Enolase from Candida albicans — purification and characterization

Comparative Biochemistry and Physiology Part B 126 (2000) 109 – 120 www.elsevier.com/locate/cbpb

Enolase from Candida albicans — purification and characterization Irena Kustrzeba-Wo´jcicka *, Marcin Golczak Department of Biochemistry, Medical Uni6ersity of Wrocl*aw, Chal*ubinskiego 10, 50 -368 Wrocl*aw, Poland Received 15 May 1999; received in revised form 23 September 1999; accepted 10 January 2000

Abstract This paper describes isolation of electrophoretically homogenous enolase from Candida albicans. The purification involved: disintegration of C. albicans cells in a Braun’s mill (67 – 100%) ammonium sulfate precipitation, chromatography on DEAE-Sephadex A-50 at pH 9.0 and chromatography on CM-Sephadex A-50 at pH 6.2. The procedure resulted in a 30-fold purification of the enzyme with a recovery rate of 6% and a specific activity 35 U mg − 1. The subunit molecular weight was 46 kDa and the pH optimum of the enzyme was 6.8. Km and Vmax values for the 2PGA “PEP reaction were determined (Km =0.95 mM, Vmax =4200 mmol min − 1 mmol − 1). In the absence of orthophosphate, inhibition by fluoride was competitive, which became noncompetitive in the presence of phosphate. It was confirmed that Mg2 + is the most potent activator (Km =0.286 mM); Mn2 + gave less activity and Zn2 + less still. It was also demonstrated that the presence of two types of cations in the reaction mixture nullified the activatory effect of the stronger agent. Properties of the enzyme from C. albicans are compared with those of enolases from other sources. © 2000 Elsevier Science Inc. All rights reserved. Keywords: Enolase; Candida albicans; Enzyme purification; Kinetics; Allergy

1. Introduction Enolase (2-phospho-D-glycerate hydrolyase, EC 4.2.1.11), an enzyme of the glycolytic pathway, catalyzes a reversible conversion of 2-phosphoglycerate to phosphoenolpyruvate. Enolases from several organisms and tissues have been isolated and characterized. Enolase is a metalloenzyme activated by cations of bivalent metals. Mg2 + serves as its natural cofactor. Enolase is a dimeric enzyme, found in mammals as a homodimer or heterodimer consisting of three subunits: a, b and g encoded by three distinct loci. Molecular weights of most examined enolases are in the * Corresponding author. Tel.: + 48-71-3209750; fax: +4871-3225415.

range of 80–100 kDa, therefore the mass of a single subunit ranges usually from 40 to 50 kDa. Enzymes from prokaryotic cells seem to be an exception, e.g. enolase from termophylic bacteria is an octamer, and enolase isolated from Streptococcus rattus has a molecular weight of about 49 kDa (the weight of subunit in this case is about 22 kDa) (Hu¨ther et al., 1990). The knowledge of enolases from fungal cells is limited, Saccharomyces cere6isiae being an exception. The classical procedure for isolation of enolase from baker’s yeast was devised in 1942, by Warburg and Christian. The enzyme obtained by this method is relatively pure. Yeast enolase has been crystallized and the tertiary structure of the enzyme determined (Lebioda and Stec, 1988; Lebioda et al., 1989; Stec and Lebioda, 1990;

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Lebioda and Stec, 1991; Zhang et al., 1994, 1997). Each monomer consists of two different domains. The carboxyl-terminal end of the main domain creating a catalytic site has a highly conservative character. There are several papers concerning detailed research on the catalytic mechanism of enolase (Hilal et al., 1995; Kornblatt, 1996; Brewer et al., 1997, 1998; Kornblatt et al., 1998). An increasing interest in enolases from fungi and moulds has occurred since Baldo’s discovery of the allergenic activity of these proteins (Baldo and Baker, 1988; Baldo, 1995). Hypersensitivity of the skin to fungi and moulds manifested by inhalant or skin allergy is often caused by spores or shreds of mycelium. Enolase is a principal allergen of fungi and moulds (Franklyn et al., 1990; Sundstrom and Aliaga, 1992, 1994; NittnerMarszalska et al., 1997; Breitenbach et al., 1997). The sequences of enolases from Candida albicans, S. cere6isiae, Alternaria alternata and Cladosporium herbarum exhibit about 80% homology (Breitenbach et al., 1997). van Deventer et al. (1994) developed a method of isolation of enolase from C. albicans and used the purified enzyme in a diagnostic test for differentiation of candidiasis cases in humans. However, besides the determination of the molecular weight of the purified enzyme, the authors did not study any other properties of this protein. We have developed a simpler method of enolase preparation from C. albicans, examined some physical, chemical and kinetic properties of the purified protein and compared the enzyme with enolases from other sources.

2. Materials and methods

2.1. Culture conditions

12 ml of water and 5 g of C. albicans cells. The inside of the Braun’s mill was cooled by liquid CO2. The total time of disintegration was 15 min (five series, 3 min each). The extent of disintegration was monitored using a light microscope. The crude extract was separated from the cell walls and glass beads by centrifugation at 40 000× g at 4°C for 20 min.

2.3. Ammonium sulfate fractionation and chromatography The crude extract was adjusted to 50% of saturation by adding a saturated solution of ammonium sulfate and subsequently to 67% by addition of solid ammonium sulfate. After centrifugation at 40 000× g at 4°C for 20 min, the supernatant was made 100% saturated and centrifuged again at 40 000× g at 4°C for 20 min. The supernatant was discarded and the pellet dissolved in 10 mM Tris–HCl buffer, pH 9.0, containing 5 mM MgSO4, 1 mM EDTA, 1 mM 2-mercaptoethanol; dialyzed overnight against the same buffer and applied to a DEAE-Sephadex A-50 column (Pharmacia, 1× 15 cm2) equilibrated with 10 mM Tris–HCl buffer, pH 9.0, containing 5 mM MgSO4, 1 mM EDTA, 1 mM 2-mercaptoethanol. The proteins were eluted with a linear NaCl gradient (0–0.5 M). The flow rate was 8 ml h − 1. Fractions with enolase activity were collected, concentrated by water evaporation in vacuum and dialyzed overnight against 15 mM NaH2PO4/ Na2HPO4, pH 6.2 containing 3 mM MgSO4. The dialyzate was applied on CM-Sephadex C-50 column (Pharmacia, 1× 15 cm2) equilibrated with 15 mM NaH2PO4/Na2HPO4 buffer, pH 6.2 containing 3 mM MgSO4. The protein was eluted using the same buffer, with the flow rate 8 ml h − 1.

2.4. Electrophoresis

C. albicans was grown in liquid Saboraud medium (4% glucose, 1% peptone, 0.5% NaCl) at 37°C with shaking for 48 h. Growth was terminated at the stationary phase. The cells were centrifuged at 3000 rpm at 4°C for 20 min and washed twice to remove the culture medium.

Electrophoresis was conducted in the Laemmli system (Laemmli, 1970) using a 4% stocking gel and a 10% separation gel. Samples containing 10–40 mg of the protein were applied.

2.2. Disintegration of cells

2.5. Molecular weight determination

Fragmentation of cells was performed in a Brauns’s mill using glass beads, F = 0.5 mm (Merck). The mixture contained: 7.5 g of beads,

The molecular weight of enolase was determined electrophoretically in the Laemmli system. For the molecular weight determination the fol-

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lowing markers (Serva) were used: cytochrome C (12.3 kDa), myoglobin (17.8 kDa), chymotrypsinogen A (25.0 kDa), albumin-egg (45.5 kDa), bovine albumin (67.0 kDa), rabbit muscle aldolase (160.0 kDa). Samples containing 10 mg of standard proteins or enolase were electrophoresed.

2.6. Protein concentration determination Protein was determined spectrophotometrically from the absorption at 280 nm. The concentration of enzyme was calculated using an absorption coefficient of 0.9 ml mg − 1 cm − 1 determined for rabbit muscle enolase.

2.7. Measurement of enolase acti6ity Enolase activity was determined spectrophotometrically measuring the absorption of phosphoenolpyruvate, a reaction product. The reaction mixture contained 0.05 M imidazole buffer, pH 6.8, 0.4 M KCl, 1 mM 2-PGA and 3 mM MgSO4 in a final volume of 1.5 ml. The reaction was monitored by measuring the changes in absorbance at 240 nm, at 30°C, for 1 min, after the addition of enzyme. In these conditions, an increase in absorbance by 0.2 corresponds to the conversion of 0.226 mmol of substrate. One unit of activity was defined as that which catalyzes the conversion of 1 mmol of the substrate in 1 min. Specific activity was expressed as activity units per 1 mg of protein.

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3. Results

3.1. Preparation of the enzyme A cell-free extract from C. albicans was subjected to the procedure described in Section 2. The DEAE-Sephadex adsorbed the enolase, which was subsequently eluted using a linear gradient of NaCl. Fractions with enolase activity displaying a clear, sharp peak outside of the main protein peak (Fig. 1) were collected. Fractions with high activity were pooled, concentrated, dialyzed and applied to CM-Sephadex at pH 6.2. A part of the protein was adsorbed, but enolase did not bind and was washed out by the buffer. The fractions with enolase activity formed a wide plateau (Fig. 2), but only the fractions outside the main protein peak contained electrophoretically homogenous protein (Fig. 3). This protein was the subject of further studies. The enzyme was purified 27-fold and had a specific activity of 35 U mg − 1 protein (Table 1). The enolase preparation was dialyzed against 0.05% MgSO4 and stored at 4°C. 4. Molecular properties of enolase from C. albicans

4.1. Determination of enolase subunit molecular weight The subunit molecular weight of enolase was determined to be 46.0 kDa using SDS-gel electrophoresis (Fig. 3).

4.2. Thermostability of enolase 2.8. Thermostability studies −1

Some 1 mg ml solution of enolase in 0.05% MgSO4 was incubated at 50°C. After certain periods aliquots of 0.5 nmol of protein were taken for enzymatic activity determination. A sample of protein with the same concentration incubated at room temperature served as a control.

After 1 h of incubation at 50°C, a 50% reduction of the enzyme activity was caused. After 2 h of incubation, the enzyme retained 30% of the initial activity. 5. Kinetic properties of enolase from C. albicans

5.1. Effect of pH on the enzyme acti6ity 2.9. Reagents Molecular weight standards were purchased from Serva, DEAE-Sephadex A-50 and CM-Sephadex C-50 were obtained from Pharmacia. All the other reagents were from Sigma and were of analytical grade.

The effects of pH on enolase activity was studied in the pH range from 6.0 to 8.4 in 0.05 M imidazole buffer containing 0.4 M KCl, 3 mM MgSO4 and 1 mM 2-PGA. The reaction was started by the addition of 0.05 nmol of the enzyme to 1.5 ml of reaction mixture.

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Crude extract 67% ammonium sulfate saturation supernatant 100% ammonium sulfate saturation pellet DEAE-Sephadex A-50 CM Sephadex C-50

Volume (ml)

Protein concentration (mg ml−1)

Total protein (mg)

30.0 70.0

15.96 0.78

558.60 260.40

4.8

13.76

7.0 5.0

0.76 0.24

Specific activity (Units mg−1)

Total activity (Units)

Yield %

Purification factor

1.31 2.11

731.80 549.40

100 75.1

1.00 1.61

66.03

2.33

153.80

21.0

1.78

5.56 1.20

8.55 35.31

47.52 42.37

6.5 5.8

6.53 27.00

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Table 1 Purification of enolase from C. albicans

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Fig. 1. DEAE-Sephadex A-50 chromatography. Elution profile. The column was equilibrated with 10 mM Tris – HCl buffer, pH 9.0 containing 5 mM MgSO4, 1 mM EDTA, 1 mM 2-mercaptoethanol. Open triangles, enolase activity. Open squares, protein concentration.

Fig. 2. CM-Sephadex C-50 chromatography. Elution profile. The column was equilibrated with 15 mM NaH2PO4/Na2HPO4 buffer, pH 6.2 containing 3 mM MgSO4. Open triangles, enolase activity. Open squares, protein concentration.

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2-PGA than for PEP. This means that, under our experimental conditions, the enzyme possessed a higher catalytic capability for dehydratation of 2-PGA to PEP. The kinetic parameters are presented in Table 2.

5.3. Inhibition of enolase acti6ity by fluoride ions The effect of fluoride ions on the kinetics of enolase from C. albicans was studied in the presence or absence of orthophosphate. We measured the initial rates of the reaction in the standard reaction mixture containing 0.5–3 mM 2-PGA, 0–80 mM fluoride and 0–30 mM phosphate. The inhibition of enolase from C. albicans by fluoride was competitive in the absence of phosphate (Fig. 5(a)), and noncompetitive in its presence (Fig. 5(b)). The Ki for fluoride ions, calculated using a graphic method of Dixon (Dixon and Webb, 1979), was 10 mM in the absence of phosphate.

5.4. Effect of bi6alent metal ions on enzymatic acti6ity of enolase from C. albicans Fig. 3. The determination of molecular weight of enolase subunit. Purified enolase from C. albicans. Molecular weight markers.

Enolase from C. albicans exhibited the highest activity at pH 6.8 (Fig. 4). The pH curve was not symmetrical. At the lower values of pH (pH be- low 6.5) the enzyme displayed a steep decrease in catalytic activity; above pH 7.0, the enzymatic activity remained almost constant at 50% of the maximal activity.

5.2. Michaelis–Menten constants for 2 -PGA and PEP Km and Vmax values were determined from measurements of initial rates of reaction using the method of Lineweaver – Burk. The standard reaction mixture contained 0.4 M KCl and 3 mM MgSO4 in 0.05 M imidazole buffer, pH 6.8, in a total volume of 1.5 ml. The 2-PGA concentrations varied from 0.3 to 1 mM; PEP concentrations from 0.5 to 3 mM. The reactions were initiated by the addition of 0.05 nmol of enzyme. Enolase from C. albicans displayed classical Michaelis–Menten kinetics for both substrates. The enzyme had a higher Vmax and lower Km for

The effect of bivalent metals-magnesium, zinc and manganese-on enolase was studied by measuring enzymatic activity in 0.05 M imidazole buffer, pH 6.8 containing 0.4 M KCl and 1 mM 2-PGA. The enzyme solution was previously dialyzed against deionized water; enzyme prepared this way did not express any catalytic activity. Concentrations of bivalent cations employed varied from 20 to 1800 mM. In the presence of Mg2 + , Vmax was 507 (mmol of substrate min − 1 mmols − 1 of enzyme) and was the highest among those obtained (Fig. 6). Mn2 + had a lower Vmax but also a lower Km than Mg2 + had; at lower concentrations, Mn2 + gave more activity than Mg2 + (Fig. 6). Maximal activity was observed at 80 mM Mn2 + , but at higher concentrations of Mn2 + a hyperbolic decrease in activity occurred. Zn2 + gave a still lower Vmax = 300 (mmol of substrate min − 1 mmols − 1 of enzyme); the optimal concentration of Zn2 + was 40 mM, and again, higher concentrations caused a steep hyperbolic decrease in activity. The kinetic data obtained are presented in Table 3. To examine mutual interactions of these cations and enolase in the presence of high (close to saturation) Mg2 + concentrations, increasing concentrations Mn2 + and Zn2 + were added to the

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Fig. 4. pH profile of enolase from C. albicans. The activity was determined in 0.05 M. imidazole buffer, containing 0.4 M. KCl, 3 mM MgSO4, 1 mM 2-PGA.

solution. Synergism between the metal ions was not observed. In the presence of Mg2 + added Mn2 + produced lower enzyme activities, to the level corresponding to the presence of Zn2 + alone (Fig. 7). We infer from these observations that the displacement of Mg2 + by Mn2 + and Zn2 + from the catalytic site is more a probable explanation than binding of these ions at an inhibitory site of the enzyme.

6. Discussion To date, isolation of enolase from two species of fungi have been described. The older, dating from the forties, describes the purification of the enzyme from S. cere6isiae (Warburg and Christian, 1941). The method has been modified many times. Westhead and McLain (1964) proposed a method, which consists of many stages, such as: toluene autolysis of the cells, acetone fractionation followed by ethanol precipitation. Lee and Nowak (1992b) reported a new method, which replaced the acetone and ethanol fractionation steps by milder procedures to avoid instability of the enzyme (Wedekind et al., 1994). One of the most recent methods of preparation of enolase

pertains to C. albicans cells and was published in 1994 (van Deventer et al., 1994). This method is simple but requires the use of an FPLC apparatus, which limits its use. Moreover, the yield and purification were similar as we report, and no information on homogeneity was presented. Our method is easy to conduct (four stages only), and uses commonly accessible reagents, reducing the cost of preparation of the enzyme. It provides electrophoretically homogenous protein. Enolases from mammalian muscle precipitate at 60–70% of ammonium sulfate saturation (Holt and Wold, 1961; Baranowski and Wolna, 1975; Lee and Nowak, 1992b)) from carp muscles at 80–90% (Pietkiewicz and Kustrzeba-Wo´jcicka, 1983). In isolation of enolase from vertebrate tissues, thermal denaturation of other proteins was applied at early stages. In the case of C. albicans, this step could be omitted. The final step Table 2 Kinetic parameters of enolase from C. albicans Substrate

Km (mM)

Vmax (mmol of substrate min−1 mmol−1 of enzyme)

2-PGA PEP

0.38 0.95

4200 910

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Fig. 5. (a) Determination of the mode for fluoride ions inhibition. Enolase activity was measured in 0.05 M imidazole buffer, pH 6.8 containing 0.4 M KCl and 3 mM MgSO4. 2-PGA concentration was changed in the range 0.5 – 3 mM, and the concentration of fluoride ions was 0 (closed squares), 40 (closed circles) or 80 mM (closed triangles). The reaction was started by the addition of 0.05 nmol of enzyme. (b) Determination of the mode for fluoride ions inhibition in the presence of orthophosphate ions. Enolase activity was measured in 0.05 M imidazole buffer, pH containing 0.4 M KCl and 3 mM MgSO4. 2-PGA concentration was changed in the range 0.5–3 mM, and the concentration of fluoride ions was 0 (closed squares), 10 (closed circles) or 30 mM (closed triangles). The concentration of PO34 − ions was 30 mM. The reaction was started by the addition of 0.05 nmol of enzyme.

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Fig. 6. Km determination for Mg2 + , Mn2 + , Zn2 + ions. Lineweaver-Burk plot. The activity was determined in 0.05 M imidazole buffer, pH 6.8 containing 0.4 M KCl and 1 mM 2-PGA. Ions concentration was varied in the range of 20 – 1200 mM. The reaction was started by the addition of 0.05 nmol of enzyme.

in preparation of enolase from carp muscle involved chromatography on QAE-Sephadex at pH 9.0. This step did not result in further purification of the enzyme from C. albicans. The subunit molecular weight of C. albicans enolase was 46 kDa, which is close to the subunit molecular weight of this enzyme reported previously (van Deventer et al., 1994). The structure of enolase, a key enzyme of the glycolytic pathway, did not change much in evolution, therefore enzymes isolated from various sources have similar subunit molecular weights: Escherichia coli Mw = 46 kDa (Wold and Ballou, 1957), Pyrococcus furiousus Mw = 45 kDa (Peak et al., 1994), S. cere6isiae Mw =46.691 kDa, rabbit muscle Mw = 41 kDa (Wold and Ballou, 1957), carp muscle

Mw = 49 kDa (Pietkiewicz and Kustrzeba-Wo´jcicka, 1983). Since enolase is a heat shock protein, it is not unusual that it has high thermostability. Similarly high thermostability characterizes yeast enolase and enolase from S. rattus. Enolases from higher organisms (carp and rabbit muscle, bovine brain) are less resistant to thermal denaturation (Wold and Ballou, 1957; Pietkiewicz and Kustrzeba-Wo´jcicka, 1983; Kustrzeba-Wo´jcicka et al., 1986; Nazarian et al., 1992). The pH optima of enolases from various organisms are similar. For the protein from C. albicans it is at pH 6.8 and is similar to those from carp muscle (pH 6.8) (Pietkiewicz and Kustrzeba-Wo´jcicka, 1983) and rabbit muscle (pH 7.0) (Wold,

Table 3 Influence of magnesium, manganese and zinc ions on kinetic parameters of enolase from C. albicans Ion

Optimal concentration (mM)

Vmax (mmol of substrate min−1 mmols of enzyme−1)

Km (mM)

Mg2+ Mn2+ Zn2+

0.10 0.04

507 394 300

0.286 0.057 0.015

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furiousus has a still higher pH optimum (8.1) (Peak et al., 1994). The pH profile of activity for enolases seems characteristic; activity decreases steeply for pH values below the pH optimum while for pH values above the optimum the activity decrease but not so sharply. The Km value for 2-PGA as a substrate for enolase from C. albicans is 0.38 mM. Almost identical Km values characterize enolases from P. furiousus (Km =0.4 mM) (Peak et al., 1994) and from carp (Km = 0.31 mM) (Pietkiewicz and Kustrzeba-Wo´jcicka, 1983). The Km value for yeast enolase is 0.12 mM (Wang and Himoe 1974). Enolase from S. rattus, an evolutionarily distant organism, has Km =4.35 mM (Hu¨ther et al., 1990) which is one order of magnitude higher than the others. All known enolases are inhibited by fluoride. For the protein from C. albicans, Ki equals 10 mM in the absence of phosphate. This value is rather high, compared to inhibition constants for the enzymes from other sources. For example Ki for the protein from S. rattus is 0.85 mM (Hu¨ther et al., 1990) and for carp muscle Ki =0.24 mM in

the absence of phosphate (Pietkiewicz and Kustrzeba-Wo´jcicka, 1983). The type of inhibition depends on whether phosphate is present. In the case of enolases from C. albicans and S. rattus in the absence of phosphate a competitive inhibition is displayed, but in the presence of milimolar phosphate concentrations the character of inhibition by fluoride changes to noncompetitive (Hu¨ther et al., 1990). This could indicate conformational changes of the enzyme due to the presence of PO34 − ions, but this hypothesis requires verification in further studies. Some bivalent metal ions are activators of enolase. Mg2 + is the strongest activator for all known enolases (Wang and Himoe, 1974; Pietkiewicz and Kustrzeba-Wo´jcicka, 1983; Lee and Nowak, 1992a,b). Vinarov and Nowak, 1998 kinetic studies of yeast enolase show that activation with Mg2 + is pH independent for the activator constant Ka but at higher concentrations of Mg2 + the pH dependent inhibition is observed. Other bivalent ions of metals activate the enzyme from various sources to different degrees. Mn2 + strongly activates the activity of enolase from C.

Fig. 7. The effect of bivalent cations on C. albicans enolase activity. The activity was determined in 0.05 M imidazole buffer, pH 6.8 containing 0.4 M KCl and 1 mM 2-PGA. Both the kind and concentration of metal ions were changed as indicated on the graph. The reaction was started by the addition of 0.05 nmol of enzyme.

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albicans and S. cere6isiae (Lee and Nowak, 1992a,b), but they are weaker activators for the enzyme from carp muscle. Zn2 + gives higher activities than Mn2 + in the case of carp enolases but the reverse is true for enolase from yeast and C. albicans (Pietkiewicz and Kustrzeba-Wo´jcicka, 1983; Lee and Nowak, 1992b). Transition metal ions Km values for enolase are much lower than Km values for Mg2 + (Wang and Himoe, 1974; Pietkiewicz and Kustrzeba-Wo´jcicka, 1983; Lee and Nowak, 1992a,b). Typically above the optimal concentrations of transition metal ions, a hyperbolic decline in the enzyme activity occurs (Lee and Nowak, 1992a,b). Synergism of various ions affecting the enzyme activity occurs only when the catalytic center of the enzyme has at least two sites binding metal cations and each of them plays a different role in catalytic process. We can speak of synergism when the activity of enzyme resulting from the action of two metals simultaneously is different from the activity resulting from the action of each of them separately or from the sum of activities. A lack of synergism between Mg2 + and Mn2 + , and Mg2 + and Zn2 + ions was observed in the case of enolase from C. albicans: Data obtained from the enzymatic reaction conducted in solutions containing a mixture of ions indicate a hyperbolic decline in protein catalytic activity to the level corresponding to the presence of only one of the ions Mn2 + or Zn2 + . This was observed earlier for enolase from S. cere6isiae. The fact that Mn2 + binds to the enzyme in a 1:1 molar ratio in the absence of substrate (Lee and Nowak, 1992a,b) suggests that only one of two binding sites directly participates in enzymatic catalysis or controls the rate limiting step. The effects of conducting enzymatic reaction in the presence of Mn2 + and Mg2 + ions, and Zn2 + and Mn2 + ions, indicate that the affinity of enolase for Zn2 + and Mn2 + ions is higher than for magnesium ions. This phenomenon causes displacement of Mg2 + ions from catalytic and conformational site of enolase (Lee and Nowak, 1992a,b).

Acknowledgements We thank Dr Urszula Nawrot from the Department of Microbiology, Medical University of Wrocl*aw, from whom we obtained the culture of C. albicans.

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References Baldo, B.A., 1995. Allergenic crossreactivity of fungi with emphasis on yeast: strategies for further study. Clin. Exp. Allergy 25, 488 – 492. Baldo, B.A., Baker, R.S., 1988. Inhalant allergies to fungi: reactions to baker’s yeast (Saccharomyces cere6isiae) and identification of baker’s yeast enolase as an important allergen. Int. Archs Allergy Appl. Immun. 86, 201 – 208. Baranowski, T., Wolna, E., 1975. Enolase from human muscle. In: Colowick, S.P., Kaplan, N.O. (Eds.), Methods in Enzymology, vol. XVLII. Academic Press, New York, pp. 335 – 338. Breitenbach, M., Simon, B., Probst, G., Oberkofler, H., Ferreira, F., Briza, P., Achatz, G., Unger, A., Ebner, C., Kraft, D., Hirschwehr, R., 1997. Enolase are highly conserved fungal allergens. Int. Arch. Allergy Immunol. 113, 114 – 117. Brewer, J.M., Glover, C.V.C., Holland, M.J., Lebioda, L., 1997. Effect of site-directed mutagenesis of His 373 of yeast enolase on some of its physical and enzymatic properties. Biochem. Biophys. Acta 1340, 88 – 96. Brewer, J.M., Glover, C.V.C., Holland, M.J., Lebioda, L., 1998. Significance of the enzymatic properties of yeast S39A enolase to the catalytic mechanism. Biochem. Biophys. Acta 1383, 351 – 355. van Deventer, A.J.M., van Vliet, H.J.A., Hop, W.C.J., Goessens, W.H.F., 1994. Diagnostic value of antiCandida enolase antibodies. J. Clin. Microbiol. 32, 17 – 23. Dixon, M., Webb, E.C., 1979. Enzymes. Longman, London, pp. 327 – 330. Franklyn, K.M., Warmington, J.R., Ott, A.K., Ashman, R.B., 1990. An immunodominant antigen of Candida albicans shows homology to the enzyme enolase. Immunol. Cell Biol. 68, 173 – 178. Hilal, S.H., Brewer, J.M., Lebioda, L., Carreira, L.A., 1995. Calculated effects of the chemical environment of 2-Phospho-D-Glycerate on the pKa of its carbon2 and correlations with the proposed mechanism of action of enolase. Biochem. Biophys. Res. Comm. 211, 607 – 613. Holt, A., Wold, F., 1961. Enolase from rabbit muscle. J. Biol. Chem. 236, 3227 – 3236. Hu¨ther, F.J., Psarros, N., Duschner, H., 1990. Isolation, characterization and inhibition kinetics of enolase from Streptococcus rattus FA-1. Infect. Immun. 58, 1043 – 1047. Kornblatt, M.J., 1996. The mechanism of rabbit muscle enolase: identification of the rate-limiting step and the site of Li+ inhibition. Arch. Biochem. Biophys. 330, 12 – 18. Kornblatt, M.J., Lange, R., Balny, C., 1998. Can monomers of yeast enolase have enzymatic activity? Eur. J. Biochem. 251, 775 – 780.

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Kustrzeba-Wo´jcicka, I., Pietkiewicz, J., Wolna, E., 1986. Studies on immunological properties of enolase from carp muscles after chemical modification of some amino-acid residues. Arch. Immunol. Ther. Exp. 34, 93–99. Laemmli, U.K., 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685. Lebioda, L., Stec, B., 1988. Crystal structure of enolase indicates that enolase and pyruvate kinase evolved from a common ancestor. Nature 333, 683–686. Lebioda, L., Stec, B., 1991. Mechanism of Enolase: The crystal structure of enolase-Mg2 + -2-phosphoglycerate/phosphoenolopyruvate complex at 2.2-A, resolution. Biochemistry 30, 2817–2822. Lebioda, L., Stec, B., Brewer, J.M., 1989. The structure of yeast enolase at 2.25-A, resolution, an 8Fold b +a-barrel with a novel bbaa(ba)6 topology. J. Biol. Chem. 264, 3685–3693. Lee, B.H., Nowak, T., 1992a. Influence of pH on the Mn2 + activation of the binding to yeast enolase: a functional study. Biochemistry 31, 2165–2171. Lee, M.E., Nowak, T., 1992b. Metal ion specificity at the catalytic site of yeast enolase. Biochemistry 31, 2172–2180. Nazarian, K., Wajs, M., Egorian, R.U., KustrzebaWo´jcicka, I., Pietkiewicz, J., 1992. Regulation of activity of neuron-specific enolase by endogenous compounds. Biokhimia 57, 1827–1833. Nittner-Marszalska, M., Bogacka, E., Kustrzeba-Wo´jcicka, I., Patkowski, J., 1997. Enolase cutaneous test in fungal inhallator allergy. Med. Mycol. 4, 25. Peak, M.J., Peak, J.G., Stevens, F.J., Blamey, J., Mai, X., Zhou, H., Adams, M.W.W., 1994. The hyperthermophilic glycolytic enzyme enolase in the archaeon, Pyrococcus furiousus: Comparison with mesophilic enolase. Arch. Biochem. Biophys. 313, 280–286. Pietkiewicz, J., Kustrzeba-Wo´jcicka, I., 1983. Purification and properties of enolase from carp. Comparison with enolase from mammals muscles and yeast. Comp. Biochem. Physiol. C 75B, 693–698. Stec, B., Lebioda, L., 1990. Refined structure of yeast

.

apo-enolase at 2.25 A, resolution. J. Mol. Biol. 211, 235 – 248. Sundstrom, P., Aliaga, G.R., 1992. Molecular cloning of cDNA and analysis of protein secondary structure of Candida albicans enolase, an abundant, immunodominant glycolytic enzyme. J. Bacteriol. 174, 6789 – 6799. Sundstrom, P., Aliaga, G.R., 1994. A subset of proteins found in culture supernatants of Candida albicans includes the abundant, immunodominant, glycolytic enzyme enolase. J. Infect. Dis. 169, 452 – 456. Vinarov, D.A., Nowak, T., 1998. PH dependence of the reaction catalysed by yeast Mg-enolase. Biochemistry 37, 15238 – 15246. Wang, T., Himoe, A., 1974. Kinetics of the rabbit muscle enolase-catalyzed dehydration of 2-phosphoglycerate. J. Biol. Chem. 249, 3895 – 3902. Warburg, O., Christian, W., 1941. Isolierung und Kristallisation des Ga¨rungsferments Enolase. Biochem. Z. 310, 384 – 421. Wedekind, J.E., Poyner, R.R., Reed, G.H., Rayment, I., 1994. Chelation of serine 39 to Mg2 + latches a gate at the active site of enolase: structure of the bis(Mg2 + ) complex of yeast enolase and the intermediate analog phosphonoacetohydroxamate at 2.1-A, resolution. Biochemistry 33, 9333 – 9342. Westhead, E.W., McLain, G., 1964. A purification of brewers’ and baker’s yeast enolase yielding a single active component. J. Biol. Chem. 239, 2464 – 2468. Wold, F., 1971. Enolase. In: Boyer, P.D. (Ed.), The Enzymes, vol. 5. Academic Press, New York, pp. 499 – 538. Wold, F., Ballou, C.E., 1957. Studies on the enolase. J. Biol. Chem. 227, 301 – 312. Zhang, E., Hatada, M., Brewer, J.M., Lebioda, L., 1994. Catalytic metal ion binding in enolase: the crystal structure of an enolase-Mn2 + -phosphonoacetohydroxamate complex at 2.4 A, resolution. Biochemistry 33, 6295 – 6300. Zhang, E., Brewer, J.M., Minor, W., Carreira, L.A., Lebioda, L., 1997. Mechanism of enolase: the crystal structure of asymmetric dimer enolase-2-phospho-D-glycerate/enolase-phosphoenolopyruvate at 2.0 A, resolution. Biochemistry 36, 12526 – 12534.