- Email: [email protected]
The H159A Mutant of Yeast Enolase 1 Has Significant Activity
Biochemical and Biophysical Research Communications 276, 1199 –1202 (2000) doi:10.1006/bbrc.2000.3618, available online at http://www.idealibrary.com on
The H159A Mutant of Yeast Enolase 1 Has Significant Activity John M. Brewer,* ,1 Michael J. Holland,† and Lukasz Lebioda‡ *Department of Biochemistry and Molecular Biology, University of Georgia, Athens, Georgia 30602; †Department of Biological Chemistry, University of California Medical School at Davis, Davis, California 95683; ‡Department of Chemistry and Biochemistry, University of South Carolina, Columbia, South Carolina 29208
Received August 10, 2000
The function of His159 in the enolase mechanism is disputed. Recently, Vinarov and Nowak (Biochemistry (1999) 38, 12138 –12149) prepared the H159A mutant of yeast enolase 1 and expressed this in Escherichia coli. They reported minimal (ca. 0.01% of the native value) activity, though the protein appeared to be correctly folded, according to its CD spectrum, tryptophan fluorescence, and binding of metal ion and substrate. We prepared H159A enolase using a multicopy plasmid and expressed the enzyme in yeast. Our preparations of H159A enolase have 0.2– 0.4% of the native activity under standard assay conditions and are further activated by Mg 2ⴙ concentrations above 1 mM to 1–1.5% of the native activity. Native enolase 1 (and enolase 2) are inhibited by such Mg 2ⴙ concentrations. It is possible that His159 is necessary for correct folding of the enzyme and that expression in E. coli leads to largely misfolded protein. © 2000 Academic Press Key Words: enolase; expression; folding; cloning; mechanism; mutagenesis.
alogue (2-phosphoglycolate), to be the catalytic base abstracting the proton from carbon-2 of the substrate (9). In contrast, 31P-NMR measurements and the X-ray structure of the yeast enzyme complexed with substrate/product indicated that His159 initiates the catalytic cycle by protonating the phosphate of the substrate (8, 10). Recently, Vinarov and Nowak (11) prepared the H159A mutant of yeast enolase 1, expressing this in E. coli. Their preparation had ca. 0.01% of native enolase activity, supporting a function of His159 as a catalytic base. We, however, express our yeast enolase 1 mutants in the native host organism, yeast, and have routinely obtained much higher levels of activity in His159 mutants (12). Because so many workers use cloning, usually in E. coli, to overexpress proteins from other organisms, often coupling this with mutagenesis, we extended our investigation of the source of this discrepancy. MATERIALS AND METHODS
Enolase (2-phosphoglycerate hydrolyase, E.C.4.2.1.11) catalyzes the interconversion of 2-phosphoglycerate and phosphoenolpyruvate. Enolase from yeast, like most enolases, is dimeric (1). Each subunit can bind one equivalent of divalent cation at what we refer to as a “conformational” site (2). This enables substrate or analogues to bind (3); when substrate or analogues are bound, a second equivalent of divalent cation can bind to what we call the “catalytic” site, producing catalysis (2, 4). Mg2⫹ is the physiological activating cation (1, 2). During catalysis by enolase, three polypeptide loops move (5– 8). Two loops move together, one of which includes His159 (His157 in lobster enolase) (9). This residue was suggested, on the basis of the X-ray structure of lobster enolase complexed with a substrate an1
To whom correspondence should be addressed. Fax: (706) 5421738. E-mail: [email protected].
Two enolase isozymes, called 1 and 2, exist in yeast; the organism can grow with either alone (13). We prepare enolase mutants by mutating a multicopy plasmid containing the enolase 1 sequence and a promoter region, then transforming a strain of yeast whose chromosomal enolase 1 gene is largely deleted, so that it grows using the enolase 2 isozyme only (14). Before transforming the yeast strain used for expression, the protein coding sequence was determined, and only the appropriate codon was altered and altered as intended (CAC to GCC). H159A enolase was isolated as described (14 –16), using chromatography on Q Sepharose FF at pH 8.5 and CM Sepharose at pH 6.0 (17), using a Pharmacia-LKB LCC 500 Plus FPLC. Although enolase 2 elutes from Q Sepharose at a much higher ionic strength than enolase 1 (13) and its mutants, we find the enolase 1 mutants are contaminated with small amounts of native enolase 2. Such contamination would affect enzymatic properties such as relative activities but would not significantly affect calorimetric or difference spectral measurements. Contamination of enolase 1 mutants with enolase 2 varies inversely with the net expression of the mutant, in terms of enzymatic activity (16). The net expression of the H159 mutants was low (we obtained no H159Q enolase) in comparison to, for example, the E168Q and E211Q mutants (14, 15). We have found rechromatography of enolase 1 mutants on a 1 ⫻ 5 cm
1199
0006-291X/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.
Vol. 276, No. 3, 2000
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
MonoQ column using the pH 8.5 solvent system employed in the Q Sepharose chromatography to be effective in reducing enolase 2 levels: rechromatographed S39A enolase has a relative activity of 10 ⫺4 of native enolase (16). Native enolase 2 itself was prepared from the untransformed yeast strain mentioned above using the same chromatographic steps described above, together with chromatography on an S-200 Superdex column at pH 7.8 (14). Our preparations of H159A enolase appeared homogeneous on SDS-gels (not shown). The substrate analogues D-tartronate semialdehyde phosphate (TSP) was synthesized as described by Spring and Wold (18); phosphonoacetohydroxamate was synthesized as described by Anderson et al. (19). The substrate, 2-phosphoglycerate, was from Sigma (St. Louis, MO). Differential scanning calorimetry (DSC) measurements were performed using an instrument from Calorimetry Sciences Corp. (Provo, UT). Measurements of absorbance at single wavelengths were done using a Spectronic 601 spectrophoto-meter (Milton Roy). Difference spectral measurements were performed with a Hewlett-Packard 8452A Diode Array Spectrophotometer controlled by a Standard Turbo 10 computer using Specos 3.71 software. Measurements of pH values were done using an Orion 701A digital lonalyzer with a single probe microelectrode. Enzyme activities were measured at ambient temperature (20 –22°) as described by Westhead (20).
RESULTS As in our previous studies of mutant enolases (14 – 16, 20), differential scanning calorimetric (DSC) analyses of the effect of substrate and a strongly bound (19) competitive inhibitor, phosphonoacetohydroxamate, on the thermal denaturation of mutant enolases showed that our preparation of H159A enolase binds both compounds and is stabilized by them (not shown). With 2 mM Mg 2⫹ present, substrate increases the temperature of maximum excess heat capacity (T max) by 5° and the inhibitor stabilizes the H159A mutant by 21°. With native enolase, substrate and analogue increase T max values by 6° and 22°, respectively (16). All the H159A enolase appeared to bind, and be stabilized by, the substrate and inhibitor, hence much if not all the protein is correctly folded. The H159A enolase fractions from the CM columns routinely exhibited specific activities under standard assay conditions (0.05 ionic strength Tris–HCl, pH 7.8, 1 mM MgCl 2, 10 ⫺5M EDTA, 1 mM 2-phospho-Dglycerate) (20) of 1–3 units (⌬OD 230/min/OD 280) (20) which increased with increasing fraction number. These preparations after pooling and lyophilization were neither stimulated nor inhibited by Mg 2⫹ concentrations above 1 mM. Since traces of any enolase 2 present would strongly influence the activity, we rechromatographed H159A enolase preparations on the monoQ column. This treatment did not reduce the specific activity as dramatically as was seen with S39A enolase (16): specific activities of 1–2 units and, in two preparations examined, the specific activities of the column fractions were constant across the peak of OD 280 due to H159A enolase (not shown). However, the rechromatographed H159A enolase activity is strongly stimulated by Mg 2⫹ concentrations
FIG. 1. Dependence of specific activities of H159A and native enolases 1 and 2 on magnesium ion concentrations. Enolase assay medium, 0.58 –1.0 ml (0.05 ionic strength Tris–HCl, pH 7.8, 1 mM MgCl 2, 1 mM 2-phosphoglycerate, and 1 ⫺5M EDTA), was supplemented with microliter amounts of 0.5 M MgCl 2, to produce the final concentrations shown. H159A enolase (two preparations), 3– 6 g, or 0.24 g enolase 1 or 0.38 g enolase 2, were added to these solutions, mixed, and the rate of increase of absorbance at 230 nm was determined. The filled symbols are using H159A enolase, the crosses enolase 1, and the open circles enolase 2. The H159A enolase, enolase 1, and enolase 2 were rechromatographed on a MonoQ column before these measurements. Specific activities are ⌬OD 230/min/OD 280 (of enzyme in the assay solution), as described by Westhead (20).
above 1 mM (Fig. 1). Both native enolases 1 (4, 22) and 2 are inhibited by Mg 2⫹ concentrations above 1 mM. This is not the result of the rechromatography per se, as the data shown were obtained with native enolase 1 and 2 which were rechromatographed on the MonoQ column as well. The rechromatography did not affect the inhibition of the native enolase 1 or 2 by Mg 2⫹ (not shown). The figure shows results from two samples of rechromatographed H159A enolase, measured on different days. The higher activity of one of the samples may be owing to residual enolase 2, as the stimulation of activity by Mg 2⫹ of that sample seems less also. The activities of these and other rechromatographed preparations of H159A enolase varied from 0.9 –1.9 units (⌬OD 230/min/OD 280) measured under standard assay
1200
Vol. 276, No. 3, 2000
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
FIG. 2. Reactions of H159A and native enolase 1 with TSP. Spectra of solutions (1 ml) containing 20 M native or unrechromatographed H159A enolase in 0.1 ionic strength Tris–HCl, pH 7.8 and 1 or 10 mM MgCl 2 were obtained (4 scans averaged) then 20 l of 10 mM TSP was added. Spectra were then taken at 2, 6, and 20 min after addition, but the spectra shown were after 20 min after TSP addition. The spectra of TSP alone in the same solvent after the same incubation times were subtracted. No corrections for the effects of dilution of the protein were made, as these were insignificant. A, unrechromatographed H159A enolase; “native” is native yeast enolase 1.
conditions (16). This is 0.2– 0.4% of the native enolase 1 value. From Lineweaver-Burk plots of the H159A data, we found the maximum activity of H159A enolase is about 1–1.5% of the maximum activity of native enolase 1. These data show the bulk of the activity exhibited by our preparations of H159A enolase is characteristic of that mutant, and cannot be due to contamination by native enolases 1 or 2. The reaction of native and mutant enolase with the substrate analogue TSP (D-tartronate semialdehyde phosphate) (18) produces difference spectral changes in the enzyme-bound analogue (23), as shown in Fig. 2. The reaction is thought to involve removal of a proton from carbon 2 of TSP, similar to the rate-limiting removal of a proton from carbon-2 of the substrate, with production of an enzyme-bound TSP enolate (23). With native enolase, a double peak results, similar to those previously observed (14, 23). His159A enolase in 10 mM Mg 2⫹ reacts much more slowly, and gives difference spectra with maxima at 272 nm, skewed toward longer wavelengths, and of lower magnitudes. The reaction of TSP with H159A is also faster in 10 mM Mg 2⫹ than in 1mM Mg 2⫹, independently corroborating the results shown in Fig. 1 (Fig. 2). The extrapolated difference extinction coefficient is 7800 M ⫺1cm ⫺1 at 272 nm, lower than the value for native enolase: 13700 M ⫺1cm ⫺1 at 285 nm (16,23). The extrapolated difference extinction coefficient (272 nm) was obtained by plotting the reciprocals of the value of ⌬OD 272 after 2, 6, and 20 min incubation versus the reciprocals of the times at which the difference spectra were obtained (16, 21) (not shown). These results were obtained using unrechromatographed H159A enolase, but if the preparations were contaminated with, for example, 1% of native enolase 2, the maximum absorbance difference
would be only 1% of the value for native enolase 1 or 2, as the enolized TSP producing the difference spectra is enzyme-bound (23). We think the lower difference extinction coefficient value and shorter wavelength of the maximum difference in absorbance result from the double loop containing His159 not folding over the active site. Our working hypothesis is that the activation of H159A enolase by high Mg 2⫹ concentrations (Figs. 1 and 2) is due to binding a third Mg 2⫹ per active site where protonated His159 normally interacts with the substrate. This putative third Mg 2⫹ inhibits native enolases 1 and 2 but partially compensates for the absence of protonated His159 in H159A enolase. This hypothesis is under investigation. DISCUSSION The level of enzymatic activity obtained with this mutant by us (up to 1% of the native value) is higher than one would expect from loss of a catalytic base (suggested to be ca. 4 orders of magnitude) (24). This finding supports the mechanism derived from the X-ray crystallographic structures of the yeast enzyme with substrate or substrate/product bound (7, 8). Poyner et al. (25) also used the E. coli expression system to prepare mutants of the yeast enzyme. They reported that unmodified enolase had the appropriate specific activity, but did not examine the folding of their mutants. Vinarov and Nowak (11) were well aware of the possible dangers involved in expressing a mutant eukaryotic protein in a procaryote. They also found the unmodified yeast enolase, expressed in E. coli, had the appropriate specific activity, but in addition examined the CD spectrum, fluorescence emission spectrum and binding of metal ions and substrate to their preparation of H159A enolase. Their data showed their protein was not a random coil and did indeed bind metal ion (Mn 2⫹) and substrate with dissociation constants comparable to those of native enolase 1. However, the extent of binding, that is, the moles of metal ion or substrate bound per mole of protein present, was not directly determined. The data presented herein show that the possibility of misfolding of proteins using widely-employed cloning techniques must be taken seriously and that even if the native protein folds correctly, mutants may not. ACKNOWLEDGMENTS The authors thank Dr. Thomas Nowak of the University of Notre Dame for his courtesy in providing us with a copy of his paper on H159A enolase considerably in advance of publication and Dr. C. V. C. Glover of the University of Georgia for advice and use of equipment.
1201
Vol. 276, No. 3, 2000
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
REFERENCES 1. Wold, F. (1971) Enolase. In The Enzymes (Boyer, P. D., Ed.), 3rd ed., Vol. 5, pp. 499 –538, Academic Press, New York. 2. Brewer, J. M. (1981) Yeast enolase: Mechanism of activation by metal ions. Crit. Rev. Biochem. 11, 209 –254. 3. Faller, L. D., and Johnson, A. M. (1974) Calorimetric studies of the role of magnesium ions in yeast enolase catalysis. Proc. Nat. Acad. Sci. USA 71, 1083–1087. 4. Faller, L. D., Baroudy, B. M., Johnson, A. M., and Ewall, R. X. (1977) Magnesium ion requirements for enolase activity. Biochemistry 16, 3864 –3869. 5. Lebioda, L., and Stec, B. (1991) Mechanism of enolase: The crystal structure of enolase Mg 2⫹-2-phosphoglycerate/phosphoenolpyruvate complex at 2.2Å resolution. Biochemistry 30, 2817– 2822. 6. Zhang, E., Hatada, M., Brewer, J. M., and Lebioda, L. (1994) Catalytic metal ion binding in enolase: The crystal structure of enolase-Mn 2⫹-phosphono-acetohydroxamate complex at 2.4Å resolution. Biochemistry 33, 6295– 6300. 7. Larsen, T. M., Wedekind, J. E., Rayment, I., and Reed, G. H. (1996) A carboxylate oxygen of the substrate bridges the magnesium ions at the active site of enolase: Structure of the yeast enzyme complexed with the equilibrium mixture of 2-phosphoglycerate and phosphoenolpyruvate at 1.8Å resolution. Biochemistry 35, 4349 – 4358. 8. Zhang, E., Brewer, J. M., Minor, W., Carreira, L. A., and Lebioda, L. (1997) Mechanism of enolase: The crystal structure of asymmetric dimer enolase-2-phospho-D-glycerate/enolasephosphoenolpyruvate at 2.0Å resolution. Biochemistry 36, 12526 –12534. 9. Duquerroy, S., Camus, C., and Janin, J. (1995) X-ray structure and catalytic mechanism of lobster enolase. Biochemistry 34, 12513–12523. 10. Brewer, J. M., and Ellis, P. D. (1983) 31P-NMR studies of the effect of various metals on substrate binding to yeast enolase. J. Inorg. Biochem. 18, 71– 82. 11. Vinarov, D. A., and Nowak, T. (1999) The role of His159 in yeast enolase catalysis. Biochemistry 38, 12138 –12149. 12. Brewer, J. M., Glover, C. V. C., Holland, M. J., and Lebioda, L. (1999) Enzymatic function of loop Movement in enolase: H159N/ S307Y, H159A, H159F and N207A enolases. FASEB J. 13, A1363.
13. McAlister, L., and Holland, M. J. (1982) Targeted deletion of a yeast enolase structural gene-identification and isolation of yeast enolase isozymes. J. Biol. Chem. 257, 7181–7188. 14. Brewer, J. M., Robson, R. L., Glover, C. V. C., Holland, M. J., and Lebioda, L. (1993) Preparation and characterization of the E168Q site-directed mutant of yeast enolase 1. Proteins Struct. Funct. Genet. 17, 426 – 434. 15. Sangadala, V. S., Glover, C. V. C., Robson, R. L., Holland, M. J., Lebioda, L., and Brewer, J. M. (1995) Preparation by sitedirected mutagenesis and characterization of the E211Q mutant of yeast enolase 1. Biochim. Biophys. Acta 1251, 23–31. 16. Brewer, J. M., Glover, C. V. C., Holland, M. J., and Lebioda, L. (1998) Significance of the enzymatic properties of yeast S39A enolase to the catalytic mechanism. Biochim. Biophys. Acta 1383, 351–355. 17. Lee, B. H., and Nowak, T. (1992) The influence of pH on the Mn 2⫹ activation of and binding to yeast enolase: A functional study. Biochemistry 31, 2165–2171. 18. Spring, T. G., and Wold, F. (1971) Studies on two high-affinity enolase inhibitors, chemical characterization. Biochemistry 10, 4649 – 4654. 19. Anderson, V. E., Weiss, P. M., and Cleland, W. W. (1984) Reaction intermediate analogues for enolase. Biochemistry 23, 2779 – 2786. 20. Westhead, E. W. (1966) Enolase from yeast and rabbit muscle. Methods Enzymol. 9, 670 – 679. 21. Brewer, J. M., Glover, C. V. C., Holland, M. J., and Lebioda, L. (1997) Effect of site-directed mutagenesis of His373 of yeast enolase on some of its physical and enzymatic properties. Biochim. Biophys. Acta 1340, 88 –96. 22. Wold, F., and Ballou, C. E. (1957) Studies on the enzyme enolase: II, Kinetic studies. J. Biol. Chem. 227, 313–328. 23. Spring, T. G., and Wold, F. (1971) Studies on two high-affinity enolase inhibitors, reaction with enolases. Biochemistry 10, 4655– 4660. 24. Serpersu, E. H., Shortle, D., and Mildvan, A. S. (1987) Kinetic and magnetic resonance studies of active-site mutants of staphylococcal nuclease: Factors contributing to catalysis. Biochemistry 26, 1289 –1300. 25. Poyner, R. R., Langhlin, L. T., Sowa, G. A., and Reed, G. H. (1996) Toward identification of acid base catalysts in the active site of enolase: Comparison of the properties of K345A, E168Q and E211Q variants. Biochemistry 35, 1692–1699.
1202
The H159A Mutant of Yeast Enolase 1 Has Significant Activity John M. Brewer,* ,1 Michael J. Holland,† and Lukasz Lebioda‡ *Department of Biochemistry and Molecular Biology, University of Georgia, Athens, Georgia 30602; †Department of Biological Chemistry, University of California Medical School at Davis, Davis, California 95683; ‡Department of Chemistry and Biochemistry, University of South Carolina, Columbia, South Carolina 29208
Received August 10, 2000
The function of His159 in the enolase mechanism is disputed. Recently, Vinarov and Nowak (Biochemistry (1999) 38, 12138 –12149) prepared the H159A mutant of yeast enolase 1 and expressed this in Escherichia coli. They reported minimal (ca. 0.01% of the native value) activity, though the protein appeared to be correctly folded, according to its CD spectrum, tryptophan fluorescence, and binding of metal ion and substrate. We prepared H159A enolase using a multicopy plasmid and expressed the enzyme in yeast. Our preparations of H159A enolase have 0.2– 0.4% of the native activity under standard assay conditions and are further activated by Mg 2ⴙ concentrations above 1 mM to 1–1.5% of the native activity. Native enolase 1 (and enolase 2) are inhibited by such Mg 2ⴙ concentrations. It is possible that His159 is necessary for correct folding of the enzyme and that expression in E. coli leads to largely misfolded protein. © 2000 Academic Press Key Words: enolase; expression; folding; cloning; mechanism; mutagenesis.
alogue (2-phosphoglycolate), to be the catalytic base abstracting the proton from carbon-2 of the substrate (9). In contrast, 31P-NMR measurements and the X-ray structure of the yeast enzyme complexed with substrate/product indicated that His159 initiates the catalytic cycle by protonating the phosphate of the substrate (8, 10). Recently, Vinarov and Nowak (11) prepared the H159A mutant of yeast enolase 1, expressing this in E. coli. Their preparation had ca. 0.01% of native enolase activity, supporting a function of His159 as a catalytic base. We, however, express our yeast enolase 1 mutants in the native host organism, yeast, and have routinely obtained much higher levels of activity in His159 mutants (12). Because so many workers use cloning, usually in E. coli, to overexpress proteins from other organisms, often coupling this with mutagenesis, we extended our investigation of the source of this discrepancy. MATERIALS AND METHODS
Enolase (2-phosphoglycerate hydrolyase, E.C.4.2.1.11) catalyzes the interconversion of 2-phosphoglycerate and phosphoenolpyruvate. Enolase from yeast, like most enolases, is dimeric (1). Each subunit can bind one equivalent of divalent cation at what we refer to as a “conformational” site (2). This enables substrate or analogues to bind (3); when substrate or analogues are bound, a second equivalent of divalent cation can bind to what we call the “catalytic” site, producing catalysis (2, 4). Mg2⫹ is the physiological activating cation (1, 2). During catalysis by enolase, three polypeptide loops move (5– 8). Two loops move together, one of which includes His159 (His157 in lobster enolase) (9). This residue was suggested, on the basis of the X-ray structure of lobster enolase complexed with a substrate an1
To whom correspondence should be addressed. Fax: (706) 5421738. E-mail: [email protected].
Two enolase isozymes, called 1 and 2, exist in yeast; the organism can grow with either alone (13). We prepare enolase mutants by mutating a multicopy plasmid containing the enolase 1 sequence and a promoter region, then transforming a strain of yeast whose chromosomal enolase 1 gene is largely deleted, so that it grows using the enolase 2 isozyme only (14). Before transforming the yeast strain used for expression, the protein coding sequence was determined, and only the appropriate codon was altered and altered as intended (CAC to GCC). H159A enolase was isolated as described (14 –16), using chromatography on Q Sepharose FF at pH 8.5 and CM Sepharose at pH 6.0 (17), using a Pharmacia-LKB LCC 500 Plus FPLC. Although enolase 2 elutes from Q Sepharose at a much higher ionic strength than enolase 1 (13) and its mutants, we find the enolase 1 mutants are contaminated with small amounts of native enolase 2. Such contamination would affect enzymatic properties such as relative activities but would not significantly affect calorimetric or difference spectral measurements. Contamination of enolase 1 mutants with enolase 2 varies inversely with the net expression of the mutant, in terms of enzymatic activity (16). The net expression of the H159 mutants was low (we obtained no H159Q enolase) in comparison to, for example, the E168Q and E211Q mutants (14, 15). We have found rechromatography of enolase 1 mutants on a 1 ⫻ 5 cm
1199
0006-291X/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.
Vol. 276, No. 3, 2000
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
MonoQ column using the pH 8.5 solvent system employed in the Q Sepharose chromatography to be effective in reducing enolase 2 levels: rechromatographed S39A enolase has a relative activity of 10 ⫺4 of native enolase (16). Native enolase 2 itself was prepared from the untransformed yeast strain mentioned above using the same chromatographic steps described above, together with chromatography on an S-200 Superdex column at pH 7.8 (14). Our preparations of H159A enolase appeared homogeneous on SDS-gels (not shown). The substrate analogues D-tartronate semialdehyde phosphate (TSP) was synthesized as described by Spring and Wold (18); phosphonoacetohydroxamate was synthesized as described by Anderson et al. (19). The substrate, 2-phosphoglycerate, was from Sigma (St. Louis, MO). Differential scanning calorimetry (DSC) measurements were performed using an instrument from Calorimetry Sciences Corp. (Provo, UT). Measurements of absorbance at single wavelengths were done using a Spectronic 601 spectrophoto-meter (Milton Roy). Difference spectral measurements were performed with a Hewlett-Packard 8452A Diode Array Spectrophotometer controlled by a Standard Turbo 10 computer using Specos 3.71 software. Measurements of pH values were done using an Orion 701A digital lonalyzer with a single probe microelectrode. Enzyme activities were measured at ambient temperature (20 –22°) as described by Westhead (20).
RESULTS As in our previous studies of mutant enolases (14 – 16, 20), differential scanning calorimetric (DSC) analyses of the effect of substrate and a strongly bound (19) competitive inhibitor, phosphonoacetohydroxamate, on the thermal denaturation of mutant enolases showed that our preparation of H159A enolase binds both compounds and is stabilized by them (not shown). With 2 mM Mg 2⫹ present, substrate increases the temperature of maximum excess heat capacity (T max) by 5° and the inhibitor stabilizes the H159A mutant by 21°. With native enolase, substrate and analogue increase T max values by 6° and 22°, respectively (16). All the H159A enolase appeared to bind, and be stabilized by, the substrate and inhibitor, hence much if not all the protein is correctly folded. The H159A enolase fractions from the CM columns routinely exhibited specific activities under standard assay conditions (0.05 ionic strength Tris–HCl, pH 7.8, 1 mM MgCl 2, 10 ⫺5M EDTA, 1 mM 2-phospho-Dglycerate) (20) of 1–3 units (⌬OD 230/min/OD 280) (20) which increased with increasing fraction number. These preparations after pooling and lyophilization were neither stimulated nor inhibited by Mg 2⫹ concentrations above 1 mM. Since traces of any enolase 2 present would strongly influence the activity, we rechromatographed H159A enolase preparations on the monoQ column. This treatment did not reduce the specific activity as dramatically as was seen with S39A enolase (16): specific activities of 1–2 units and, in two preparations examined, the specific activities of the column fractions were constant across the peak of OD 280 due to H159A enolase (not shown). However, the rechromatographed H159A enolase activity is strongly stimulated by Mg 2⫹ concentrations
FIG. 1. Dependence of specific activities of H159A and native enolases 1 and 2 on magnesium ion concentrations. Enolase assay medium, 0.58 –1.0 ml (0.05 ionic strength Tris–HCl, pH 7.8, 1 mM MgCl 2, 1 mM 2-phosphoglycerate, and 1 ⫺5M EDTA), was supplemented with microliter amounts of 0.5 M MgCl 2, to produce the final concentrations shown. H159A enolase (two preparations), 3– 6 g, or 0.24 g enolase 1 or 0.38 g enolase 2, were added to these solutions, mixed, and the rate of increase of absorbance at 230 nm was determined. The filled symbols are using H159A enolase, the crosses enolase 1, and the open circles enolase 2. The H159A enolase, enolase 1, and enolase 2 were rechromatographed on a MonoQ column before these measurements. Specific activities are ⌬OD 230/min/OD 280 (of enzyme in the assay solution), as described by Westhead (20).
above 1 mM (Fig. 1). Both native enolases 1 (4, 22) and 2 are inhibited by Mg 2⫹ concentrations above 1 mM. This is not the result of the rechromatography per se, as the data shown were obtained with native enolase 1 and 2 which were rechromatographed on the MonoQ column as well. The rechromatography did not affect the inhibition of the native enolase 1 or 2 by Mg 2⫹ (not shown). The figure shows results from two samples of rechromatographed H159A enolase, measured on different days. The higher activity of one of the samples may be owing to residual enolase 2, as the stimulation of activity by Mg 2⫹ of that sample seems less also. The activities of these and other rechromatographed preparations of H159A enolase varied from 0.9 –1.9 units (⌬OD 230/min/OD 280) measured under standard assay
1200
Vol. 276, No. 3, 2000
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
FIG. 2. Reactions of H159A and native enolase 1 with TSP. Spectra of solutions (1 ml) containing 20 M native or unrechromatographed H159A enolase in 0.1 ionic strength Tris–HCl, pH 7.8 and 1 or 10 mM MgCl 2 were obtained (4 scans averaged) then 20 l of 10 mM TSP was added. Spectra were then taken at 2, 6, and 20 min after addition, but the spectra shown were after 20 min after TSP addition. The spectra of TSP alone in the same solvent after the same incubation times were subtracted. No corrections for the effects of dilution of the protein were made, as these were insignificant. A, unrechromatographed H159A enolase; “native” is native yeast enolase 1.
conditions (16). This is 0.2– 0.4% of the native enolase 1 value. From Lineweaver-Burk plots of the H159A data, we found the maximum activity of H159A enolase is about 1–1.5% of the maximum activity of native enolase 1. These data show the bulk of the activity exhibited by our preparations of H159A enolase is characteristic of that mutant, and cannot be due to contamination by native enolases 1 or 2. The reaction of native and mutant enolase with the substrate analogue TSP (D-tartronate semialdehyde phosphate) (18) produces difference spectral changes in the enzyme-bound analogue (23), as shown in Fig. 2. The reaction is thought to involve removal of a proton from carbon 2 of TSP, similar to the rate-limiting removal of a proton from carbon-2 of the substrate, with production of an enzyme-bound TSP enolate (23). With native enolase, a double peak results, similar to those previously observed (14, 23). His159A enolase in 10 mM Mg 2⫹ reacts much more slowly, and gives difference spectra with maxima at 272 nm, skewed toward longer wavelengths, and of lower magnitudes. The reaction of TSP with H159A is also faster in 10 mM Mg 2⫹ than in 1mM Mg 2⫹, independently corroborating the results shown in Fig. 1 (Fig. 2). The extrapolated difference extinction coefficient is 7800 M ⫺1cm ⫺1 at 272 nm, lower than the value for native enolase: 13700 M ⫺1cm ⫺1 at 285 nm (16,23). The extrapolated difference extinction coefficient (272 nm) was obtained by plotting the reciprocals of the value of ⌬OD 272 after 2, 6, and 20 min incubation versus the reciprocals of the times at which the difference spectra were obtained (16, 21) (not shown). These results were obtained using unrechromatographed H159A enolase, but if the preparations were contaminated with, for example, 1% of native enolase 2, the maximum absorbance difference
would be only 1% of the value for native enolase 1 or 2, as the enolized TSP producing the difference spectra is enzyme-bound (23). We think the lower difference extinction coefficient value and shorter wavelength of the maximum difference in absorbance result from the double loop containing His159 not folding over the active site. Our working hypothesis is that the activation of H159A enolase by high Mg 2⫹ concentrations (Figs. 1 and 2) is due to binding a third Mg 2⫹ per active site where protonated His159 normally interacts with the substrate. This putative third Mg 2⫹ inhibits native enolases 1 and 2 but partially compensates for the absence of protonated His159 in H159A enolase. This hypothesis is under investigation. DISCUSSION The level of enzymatic activity obtained with this mutant by us (up to 1% of the native value) is higher than one would expect from loss of a catalytic base (suggested to be ca. 4 orders of magnitude) (24). This finding supports the mechanism derived from the X-ray crystallographic structures of the yeast enzyme with substrate or substrate/product bound (7, 8). Poyner et al. (25) also used the E. coli expression system to prepare mutants of the yeast enzyme. They reported that unmodified enolase had the appropriate specific activity, but did not examine the folding of their mutants. Vinarov and Nowak (11) were well aware of the possible dangers involved in expressing a mutant eukaryotic protein in a procaryote. They also found the unmodified yeast enolase, expressed in E. coli, had the appropriate specific activity, but in addition examined the CD spectrum, fluorescence emission spectrum and binding of metal ions and substrate to their preparation of H159A enolase. Their data showed their protein was not a random coil and did indeed bind metal ion (Mn 2⫹) and substrate with dissociation constants comparable to those of native enolase 1. However, the extent of binding, that is, the moles of metal ion or substrate bound per mole of protein present, was not directly determined. The data presented herein show that the possibility of misfolding of proteins using widely-employed cloning techniques must be taken seriously and that even if the native protein folds correctly, mutants may not. ACKNOWLEDGMENTS The authors thank Dr. Thomas Nowak of the University of Notre Dame for his courtesy in providing us with a copy of his paper on H159A enolase considerably in advance of publication and Dr. C. V. C. Glover of the University of Georgia for advice and use of equipment.
1201
Vol. 276, No. 3, 2000
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
REFERENCES 1. Wold, F. (1971) Enolase. In The Enzymes (Boyer, P. D., Ed.), 3rd ed., Vol. 5, pp. 499 –538, Academic Press, New York. 2. Brewer, J. M. (1981) Yeast enolase: Mechanism of activation by metal ions. Crit. Rev. Biochem. 11, 209 –254. 3. Faller, L. D., and Johnson, A. M. (1974) Calorimetric studies of the role of magnesium ions in yeast enolase catalysis. Proc. Nat. Acad. Sci. USA 71, 1083–1087. 4. Faller, L. D., Baroudy, B. M., Johnson, A. M., and Ewall, R. X. (1977) Magnesium ion requirements for enolase activity. Biochemistry 16, 3864 –3869. 5. Lebioda, L., and Stec, B. (1991) Mechanism of enolase: The crystal structure of enolase Mg 2⫹-2-phosphoglycerate/phosphoenolpyruvate complex at 2.2Å resolution. Biochemistry 30, 2817– 2822. 6. Zhang, E., Hatada, M., Brewer, J. M., and Lebioda, L. (1994) Catalytic metal ion binding in enolase: The crystal structure of enolase-Mn 2⫹-phosphono-acetohydroxamate complex at 2.4Å resolution. Biochemistry 33, 6295– 6300. 7. Larsen, T. M., Wedekind, J. E., Rayment, I., and Reed, G. H. (1996) A carboxylate oxygen of the substrate bridges the magnesium ions at the active site of enolase: Structure of the yeast enzyme complexed with the equilibrium mixture of 2-phosphoglycerate and phosphoenolpyruvate at 1.8Å resolution. Biochemistry 35, 4349 – 4358. 8. Zhang, E., Brewer, J. M., Minor, W., Carreira, L. A., and Lebioda, L. (1997) Mechanism of enolase: The crystal structure of asymmetric dimer enolase-2-phospho-D-glycerate/enolasephosphoenolpyruvate at 2.0Å resolution. Biochemistry 36, 12526 –12534. 9. Duquerroy, S., Camus, C., and Janin, J. (1995) X-ray structure and catalytic mechanism of lobster enolase. Biochemistry 34, 12513–12523. 10. Brewer, J. M., and Ellis, P. D. (1983) 31P-NMR studies of the effect of various metals on substrate binding to yeast enolase. J. Inorg. Biochem. 18, 71– 82. 11. Vinarov, D. A., and Nowak, T. (1999) The role of His159 in yeast enolase catalysis. Biochemistry 38, 12138 –12149. 12. Brewer, J. M., Glover, C. V. C., Holland, M. J., and Lebioda, L. (1999) Enzymatic function of loop Movement in enolase: H159N/ S307Y, H159A, H159F and N207A enolases. FASEB J. 13, A1363.
13. McAlister, L., and Holland, M. J. (1982) Targeted deletion of a yeast enolase structural gene-identification and isolation of yeast enolase isozymes. J. Biol. Chem. 257, 7181–7188. 14. Brewer, J. M., Robson, R. L., Glover, C. V. C., Holland, M. J., and Lebioda, L. (1993) Preparation and characterization of the E168Q site-directed mutant of yeast enolase 1. Proteins Struct. Funct. Genet. 17, 426 – 434. 15. Sangadala, V. S., Glover, C. V. C., Robson, R. L., Holland, M. J., Lebioda, L., and Brewer, J. M. (1995) Preparation by sitedirected mutagenesis and characterization of the E211Q mutant of yeast enolase 1. Biochim. Biophys. Acta 1251, 23–31. 16. Brewer, J. M., Glover, C. V. C., Holland, M. J., and Lebioda, L. (1998) Significance of the enzymatic properties of yeast S39A enolase to the catalytic mechanism. Biochim. Biophys. Acta 1383, 351–355. 17. Lee, B. H., and Nowak, T. (1992) The influence of pH on the Mn 2⫹ activation of and binding to yeast enolase: A functional study. Biochemistry 31, 2165–2171. 18. Spring, T. G., and Wold, F. (1971) Studies on two high-affinity enolase inhibitors, chemical characterization. Biochemistry 10, 4649 – 4654. 19. Anderson, V. E., Weiss, P. M., and Cleland, W. W. (1984) Reaction intermediate analogues for enolase. Biochemistry 23, 2779 – 2786. 20. Westhead, E. W. (1966) Enolase from yeast and rabbit muscle. Methods Enzymol. 9, 670 – 679. 21. Brewer, J. M., Glover, C. V. C., Holland, M. J., and Lebioda, L. (1997) Effect of site-directed mutagenesis of His373 of yeast enolase on some of its physical and enzymatic properties. Biochim. Biophys. Acta 1340, 88 –96. 22. Wold, F., and Ballou, C. E. (1957) Studies on the enzyme enolase: II, Kinetic studies. J. Biol. Chem. 227, 313–328. 23. Spring, T. G., and Wold, F. (1971) Studies on two high-affinity enolase inhibitors, reaction with enolases. Biochemistry 10, 4655– 4660. 24. Serpersu, E. H., Shortle, D., and Mildvan, A. S. (1987) Kinetic and magnetic resonance studies of active-site mutants of staphylococcal nuclease: Factors contributing to catalysis. Biochemistry 26, 1289 –1300. 25. Poyner, R. R., Langhlin, L. T., Sowa, G. A., and Reed, G. H. (1996) Toward identification of acid base catalysts in the active site of enolase: Comparison of the properties of K345A, E168Q and E211Q variants. Biochemistry 35, 1692–1699.
1202