutation at position 49 thus suggest that the hydrogen bond between T rp-49 and Asp-339 or/and the hydrophobic side chain provided by Trp-49 are involved in maintaining the active site structure for substrate binding (see effect on KM in Table 3); this bond is also important in the resistance to thermoinactivation. Substitution of Trp-139 by smaller hydrophobic amino acids enhanced thermostability, indicating that the indole group of T rp-139 does not contribute to the maintenance of the active site structure. In fact, the bulky indole group of Trp-139 has adverse effect on protein thermostability. DISCUSSION Comparison of the catalytic efficiency, keg/KM, for the two substrates indicated that D—xylose is a much better substrate than D-glucose, and this difference is due mainly to the differences in the Michaelis constant. One of the important mechanisms for Thermoanaerobacterium D—xylose isomerase to discriminate between xylose and glucose is the steric hindrance offered by the indole group of Trp-139 against the C6-methanolic group of glucose. When Trp- 139 was substituted by amino acids with smaller side chains the catalytic efficiency for glucose increased. We believe that the enlarged active site pocket did not accommodate xylose as well as the pocket of the wild- type enzyme and this resulted in the decrease of the catalytic efficiency for xylose. The correlation of Kuwmw) with the water accessible surface area of the side chain of amino acid at position 139 indicates that this side chain protrudes into the solvent and constitutes the steric hindrance for the binding of glucose. The indole group of Trp-188 is parallel to the hydrocarbon backbone of the pyranose ring and, in the Arthrobacter isomerase, it is in the hydrophobic interaction with this part of the substrate molecule. The 800-fold increase in KM(mM) caused by the substitution of Trp-188 with His suggests that this hydrophobic interaction contributes very significantly to substrate binding. The indole group may also orient the pyranose ring in a position such that the hydroxyl 96 97 groups of substrate can hydrogen bond to the hydrophilic residues of the active site. In xylose isomerase from Actinoplanes missouriensis Trp-137 (corresponding to Trp-188 of T hermoanaerobacterium enzyme) has been changed to Phe and the KM for xylose and glucose have been found to increase 4.4 and 2.7 fold, respectively (Lambeir et al., 1992). The importance of the hydrophobic interaction between tryptophan and sugar for substrate binding has also been found in maltose-binding protein of E. coli. Substitution of one of the tryptophan residues in the active site of the maltose binding protein by alanine increased dissociation constant of the enzyme-substrate complex, Kd, 67 fold, similar substitution of another tryptophan at this site resulted in a 300 fold increase in Kd (Martineau et al., 1990). In this work substitution of Phe-145 with Lys increased the K ”We”, 50 fold. Predictions based on the structure of the Arthrobacter isomerase active center indicate that the phenyl group of Phe-145 is perpendicular to the indole group of Trp-188 (Figure 1). Phe-145 might help Trp-188 to maintain a proper position for the interaction with the substrate. Although KM(glme) increased in both Trp-49 —> Phe and Trp-49 -—> Ala mutant proteins, Trp—49 -) Arg exhibited kinetic constants similar to those of the wild type protein. We speculate that Arg-49 may hydrogen bond to Asp-339 which coordinates to metal [I] and thus provide the same function as Trp-49. The primary functional role of Trp-49, therefore, might be to hydrogen bond to Asp-339 and contribute to substrate binding indirectly. Argos et al. (Argos etal., 1979) have proposed that protein thermal stability 98 is enhanced when the area of hydrophobic surface in contact with the aqueous solvent is reduced. The thermal stability of lactate dehydrogenase from Bacillus stearothermophilus has been enhanced by reduction of the area of a water- accessible hydrophobic surface (Wigley et al., 1987). In this work we examined thermostabilities of Trp-139 —> Phe, Trp-139 —> Met and Trp-139 —> Ala mutant enzymes and found that all of them are more stable than the wild-type enzyme. Since the indole group of Trp-139 protrudes into the solvent and does not contribute to the architecture of the active site, replacement of Trp-139 with Phe, Met or Ala reduced the area of active site hydrophobic surface which is expected to expose to water (Figure 4). This is, therefore, responsible for the enhancement of thermostability in Trp-139 —) Phe, Trp-139 -) Met and Trp-139 —-> Ala mutant enzymes. Enhancement of the thermostability in Trp-49 —> Arg could be brought about by the same mechanism since Arg-49 could presumably fulfill the function of hydrogen bonding to Asp-339 and thus leave the active site structure unchanged. This explanation is in good agreement with the properties of Trp-49 —-> Phe mutant which exhibited a reduced thermostability. Phenylalanine would be unable to hydrogen bond with Asp-339 and this substitution would, therefore, be expected to disturb the architecture of the active site. The reduction of thermostability in Trp- 188 -) His, Trp-139 —> Lys and Phe-145 —-> Lys, observed in this work, may be due to the perturbation of the active site structure. Substitution of the hydrophobic residues involved in architecture of the active site with charged residues could 99 Figure 4: Schematic illustration of the reduction of hydrophobic surface area as Trp-139 changed to Phe or Ala. 100 t”2 = 35 min t”2 = 67 min 101 certainly be expected to destabilize this region of the protein. REFERENCES Argos, P., Rossman, M. G., Grau, U., Zuber, H., Frank, G. & Tratschin, J. D. (1979) Biochemistry 18, 5698-5703. Bone, R., Silen, J.L. & Agard, DA. (1989) Nature (London) 339, 191-195. Collyer, C. A., Henrick, K. & Blow, D. M. (1990) J. Mol. Biol. 212, 211-235. Craik, C.S., Largman, C., Fletcher, T., Roczniak, S., Barr, P.J., Fletterick, R. & Rutter, W. (1985) Science 228, 291-297. Dean, A.D. & Koshland, Jr., DE. (1990) Science 249, 1044-1046. Estell, D.A., Graycar, T.P., Miller, J.V., Powers, D.B., Bumier, J.P., Ng, P.G. & Wells, J.A. (1986) Science 233, 659-663. Evnin, L.B., Vésquez, J.R. & Craik, CS. (1990) Proc. Natl. Acad. Sci. USA 87, 6659-6663. Higaki, J.N., Haymore, B.L., Chen, S., Fletterick, R. & Craik, CS. (1990) Biochemistry 29, 8582-8586. Hurley, J.H., Dean, A.M., Sohl, J.L., Koshland, Jr., D.E. & Stroud, RM. (1990) Science 249, 1012-1016. Lambeir, A.-M., Lauwereys, M., Stanssens, P., Mrabet, N. T., Snauwaert, J ., van Tilbeurgh, H., Matthyssens, G., Lasters, 1., Maeyer, M. D., Wodak, S. J ., Jenkins, J ., Chiadmi, M. & Janin, J. (1992) Biochemistry 31, 5459-5466. Lee, C., Bagdasarian, M., Meng, M. & Zeikus, J. G. (1990) J. Biol. Chem. 265, 19082-19090. Lowry, O. H., Rosebrough, N. J., Farr, A. L., & Randall, R. J. (1951) J. Biol. Chem. 193, 265-275. Martineau, P., Szmelcman, S., Spurlino, J. C., Quiocho, F. A. & Hofnung, M. (1990) J. Mol. Biol. 214, 337-352. Quiocho, F. A., (1989) Pure Appl. Chem. 61, 1293-1306. 102 103 Scrutton, N.S., Berry, A. & Perham, RN. (1990) Nature (London) 343, 3843. Wells, J.A., Powers, D.B., Bott, R.R., Graycar, T .P. & Estell, DA. (1987) Proc. Natl. Acad. Sci. USA 84, 1219-1223. Wigley, D. B., Clarke, A. R., Dunn, C. R., Barstow, D. A., Atkinson, T., Chia, W. N., Muirhead, H. & Holbrook, J. J. (1987) Biochim. Biophysica. Acta 916, 145-148. Wilkinson, A.J., Fersht, A.R., Blow, D.M., Carter, P. & Winter, G. (1954) Nature (London) 307, 187-188. Wilks, H.M., Hart, K.W., Feeney, R., Dunn, C.R., Muirhead, H., Chia, W.N., Barstow, D.A., Atkinson, T., Clarke, A.R. & Holbrook, J .J . (1988) Science 242, 1541-1544. CHAPTER IV EFFECT OF SALTS ON THERMOSTABILITY OF D-XYLOSE ISOMERASE: THE DOMINANT FACTOR GOVERNING THE PROCESS OF IRREVERSIBLE THERMOINACTIVATION OF THERMOANAEROBACTERIUM XYLOSE ISOMERASE 104 ABSTRACT The kinetics of thermoinactivation of D-xylose isomerase from Thermoanaerobacterium thermosulfurigenes in aqueous solution was investigated. Here we report for the first time that besides well known divalent cations, monovalent cations, particularly K“, also protects the enzyme against thermoinactivation. The kinetic data suggest that the rate of formation of incorrect conformation of the enzyme ("scrambled structure") is the dominant factor governing the process of thermoinactivation. 105 INTRODUCTION D-xylose isomerase (EC. 5.3.1.5), often referred to glucose isomerase, has been utilized to produce high fructose corn syrup for three decades. The industrial process for this enzymatic conversion is performed at 60-65°C; therefore, the enhancing half-life of this enzyme at elevated temperatures is desirable for practical applications. Xylose isomerase is a homotetramer; each subunit contains an or/B barrel domain. The active site pocket is located at carboxyl end of the B—strand, and involves amino acids from neighboring subunit (Henrick et al., 1989 & Whitlow et al., 1991). Therefore, the basic functional unit of xylose isomerase is a dimer. It was proposed that strengthen interaction in the interface of active dimer may enhance thermostability of the enzyme (Rangarajan, et al., 1992). Divalent cations, such as Co”, Mn+2 or Mg+2 are required for both the catalytic activity and the thermostability of D-xylose isomerase. The extent of the effects by these metal ions depends on the origin of the enzyme and the substrate (Chen, 1980). For the enzyme from Thermoanaerobacterium thermosulfurigenes Co+2 is the best protector (Lee & Zeikus, 1991). To find the optimal conditions for the stabilization of the Thermoanaerobacterium xylose isomerase we have examined the effect of different ions on thermoinactivation. The results have indicated some stages of the thermoinactivation process. 106 MATERIALS AND METHODS Enzymes. The Thermoanaerobacterium thermosulfurigenes gene coding for D- xylose isomerase was expressed in E .coli HB 101 cells as described previously (Lee et al., 1990). Mutant enzyme, Phe-60 —> His, was created and expressed as described in Materials and Methods in Chapter III. The wild type and mutant enzymes were purified through the steps of heat treatment (75°C, 20 min), DEAE- Sepharose and Sephacryl-300 chromatography to the stage of homogeneity in SDS-PAGE ( Lee et al., 1990). The metal-free enzyme was prepared by dialyzing the pure enzyme against 100 time volume of 10 mM MOPS buffer (pH 7.0) containing 10 mM EDTA; subsequently, dialyzing against 10 mM MOPS buffer (pH 7 .0) to remove EDTA. Dialysis was performed at 4°C and the dialysis buffer was changed every 12 hours for three times. The metal-free enzyme was used for all the experiments. Protein concentration was determined by the method of Lowry et al. (1951) with bovine serum albumin as standard (from Pierce). Buffer. MOPS buffer was used in all the experiments. Solution was adjusted at room temperature to the pH values that would result in pH 7.0 at the temperature of the experiments according to ApH/At of MOPS buffer which is -0.011 (Dawson, et al., 1986). 107 108 Kinetics of Irreversible Thermoinactivation. The time course of irreversible thermoinactivation of D-xylose isomerase was measured by incubating 0.6 ml enzyme solution (in the range of 0.05-0.5 mg/ml in 10 mM MOPS pH 7.0 ) at the desired temperature for various periods of time and then determining the residual activity at 65 °C. In wild type enzyme the inactivation followed first order reaction and the rate constant, k, of irreversible thermoinactivation was obtained by linear regression in semilogarithmic coordinates. In the Phe-60 —-> His mutant enzyme the activity was persistent within the initial 20 minute then it followed first order inactivation. The rate constant of Phe-60 -> His was calculated by taking the data from the first order reaction range. Half-life of enzyme was calculated from the equation: f’”’=(ln2)/k. Enzyme activity assay. Reaction was started by the addition of glucose (at final concentration of 800 mM) to enzyme solution and runing the reaction at 65°C for 30 min. Fructose formed was determined by cysteine/carbazole/sulfuric acid method (Dishe & Borenfreund, 1951); pure fructose was used as standard. Enzyme concentrations were adjusted such that less than 5% of the original substrate was converted within 30 min, which allowed the determination of initial reaction velocities. SDS-Polyacrylamide gel electrophoresis. SDS-PAGE was canied out according 109 to the method of Laemmli (Laemmli, 1970) on 7% separation and 3% stacking gel. Protein samples were dissolved in 56 mM Tris buffer, pH 6.8, containing 9% glycerol and 0.1% SDS and were loaded on the gel without prior heating Step at 100°C. After electrophoresis, the protein bands were stained with 0.1% Coomassie blue R-250 in 40% methanol and 10% acetic acid solution. Scanning calorimetry. Ultrasensitive scanning calorimeter MC—2 (MicroCal, Inc. Northampton, MA) was used in this study. Enzyme samples, (1 mg/ml) in 10 mM MOPS buffer (pH 7.7 at room temperature), containing 50 11M Co+2 or 50 11M Co+2 + 10 mM KCl, were loaded into the sample cell. Buffer of the same composition without the enzyme was loaded into reference cell as blank. Samples were degassed before loading as described in the manual. The temperature rising rate was 60°C/hr. A buffer baseline was stored and subtracted from the displayed data to obtain the normalized excess-heat-capacity function (NEF) curve. RESULTS Irreversible thermoinactivation of Thermoanaerobacterium thermosulfurigenes D-xylose isomerase at pH 7.0. Upon heating at elevated temperature, e.g., 56°C, metal-free D-xylose isomerase lost activity progressively. This inactivation followed a first order reaction with t‘m’(half-life)=8 min. An overnight incubation of this inactivated enzyme at 4°C did not restore any of the lost activity. Co+2 was known as the best protective agent against thermoinactivation (Lee & Zeikus 1991). Incubation of xylose isomerase, in the presence of Co”, at pH 7.0 and 85°C also resulted in a progressive inactivation of the enzyme. Such inactivation was accompanied by a significant precipitation of the enzyme and was irreversible. The optimum concentration of Co+2 for thermal protection was 50 uM (Table 1). Two putative divalent metal binding sites were assumed in the active site pocket of Thermoanaerobacterium enzyme according to the crystal structures of enzymes from Arthrobacter and Streptomyces (Henrick et al., 1989, & Carrelleta1., 1989). Occupancy of these two metal binding sites is believed to be responsible for the enhanced thermostability of the enzyme. Excess amount of Co+2 actually has an adverse effect and this may be due to the binding of excess Co+2 to other potential binding sites. The events occurring during irreversible thermoinactivation of enzymes can be classified into (1) covalent changes such as hydrolysis of disulfide bonds, 110 1 1 1 Table 1: Effect of CoCl2 on the half-life of D—xylose isomerase at 85°C CoCl2 (uM) t"2 (min) 12.5 29 25 37 50 39 200 17 800 4 Aqueous enzyme solution (200ttg/ml) in 10 mM MOPS buffer (pH 7.0 at 85°C) with various concentration of CoCl2 was incubated at 85°C. Half-life of the enzyme in each concentration of CoCl2 was calculated form the inactivation constant of the first ordered thermoinactivation curve. 112 peptide bonds and amides (asparagine and glutamine), (2) noncovalent changes such as aggregation of protein and formation of incorrect folded protein (Klibanov, 1983). In a preliminary test the optimum concentration of Co”, 50 MM, was found to be independent of the initial protein concentration. In order to test whether the precipitation (aggregation) is the dominant factor causing irreversible inactivation of the enzyme at 85°C in the presence of Co”, the thermoinactivation curves for the enzyme at different initial protein concentrations (50-500 rig/m1) were determined. The results are shown in Figure 1. The inactivation curves all obeyed the first order kinetics with correlation coefficients greater than 0.98. The almost identical inactivation constants (slopes of the lines) for different initial protein concentrations suggest that the dominant factor causing thermoinactivation is monomolecular event. The inactivation constant would have been dependent on the initial concentration if aggregation were the main factor governing thermoinactivation, since aggregation is a multiple molecular event. The temperature is a critical parameter. Therefore, the temperature dependence of the rate constant of irreversible thermoinactivation of xylose isomerase in the presence of Co“2 was examined within the temperature range of 80—90°C. Arrhenius plot (Figure 2) shows an activation energy of 120 kcal/mol. This high activation energy is totally uncharacteristic of a covalent reaction (Tomazic, & Klibanov, 1988). Since aggregation and covalent changes of the enzyme are unlikely to be the dominant factor accounting for the irreversible 113 Figure 1: Effect of initial protein concentration on the inactivation process of D- xylose isomerase at 85°c activity) Ln(residual 114 40 60 80 Tlme(mln) 115 Figure 2: Arrhenius plot of thermoinactivation of D-xylose isomerase Thermoinactivation constant, k, of xylose isomerase at desired temperature was determined from slope of the irreversible thermoinactivation plot of the enzyme at that temperature. Temperature was in the range of 80-90°C. 116 d 1 Q A w c222; no; -2q o -1 )x10 1/T( K 117 thermoinactivation of xylose isomerase, the main factor could be the formation of incorrectly folded enzyme which is followed by the precipitation of protein. Effect of salts on thermostability of D-xylose isomerase. Based on the empirical observation that the enzyme in crude cell extract is more stable than in homogeneous buffer solution, we decided to test the effect of salts on thermostability of the enzyme. Xylose isomerase (100 rig/ml) in 10 mM MOPS buffer (pH 7.0) containing 50 uM Co+2 and various salts was incubated at 85°C for 45 min. The residual activities were determined and the results are shown in Table 2. Except for A12(SO.,)3 and tetrarnethylarnmonium chloride, all salts added had positive effect on thermostability. However, the most prominent effects came from KCI, KNO3, CsCI, (NH4)ZSO,, and NH4C1. This enhancement of thermostability could not be the result of a general salt effect because (1) different salts protected to different extent, e. g. the effect of K+ was more prominent than that of Na”, (2) As low as 10 mM of salts was enough for protection, (3) tetramethylarnmonium chloride did not show protection effect. If enhancement of thermostability were due to general salt effect, tetramethylarnmonium chloride should have had the same effect as K‘. The dependence of half-life of xylose isomerase on K+ concentration at 88°C is shown in Figure 3. The relationship of K+ and half-life of the enzyme at 88°C is sigmoid. Seven-fold enhancement in thermostability was achieved at 10- 100 mM of K‘. The stabilizing effect of K+ could also be demonstrated on the 118 Table 1: Effect of salt on thermostability of wild-type D-xylose isomerase Salt Residual activity (%)° Control" 38 LiCl (10mM) 62 NaCl (10mM) 50 NaCl (100mM) 69 NaQSO, (3.3mM) 42 KC] (10mM) 80 KC] (100mM) 84 KNO3 (10mM) 82 CsCl (10mM) 79 (NH,,)ZSO4 (3.3mM) 81 NH4C1 (10mM) 79 MgCl2 (3.3mM) 57 A12(SO4)3 (0.33mM) 15 Tetramethylammonium chloride (10mM) 33 Aqueous enzyme solution (100ttg/ml) containing 50ttM CoClz, 10mM MOPS buffer (pH 7.0 at 85°C) and various salt was incubated at 85°C for 45 min. Enzyme activities (before heating and after heating) were assayed at 65°C for 30 min as described in Materials and Methods. a: Residual activity is expressed as the percentage of activity left after 85°C incubation. b: Control means what only contained CoCl2 and MOPS buffer. 119 Figure 3: Effect of KCl on half-life of D-xylose isomerase at 88°C 120 60 50- . _ 0 0 4 3 E5222}: 20" 101 Ln KCl(mM) 121 apoenzyme (the enzyme containing no Co“). Half-life increased from 8 min to 75 min when the apoenzyme, in 10 mM MOPS buffer (pH 7.0), was incubated at 56°C in 100mM K‘. To determine the changes of energy constants during the process of themoinactivation, purified enzyme was subjected to scanning microcalorimetry. The temperature dependence of specific heat capacity is shown in Figure 4. Xylose isomerase, instead of showing a typical unfolding peak, precipitated at a certain temperature; therefore, it was impossible to calculate the changes of energy constants during themoinactivation. However, the precipitation temperature still provides some information about relative thermostability of the enzyme. In the presence of 50 uM of Co+2 the enzyme started to precipitate at 84°C; whereas, it became more resistent to heat in the presence of 10 mM of K+ + 50 11M of Co+2 and started to precipitate at 96°C. Since K+ is not expected to change the rate of covalent changes of the protein, the effect of K” on thermostability also suggests that the rate-determining event in the process of thermoinactivation is the formation of incorrectly folded protein. Effect of substitution of Phe-60 by His on thermostability. A cluster of aromatic amino acids, Trp-15, Phe-93, Trp-136 and Phe-25 (from the neighboring subunit) is present in the active site pocket of xylose isomerase from Streptomyces ( Whitlow et al., 1991) and Arthrobacter (Collyer et al., 1990). These amino acids 122 Figure 4: Differential scanning microcalorimetric plots of D-xylose isomerase Inol O O KcaVK Inol KcaVK 400 123 300 - 200 r- 100_ 10 mM MOPS (pH 7.7) 50 ILM 00012 10 mM KCI 200 10 mM MOPS (pH 7.7) 50 ILM CoCl2 -—1 00 __ —4OO _. -700 1 I l I 50 60 7O 8O 90 1 00 Temp (°C) 124 are conserved in all known xylose isomerases. The corresponding amino acids in Thermoanaerobacterium enzyme are Trp-49, Phe-145, Trp-188 and Phe-60 (from the neighboring subunit). The hydrophobic interaction among these aromatic amino acids was postulated to be one of the important forces that keep the monomers associated in an active dimer. Rangarajan et al., working on stability of Arthrobactor xylose isomerase, proposed that strengthening the interactions at the interface of active dimer by protein engineering should increase thermostability (Rangarajan, et al., 1992). To test the significance of these putative hydrophobic forces at the interface of active dimer, Phe-60 of the Thermoanaerobacterium enzyme was changed to His. The activity of this mutant enzyme, Phe-60 —> His, dropped to approximately 20% of the wild type enzyme (see Table 3 in Chapter III). Preincubation of Phe-60 —> His mutant enzyme in 10 mM MOPS buffer at 85°C for 5 min increased the activity two-fold. The strength of active dimer was tested by incubation of the enzyme in 0.1% SDS at 50°C followed by SDS-PAGE. Surprisingly, it was found that the interaction of monomers in the dimer was stronger in Phe-60 —) His mutant than in wild-type enzyme (Figure 5). The reason for this difference is difficult to interpret without crystal structures of the enzymes. Besides the cluster of aromatic amino acids, there are several hydrophilic amino acids, such as glutamic and aspartic acid that line up the active site surface. His-60 (from neighboring subunit) may interact with one of these hydrophilic amino acid residues. Time course of thermoinactivation of Phe-60 —9 His at 85°C is shown in 125 Figure 5: SDS-PAGE of wild-type and Phe-60 -—> His mutant xylose isomerase. Enzyme (13 ug) was incubated in 0.1% SDS at 50°C for 0, 10, 20 min. Afterward, it was loaded into gel without prior boiling at 100°C. Maker is the high molecular weight standards from Biolab. Letter A, B and C represent wild- -type, Phe- 60 —) His and Trp-139 —> Phe, respectively. Number 1,2 and 3 represent incubation time 0, 10 and 20 min, respectively. 126 127 Figure 6. Unlike wild-type enzyme, this mutant enzyme did not obey the first order kinetics. It is resistant to 85°C during the initial 20 min then it follows the first order inactivation with a half-life 25 min. The stronger interaction of active dimer does not seem to Slow down the rate of inactivation in the stage which proceeds with the first order kinetics. However, it does delay start of the process of thermoinactivation. 128 Figure 6: Time course of irreversible thermoinactivation of wild-type and Phe-60 —>His mutant enzymes at 85°C. Ln(Residual activity) 129 -L00‘ -t50‘ -2.00"1 -250 20 4O 60 Time(min) 80 100 DISCUSSION It has been well documented that the binding of divalent cations, such as Mg*2 and Co”, to xylose isomerase remarkably enhance stability of the enzyme against heat and other denaturing agents (Kasumi, et al., 1982; Callens, et al., 1988; Lee, & Zeikus, 1991; Gaikwad, et al., 1992; Rangarajan, et al., 1992). In this study we report for the first time that some monovalent ions, such as K“, can enhance thermostability of Thermoanaerobacterium xylose isomerase and this stabilization is not due to a general salt effect. Such effect of K” was also shown on a commercial glucose isomerase (Spezyme GI); 100 mM KCl increased half-life of Spezyme (in the presence of 50 11M Co”) from 350 min to 590 min at 85°C. Upon heating at elevated temperature Thermoanaerobacterium xylose isomerase undergoes an irreversible thermoinactivation that runs with the first order kinetics. The independence of inactivation constant on the initial protein concentration and a very high activation energy suggest that the rate-determining step during the process of thermoinactivation is the formation of incorrectly folded enzyme. K” ions probably bind to the enzyme and stabilize the correct folding. Substitution of Phe-60 with His made the active dimer more resistent to thermal denaturation. However, such Stronger monomer interaction did not slow down the rate of thermoinactivation. This indicates that dissociation of monomers is not involved in the rate-determining event. 130 131 It is impossible to conclusively define a pathway describing the events during thermoinactivation process with this limited information. However, a simplified model is proposed based on results obtained in this work. tetramer '- active dimer - monomer —> incorrect folded monomer —-> aggregation Dissociations of the tetramer to active dimer and active dimer to monomer are reversible. The irreversible formation of incorrectly folded monomer from the native monomer is the rate-determining event and it is followed by aggregation of protein and precipitation. K‘ might bind to the protein and stabilize the correct form of monomer and, therefore, reduce the rate of formation of incorrectly folded polypeptide. Strengthened interaction at the subunit interface of the dimer could not slow down the overall rate of inactivation as indicated by Phe-60 ——> His mutant enzyme. REFERENCES Callens, M., Kersters-Hilderson, H., Vangrysperre, W. & De Bruyne, C. K. (1988) Enzyme Microb. Technol. 10, 695-700. Carrell, H. L., Glusker, J. P., Burger, V., Manfre, F., Tritsch. D., & Biellmann, J.- F. (1989) Proc. Natl. Aced. Sci. USA 86, 4440-4444. Chen, W. P. (1980) Process Biochem 15, 36—41. Collyer, C. A., Henrick, K. & Blow, D. M. (1990) J. Mol. Biol. 212, 211-235. Dawson, R. M. C., Elliott, D. C., Elliott, W. H. & Jones, K. M. (1986) Data for Biochemical Research (Clarendon Press, Oxford) 3rd Ed. Dische, Z. & Borenfreund, E. (1951) J. Biol. Chem. 192, 583-587. Gaikwad, S. M., Rao, M. B. & Deshpande, V. V. (1992) Enzyme Micro. Technol. 14, 317-320. Henrick, K., Collyer, C. A. & Blow, D. M. (1989) J. Mol. Biol. 208, 129-157. Kasumi, T., Hayashi, K. & Tsumura, N. (1982) Agric. Biol. Chem. 46, 31-39. Klibanov A. M. (1983) Advan. Appl. Microbiol 29, 1-28. Laemmli, U. K. (1970) Nature 227, 680-685. Lee, C., Bhatnagar, L., Saha, B. C., Lee, Y.-E., Takagi, M., Imanaka, T., Eggasarian, M. & Zeikus, J. G. (1990) Appl. Environ. Microbiol 56, 2638- Lee, C., Bagdasarian, M., Meng, M. & Zeikus, J. G. (1990) J. Biol. Chem. 265, 19082-19090. Lee, C. & Zeikus, J. G. (1991) Biochem. J. 274, 565-571. Lowry, O. H., Rosebrough, N. J., Farr, A. L., & Randall, R. J. (1951) J. Biol. Chem. 193, 265-275. 132 133 Rangarajan, M., Asboth, B. & Hartley, B. S. (1992) Biochem. J. 285, 889-898. Tomazic, S. J. & klibanov, A. M. (1988) J. Biol. Chem. 263, 3086-3091. Whitlow, M., Howard, A. J., Finzel. B. C., Poulos, T. L., Winbome, E., & Gilliland, G. L. (1991) Protein: Structure, Function, and Genetics 9, 153- 173. CHAPTER V CONCLUSION 134 135 To apply a protein engineering approach to elucidate the structure/function relationship of a protein, one of the most important requisites is the availability of structural information of this protein. Although the structure of xylose isomerase from T. thermosulfurigenes has not been available, the crystal structures of the enzyme from Arthrobacter and Streptomyces species were used as guidance to conduct site-directed mutagenesis in this study. Based on such strategy we were able to obtain following achievements : (1) proposed the mechanism of enzymatic reaction based on the understanding of the functions of active-site residues involved in catalysis and metal coordination. (2) elucidated the roles of active-site aromatic amino acids on substrate binding and understood the structural basis of the enzyme for discrimination between glucose and xylose. (3) created mutant enzymes which are more applicable to industrial process in that they are more resistant to thermoinactivation and have higher catalytic efficiencies, ken/KM, toward glucose by rational redesign of the active-site pocket. However, such strategy has its limitation due to the rare homology of amino acids outside the active-site pocket so that mutation could only be performed at the conserved active-site amino acids. Crystallographic study of T. thermosulfurigenes xylose isomerase has been undertaken at the laboratory of D. M. Blow in Imperial College (London, England). The crystal structure of the enzyme will, hopefully, be available soon. Such information will be of great advantage on the following aspects: (1) to confirm the interpretations of mutational results of this research. (2) to broad the target sites for 136 further site-directed mutagenesis experiments such as that of enhancing thermostability or changing metal specificity of the enzyme. Protein engineering has being used to increase the stability of enzymes. The common approaches include (1) reducing the difference in entropy between folded and unfolded protein, which in practice means reducing the number of conformations in the unfolded state (Matsumura et al., 1989; Matthews et al., 1987), (2) stabilizing the dipoles of or helices, (Nicholson et al., 1988) (3) increasing the number of hydrophobic interactions and packing ratio in the interior core (Sandberg & Terwilliger 1989). There is an example for enhancing thermostability of xylose isomerase by protein engineering. The enzyme from Actinoplanes missouriensis has been engineered to enhance its thermostability by substituting Arg for Lys-253 which is the major glycation site in the presence of high concentration of glucose (Quax et al., 1991). Due to the limited information regarding the structure of T. thermosulfurigenes xylose isomerase, the effort, in this study, for enhancing thermostability of the enzyme could only be focused on the mutations of active-site amino acids. With crystal structure of T. thermosulfurigenes enzyme at hand in the future, the battle field for site-directed mutagenesis will be able to expand to the whole structure. Considering the applicability of T. thermosulfurigenes xylose isomerase, another challenging, also obligatory, objective will be the alteration of metal specificity. Wild-type T. thermosulfurigenes enzyme requires Co+2 for both 137 catalytic ability and thermostability. However, Co“2 is an environmental hazard and can not be used in food process. Mg“2 and Mn+2 can only partially fulfill the function of Co+2 (Lee & Zeikus, 1991). Alteration of the metal requirement from Co“2 to Mg+2 or to Mn+2 by engineering the metal binding sites could be feasible if the detail structure is provided. Although T. thermosulfurigenes xylose isomerase (belongs to class II) is not the enzyme currently used for the production of high fructose corn syrup, the discoveries in this research are still applicable to the engineering of commercial xylose isomerases (belong to class I). We showed that the indole group of Trp-139 causes steric hindrance against glucose binding in T. thermosulfurigenes enzyme and a correlation between KM(gmsc) and the size of hydrophobic side chain of amino acid in position 139 exists. Instead of tryptophane, a methionine presents in the corresponding position in class I xylose isomerase . We believe that KM(glmn) of class I enzymes can be reduced if this methionine is replaced with a smaller hydrophobic amino acid such as valine, or alanine. Also substitution of Val-134 with threonine may introduce a hydrogen bond between enzyme and glucose and therefore could be helpful for the decrease of KM in class I (glucose) enzymes. In respect of enhancing thermostability, reducing the area of water- accessible hydrophobic surface by substituting Arg for Trp-15 in class I enzymes may be is an useful approach. REFERENCES Lee, C. & Zeikus, J. G. (1991) Biochem. J. 274, 565-571. Matsumura, M., Signor, G., & Matthews, B. W. (1989) Nature 342, 291-293. Matthews, B. W., Nicholson, H., & Becktel, W. J. (1987) Proc. Natl. Acad. Sci. USA 84, 6663-6667. Nicholson, H., Becktel, W. J., & Matthews, B. W. (1988) Nature 336, 651-656. Quax, W. J., Mrabet, N.T., Luiten, R. G. M., Schuurhuizen, P. W., Stanssens, P., & Lasters, I. (1991) Bio/Technology 9, 738-742. Sandberg, W. S., & Terwilliger, T. C. Science 245, 54-56. 138 IIIIIIIIIIIIII“