Characterization of the enolase isozymes of rabbit brain: kinetic differences between mammalian and yeast enolases

The Enzyme Research Group, Departments of Chemistry and Biology, Concordia University, Montreal, QuP., Canada H3G 1M8

Received July 12, 1988

KORNBLATT, M. J., and KLUGERMAN, A. 1989. Characterization of the enolase isozymes of rabbit brain: kinetic differences between mammalian and yeast enolases. Biochem. Cell Biol. 67: 103-107.

Two isozymes of enolase, aa and yy, have been purified from rabbit brain and characterized. The kinetic properties of aa and ry (pH optimum, K,,, for phosphoglycerate and phosphoenolpyruvate, requirement for a divalent cation) are very similar to those of rabbit enolase, form 00, and to those of enolase isozymes 'rom other species. However, several novel properties were observed. (i) All the enolases studied were inhibited by Na' and Li'. (ii) The rabbit enolases, but not yeast enolase, were activated by Kf , NH,', Cs', and Rb'. (iii) Rabbit enolase is more susceptible to inhibition by excess than is the yeast enolase; the increased inhibition by M~~~ above pH 7.1 accounts, at least in part, for the observed differences between mammalian and yeast enolases in their pH optima for activity.

Key words: enolase, isozymes, kinetics.

KORNBLATT, M. J., et KLUGERMAN, A. 1989. Characterization of the enolase isozymes of rabbit brain: kinetic differences between mammalian and yeast enolases. Biochem. Cell Biol. 67 : 103-107.

Nous avons purifik et caracterisi deux isozymes de l'enolase, aa et yy, de cerveau de lapin. Les propriktts cinetiques des isozymes aa et yy (pH optimum, K,,, pour le phosphoglyckrate et le phosphoCnolpyruvate, besoin d'un cation divalent) ressemblent beaucoup a celles de l'isozyme pp de l'enolase de lapin et a celles des isozymes de I'enolase d'autres espkces. Cependant, nous avons observe plusieurs propriktts nouvelles. (i) Toutes les enolases Ctudiees sont inhibees par Na+ et Lif . (ii) Les enolases du lapin, mais non l'enolase de levure, sont activees par K+, NH,', Cs+ et Rbf . (iii) L'knolase de lapin est lus susceptible a I'inhibition par un excks de que ne I'est l'enolase de levure; l'inhibition accrue par le M S f A un pH au-dessus de 7.1 explique, du moins partiellement, les differences observees entre les enolases mammaliennes et l'enolase de levure pour ce qui est du pH optimum de leur activitt.

Mots clPs : enolase, isozymes, proprietes cinetiques. [Traduit par la revue]

Introduction Enolase (EC 4.2.1.1 I), an enzyme of both glycolysis and

gluconeogenesis, catalyzes the interconversion of PGA and PEP. Enolases from a number of organisms have been purified and shown to have very similar kinetic properties (Wold 1971 ; Suzuki et al. 1980; Keller et al. 198 1 ; Miernyk and Dennis 1984). Most are dimers, with subunit molecular weights of 40 000 - 50 000 (Wold 1971; Suzuki et al. 1980; Keller et al. 1981; Miernyk and Dennis 1984); the octomeric enolases from thermophilic bacteria are the exception (Veronese et al. 1984). Mammals have three genes for enolase; their protein products are the a, P, and y subunits. Both homo- and hetero-dimers are formed with five of the six possible dimers reported to exist in vivo (Rider and Taylor 1974; Fletcher et al. 1976). The mammalian isozymes have very similar amino acid content; the sequences of the aa and yy forms present in rat brain are 82% identical (Sakimura et al. 1985). However, some differences in physical properties, such as susceptibility to inactivation by salt, pressure, and temperature, have been reported (Keller et al. 1981; Kornblatt et al. 1982; Marangos et al. 1978).

We are interested in using the enolase isozymes as a system in which to study structure-function relationships. Since rabbit PP, the isozyme present in muscle, has been purified,

ABBREVIATIONS: TMA, tetramethylammonium ion; PGA, 2-phosphoglycerate; PEP, phosphoenolpyruvate; M f , mono- valent cation.

' ~ u t h o r to whom correspondence should be addressed. Present address: Department of Chemistry, Concordia University, 1455 de Maisonneuve Boulevard West, Montreal, Que., Canada H3G 1M8.

extensively studied (Wold 1971; Stubbe and Abeles 1980; Shen and Westhead 1973; Dinovo and Boyer 1971; Spring and Wold 1971), and is available commercially, we decided to purify the other two homodimers, aa and yy, from rabbit. We report here their characterization and describe several novel features of their kinetic properties.

Materials and methods Assays

The standard assay used during purification and characterization contained 50 mM imidazole, pH 7.1, 1 mM magnesium acetate, 250 mM KCl, 0.1 mM EDTA, 1 mM 2-phosphoglycerate, and enzyme. Addition of salts and substrate did not change the pH. Unless otherwise stated, enolase activity was measured at 25OC by following the production of PEP at 240 nm ( c = 1.33 mM-' cm-I). Since the extinction coefficient of PEP varies with pH and the concentrations of KC1 and magnesium ions (Wold and Ballou 1957), the results of pH studies were analyzed using values of c determined in the buffers used. All assays were performed in duplicate.

For studies of activation and inhibition by divalent cations, 100 mM borate, pH 8.0, was used because of the observation (Elliot and Brewer 1980) that Tris and imidazole compete with the enzyme for binding of some divalent cations. The concentrations of KC1 and substrate were the same as in the standard assay but the EDTA and M~~~ were omitted; the M ~ " was replaced by varying con- centrations of other metal ions. For studies in which the inhibitory effects of divalent cations were examined, the enzyme was assayed in the presence of 1 mM and a second ion.

Other methods The concentration of protein was measured by a dye binding

assay (Bramhall et al. 1969). PGA was prepared enzymatically according to Shen and Westhead (1973), separated from PEP by

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104 BIOCHEM. CELL BIOL. VOL. 67, 1989

FIG. 1. SDS-PAGE of various enolases. The electrophoresis was performed on 10% Laemmli gels and the gel stained with Coornassie blue. Lane 2 contains acu; lane 3, acu and y y ; lane 4, y y ; lane 5, Po; lane 6 , yeast enolase. Lanes 1 and 7 contain, top to bottom, phosphorylase b, bovine serum albumin, and ovalbumin.

TABLE 1. Kinetic properties of enolases

(Y (Y rr PP Yeast

KmU PGA, x M 5.7k0.4 5 .8 t0 .3 8b 1 5 ~ PEP, x M 51_t10 4520.7 -

Optimum [ M ~ ~ ' ] , mM 1 1 l b l b VH/VD 2.0 1.7 1.7 1.8

NOTE: Unless otherwise stated, properties were determined in the following con- ditions: 50 mM imidazole, pH 7.1, 250 mM KCI, 1 mM Mg2+, and varying [PGA] ( K , and V,, for PGA); 1 mM PGA and varying [ M ~ ~ ' ] (optimum concentration of0M$+); 1 mM M~ '+ and 1 mM PGA (v,/v,).

Average + standard deviation of 2-5 determinations. %om Wold (1971).

chromatography on Dowex 1x2, and titrated to pH 7.0 with TMA hydroxide. When measuring the kinetic isotope effect, all assays (with H-PGA and D-PGA) contained 8 mM tetramethylam- rnonium chloride.

The molecular weight of native enzyme was determined in triplicate by gel filtration on Sephadex G-150 SF; cytochrome c, chymotrypsinogen A, ovalbumin, bovine albumin, and alcohol dehydrogenase were used as markers. Subunit molecular weights were determined in duplicate by SDS-PAGE on 10% Laemmli gels (Laemmli 1970) using trypsinogen, pepsin, ovalbumin, bovine albumin, and phosphorylase b as molecular weight markers.

Purification Enolase, forms cra and y y , was purified from frozen rabbit

brains (Pel-Freeze, U.S.A.); the initial steps of the purification are modified from the procedure of Suzuki et al. (1980) for rat brain enolases. Following separation of the isozymes by ion- exchange on DEAE-Sephacel, acr was further purified on R-Sepharose and y y on Q-Sepharose, resulting in a further 5- to 6-fold purification for each of the isozymes. Purified enzyme was dialyzed against buffer containing 7.5 mM imidazole, pH 7.4, 50 mM NaC1, 2.5 mM magnesium acetate, 0.05 mM EDTA, and

FIG. 2. Activity versus pH for rabbit brain enolases. Activities for each isozyme are expressed relative to the activity at the pH optimum. All assays contained imidazole, M ~ ~ + , KCl, and EDTA, as in the standard assay.

50% glycerol and stored at - 20°C. The purified aa and -yy show no loss of activity for at least 2 years.

Chemicals The trisodium salt of PGA, the tricyclohexylarnmonium salt of

PEP, and rabbit muscle enolase were purchased from Boehringer Mannheim (West Germany). Yeast enolase was purchased from Sigma (U.S.A.), molecular weight standards for gel filtration and SDS-PAGE, from Sigma or Boehringer Mannheim, and all chromatography resins, from Pharmacia (Sweden). "Puriss" grade KCl, NaCl, and LiCl were purchased from Fluka Chemie (Switzerland), TMA hydroxide (99.5% pure) from Alfa Products (U.S.A.), and "100%" deuterium oxide from MSD Isotopes (Canada).

Results Purification

Two isozymes of enolase, cra and y y , were purified from rabbit brains. About 6 mg of yy and 3 mg of aa were obtained from 100 g of frozen brain, with specific activities of 107 units/mg. The yield of enzyme and the specific activities are comparable to Suzuki's results for rat brain enolases (Suzuki et al. 1980). Both cra and yy were at least 90% pure, as judged by SDS-PAGE.

Molecular weights Molecular weights of native enzyme were determined by

gel filtration. The aa and y y isozymes had identical elution volumes, corresponding to a molecular weight of 91 000. Subunit molecular weights, as determined by SDS-PAGE, were 53 000 for the a subunit and 51 000 for y. As can be seen in Fig. 1, the a (lanes 2 and 3) and y (lanes 3 and 4) monomers have slightly different mobilities in a 10% gel and are slightly larger than rabbit /3 monomer (lane 5); the monomer of yeast enolase is also shown (lane 6, molecular weight, 46 500; Chin et al. 1981).

Kinetic characterization Preliminary experiments had shown that the rabbit

enolase isozymes were stimulated by KCI. Therefore, with the exception of experiments designed to study the effects of monovalent cations on enzymatic activity, all assays were

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KORNBLATT AND KLUGERMAN

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[SALT] (MI FIG. 3. Effects of KC1 and NaCL on the activity of various enolases. All assays contained imidazole, pH 7.1. EDTA, and ~ g " , as

in the standard assay, and varying concentrations of either KC1 (solid symbols) or NaCl (open symbols). Activity is expressed as the percent of activity in the absence of added salt. (A) Rabbit enolases: yy (0, 0) ; aa! (A, A); 00 (0, m). (B) Yeast enolase.

performed in 0.25 M KCl. The kinetic properties of the strength produces a slight stimulation as shown by the effects isozymes are given in Table 1; the activity vs. pH profiles of TMA ion; with further increases in concentration, for LYLY and yy are shown in Fig. 2. The four enolases exhibit enzymatic activity is decreased. very similar properties as regards K,,, for PGA and PEP, All ions were tested as the C1- salts; the differences in stimulation by magnesium ions, and the kinetic isotope the magnitude and direction (increased or decreased activity) effect. The rabbit brain isozymes have a pH optimum of of the effects produced by the various salts is evidence that 6.7-7.1, which is lower than that of yeast and other plant the results shown in Fig. 4 are due to M+ and not due to enolases (Wold 197 1). C1- or the increased ionic strength. In addition, in the con-

The ability of other divalent ions to replace magnesium centration range of 50 to 500 mM, the inhibition produced in the assay was also studied (data not shown). For the by sodium acetate was comparable to that produced by NaCl mammalian enolases, little or no stimulation is given by (data not shown). ~ i ~ ' and ca2+ , while c u 2 + , ~ n ~ ' , zn2+, and co2' a11 Figure 3B shows the effects of Na+ and K+ on the stimulate to varying extents. When added to assays contain- activity of yeast enolase. In addition to Na', Lif is also ing 1 mM ~ g ~ + , all of the above divalent ions inhibited the inhibitory. No stimulation was observed by K + (Fig. 3B). activity of enolase, with concentrations less than or equal Stimulation by Cs+ (100% of control at 0.12 M) and Rb+ to 10 pM producing 50% inhibition. All four enolases had (1 15% of control at 0.12 M) was not different from that similar specificities for stimulation and inhibition by divalent produced by TMA ion (1 12% at 0.12 M). metal ions.

Inhibition by M ~ Z + Effects of monovalent cations The activity of all enolases is inhibited by excess ~ g ~ + .

A striking difference between mammalian and yeast During a study of the effects of pH on the activity of the enolases was observed when we studied the effects of mammalian enolases we found that, under some conditions, monovalent cations on enzymatic activity. All three rabbit rabbit brain enolase is far more susceptible to this inhibition homodimers are stimulated by K+ and inhibited by Na' than is yeast enolase and that the degree of inhibition is a (Fig. 3A). A more thorough study of the specificity for function of pH. Figure 5 shows the activity of rabbit brain monovalent cations was performed using yy (Fig. 4). Li' enolase, form yy (Fig. 5A) and of yeast enolase (Fig. 5B) is an effective inhibitor, producing 50% inhibition at as a function of [ M ~ ~ ' ] at pH 6.2, 7.1, and 7.8. For yeast 25 mM. In addition to NH4+ and K+ (Fig. 4), Cs+ and enolase, no inhibition by M ~ ~ + is observed up to 2 mM Rb+ also stimulate yy, producing maximum activities of (2 mM point not shown). Rabbit enolase, at pH 7.8, shows 155 (0.20 M CsCl) and 195% (0.16 M RbCl). Increased ionic substantial inhibition by M ~ ~ + concentrations greater than

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106 BIOCHEM. CELL BI OL. VOL. 67, 1989

IMA' NH--

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FIG. 4. Effects of monovalent cations on the activity of rabbit enolase, form yy. All assays contained imidazole, pH 7.1, EDTA, and M ~ ~ + as in the standard assay plus varying concentrations of the chloride salts of the various cations. Data are expressed relative to the activity in the absence of any added salt. The data for NaCl are the same as in Fig. 3.

0.2 mM and, although less noticeable in the figure, Mg2+ concentrations of 1 mM are inhibitory at pH 7.1. By com- paring the relative activities at pH 7.1 and 7.8 at low (0.1 mM) and high (1 mM) Mg2+, it can be seen that part of the reason for the pH optimum of 7 for the mammalian enolase is the inhibition of activity by Mg2' at higher pH. It is also evident that part of the decrease in activity below pH 7, observed with both yeast and rabbit enolases, is due to an increased Km for Mg2+ as the pH is decreased.

Discussion The two enolase homodimers present in rabbit brain are

very similar to each other and to other enolases with respect to physical and catalytic properties. They have molecular weights of about 90 000. There is an absolute requirement for activity for a divalent metal ion, with ~ g ~ ' being the most effective; high concentrations of divalent metal ions inhibit the catalytic activity. The pH optimum of 7 is similar to that observed for other animal enolases (Wold 1971). Homodimers aa and yy have identical Km values for phosphoglycerate and for phosphoenolpyruvate; the K, for phosphoglycerate is in the range observed for other enolases and is independent of pH, at least in the range of pH 6-7 (data not shown).

The mammalian enolases have a slightly lower pH

Frc. 5. Activity versus for (A) rabbit enolase, form yy, and (B) yeast enolase. All assays contained 50 mM imidazole and 250 mM KCI: pH 6.23 (0); pH 7.14 (0); pH 7.8 (A).

t; >- 80- I- U a

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optimum for activity than does the yeast enzyme (pH 6.7-7.1 vs. pH 7.7). Above pH 7.1, the activity of yr decreases, at least in part, owing to a decrease in the concentration of M ~ ' + required to produce measureable inhibition of activ- ity. Thus the difference in pH optimum between yeast and mammalian enolases is due to a difference in their suscep- tibility to inhibition by M$+. These experiments were only performed with yy; however, because of the similar pH optima for a a , 00, and yy, we assume that this inhibition occurs with all rabbit enolases. Although inhibition by excess Mg2+ or other divalent cations has been observed with all enolases, its structural basis is not well understood. The pH dependence of the binding of divalent cations to yeast enolase has been previously described and implicates a group with a pK, of 6-7 (Malmstrom and Westlund 1956). It now appears that, in rabbit enolase, there is also a group with a pK, above 6 that is involved in producing inhibition by excess M$+ .

Monovalent cations also affect the activity of the enolases. All were inhibited by Li+ and Na+; the mammalian enolases, but not the yeast enzyme, were activated by K f and the other monovalent cations studied. The data suggest a correlation between the size of the ion and its effects on activity. NH4+ gives maximum stimulation; stimulation decreases as the ions become larger (Rb', Cs') or smaller (K'). Still smaller ions (Na', Li') inhibit the activity with the smallest ion being the best inhibitor. If both stimulating and inhibiting monovalent cations are binding to the same site on the enzyme, then the lack of stimulation of yeast enolase would indicate either that K + and other

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KORNBLATT AND KLUGERMAN 107

stimulatory ions bind but have n o effects o n activity or that the M + binding site of yeast enolase is more restricted in size than is the comparable site of rabbit enolase. Another possibility is that the mammalian enolases contain two different sites for M + while yeast enolases have only the inhibitory site.

Pyruvate kinase uses P E P as substrate and requires both a monovalent and a divalent cation for activity (Nowak and Mildvan 1972). NMR studies have suggested that the P E P is coordinated to the enzyme-bound K + via its carboxyl group (Nowak and Mildvan 1972). Suelter (1970) has proposed that monovalent cation activation is a general feature of enzvme reactions in which keto-en01 tautomeriza- tion occurs. Although enolase does not have a n absolute requirement for a monovalent cation, it is possible that the K + plays a similar role in pyruvate kinase and in enolase and assists in the enolization step. Studies on the mechanism of inhibition and activation by Mf are currently under way in our laboratory.

This study has shown that the enolase homodimers present in rabbit brain, aa and yy, are virtually identical in catalytic properties t o the muscle isozyme. The rabbit isozymes, in turn, are quite similar t o the yeast enzyme. This study has also shown, however, that there are some differences between the rabbit and yeast enzymes: p H dependence of M ~ ~ + inhibition, and the effects of monovalent cations. Thus, although the overall catalytic mechanism is probably the same for mammalian and yeast enzymes, there are differences in the fine details and hence, presumably, in the detailed structure of the active site.

Acknowledgement This work was supported by a grant from the Natural

Science and Engineering Research Council of Canada.

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