30min at 9000rpm in the GS3 rotor of a Sorvall RC-5C centrifuge and the pellet discarded. Solid trichloroacetic acid was added to the remaining supernatant to ...
Eur. J. Biochem. lY8, 513-519 (1991) 8: FEBS 1991 001429569100365G
Purification, characterisation and mutagenesis of highly expressed recombinant yeast pyruvate kinase Toby H. L. MURCOTT’, Teresa McNALLY’, Simon C. ALLEN’, Linda A. FOTHERGILL-GILMORE’ and Hilary MUIRHEAD’ Department of Biochemistry and Molecular Recognition Centre, School of Medical Sciences, University of Bristol, England
’ Department of Biochemistry, University of Edinburgh, Scotland (Received December 13, 1990)- EJB 90 1479
Recombinant yeast pyruvate kinase has been purified from a strain of Saccharomyces cerevisiae expressing the enzyme to very high levels. Expression was from a multicopy plasmid under the control of the yeast phosphoglycerate kinase promoter. The gene was expressed in the absence of the genomically encoded pyruvate kinase, using a strain of yeast in which the pyruvate kinase gene has been disrupted by the insertion of the yeast Ura3 gene. The purification procedure minimised proteolytic artefacts and enabled the covenient purification of 15 20 mg enzyme from 1 1 culture. The purified enzyme was characterised by a high specific activity and by a lack of proteolytic degradation. Two active-site mutants of yeast pyruvate kinase have been produced, expressed and characterised in this system and preliminary results are described.
Pyruvate kinase (ATP : pyruvate 2-0-phosphotransferase) is an enzyme of glycolysis that plays a major role in regulating the flux from fructose 1,6-bisphosphate (Fru(l,6)P2) to pyruvate. The enzyme is a homotetramer, with a subunit M , of 55 000 - 60000, and catalyses the essentially irreversible reaction, phosphoenolpyruvate
+ Mg . ADP + H +
1 Kt,Mg’+
pyruvate
+ Mg . ATP.
Pyruvate kinase has been extensively studied from a wide range of organisms and much is known about its physical and catalytic properties. It exists as four isoenzymes in mammals designated M1, M2, L and R. The M1 isoenzyme is essentially non-regulated, whereas the remaining isoenzymes (as well as the enzymes from lower organisms) exhibit a sigmoidal kinetic profile with respect to phosphoenolpyruvate concentration, and are allosterically regulated by a large number of nonsubstrate molecules. All forms of the enzyme require both monovalent and bivalent cations for activity. The former is normally potassium whereas a number of bivalent cations will suffice. Two bivalent cations are required/active site, one primarily associated with the nucleotide and the other more closely enzyme associated (see Muirhead, 1987, for a review). The three-dimensional structure of cat muscle M1 isoenzyme has been solved to a resolution of 0.26 nm (Muirhead et al., 1986). Each subunit of cat muscle M1 pyruvate kinase consists of four domains, N , A, B and C. Domain A is an eight-stranded a/B barrel, typical of a number of enzymes (Farber and Petsko, 1990). The active site lies between doCorrespondence to T. H. L. Murcott, Department of Biochemistry, School of Medical Sciences, University of Bristol, Bristol BS8 1TD, England Ahhreviafion. Fru(1 ,6)P2, fructose 1,6-bisphosphate. Enzyrnrs. Pyruvate kinase (EC 2.7.1.40); lactate dehydrogenase (EC 1.1.1.27).
mains A and B, and the putative effector site between domains A and C. To date, the relationship of structure with function for all forms of pyruvate kinase has been deduced from solution studies correlated with the cat muscle M1 structure. Some of the most interesting and important features of pyruvate kinase are its regulatory properties and the current situation is unsatisfactory in that the only crystal structure is that of the nonregulated M1 isoenzyme. One of the aims of the work described here is to produce sufficient high-quality yeast pyruvate kinase for X-ray structural analysis. Additionally it is proposed to investigate site-specific mutants that affect its regulatory properties. Pyruvate kinase from Saccharomyces cerevisiae is one of the most widely studied forms of this enzyme. Its kinetic and regulatory behaviour are well documented (Haeckel et al., 1968; Wieker and Hess, 1971;Kinderlerer et al., 1986; Rhodes et al., 1986) and the gene has been sequenced (Burke et al., 1933; McNally et al., 1989) and expressed from multicopy plasmids (Moore et al., 1990). Recombinant liver pyruvate kinase (L-type) has also been expressed in monkey COS cells (Tani et al., 1988), although the levels of expression were low and there was contamination with endogenous pyruvate kinase activity. For these reasons, yeast pyruvate kinase is an ideal candidate for investigation by site-directed mutagenesis. Previous preparations of yeast pyruvate kinase have been plagued by proteolysis (Roschlau and Hess, 1972; Bischofberger et al., 1971). Yeast is known to contain a wide range of proteases, not all of which are susceptible to inactivation by the commonly available protease inhibitors (Pringle, 1974). Therefore the prevention of proteolysis is a major consideration in designing a purification of any protein from yeast. This paper describes the production of a pyruvate-kinasedeficient strain of yeast by disruption of the chromosomal gene. The resulting strain of yeast was shown to have no constitutive wild-type pyruvate kinase activity. This is a major advantage when considering site-directed mutagenesis studies
514 as any pyruvate kinase mutants expressed would have no wildtype contamination. The lack of wild-type activity made it possible to establish the very low or abolished activity of two active-site mutants whose preliminary characterisation are described below. A purification of yeast pyruvate kinase expressed to very high levels in this yeast strain is described with an emphasis on avoiding proteolytic artefacts (see, for example, Pringle, 1974). The resulting preparation was shown to be stable over four months, free from proteolytic artefacts and exhibited the same kinetic behaviour as constitutively expressed pyruvate kinase.
MATERIALS A N D METHODS Materials Lyticase, amino acids and tissue culture grade pyruvate were obtained from the Sigma Chemical Company Ltd. Lactate dehydrogenase, the sodium salts of NADH and ADP and the tricyclohexylamine salt of phosphoenolpyruvate were obtained from Boehringer Mannheim Ltd. All other reagents were obtained from BDH Ltd or the Sigma Chemical Company Ltd and were of reagent grade. Q-Sepharose fast flow, Sephadex and Sephacryl S-300 were obtained from Pharmacia Ltd. Oligonucleotides for mutagenesis were obtained from the University of Bristol, Department of Biochemistry oligonucleotide-synthesis facility. Plasmid pMA91PYK was a generous gift from J. Mellor and J. Rathjen of the Department of Biochemistry, University of Oxford. When transformed into yeast this plasmid induces a high level of expression of an authentic yeast pyruvate kinase protein from a recombinant gene with identical codon usage to the original chromosomal gene. Yeast strains S. cerevisiae SF747: Leu2, ura3, trpl, gallo, matu. S. cerevisiae Pykl : : Ura3: as SF747, with the addition of disruption pykl: :ura3, so removing thepykl genotype and inserting ura3.
Transformation with plasmid D N A Transformation of yeast with supercoiled plasmid D N A was according to Burgers and Percival(l987) with the following modifications. The yeast growth medium contained 0.67% (mass/vol.) Difco yeast nitrogen base without amino acids, 2 % carbon source (pyruvate, dextrose o r glucose), 20 pg . mladenine sulphate, L-methionine, L-histidine hydrochloride, L-arginine monohydrochloride, L-tryptophan, 30 pg . ml-I L-lysine monohydrochloride, L-tyrosine, L-isoleucine, 50 pg . ml-' L-phenylalanine and 150 pg . ml-' L-valine (unless stated otherwise the amino acids were in the form of the free acid). Dextrose o r glucose were autoclaved with the yeast nitrogen base but pyruvate and the amino acids were filtersterilised and added after the medium had cooled. The pyruvate was prepared fresh whereas the amino acids were stored as a 100-fold concentrated stock at -20°C. The pyruvate-kinase-deficient yeast was grown in uracil-deficient selection medium with pyruvate as the carbon source. Transformed yeast was grown in uracil- and leucine-deficient medium, with dextrose or glucose as the carbon source for wildtype pyruvate kinase and with pyruvate as the carbon source
for all mutants. Lyticase, stock concentration of 10000 units . ml-', was used to prepare the spheroplasts. Production of pyruvate-kinase-deficientyeast strain Pykl ::Ura3 The wild-type, chromosomal copy of the pyruvate-kinase gene present in yeast strain SF747 was disrupted by insertion of the yeast Ura3 gene (Rothstein, 1983). A 1.2-kbp BglII restriction fragment containing the coding region of the Ura3 gene was inserted into the BglII site present within the coding sequence of the pyruvate kinase gene (see McNally et al., 1989, for a partial restriction map) carried by plasmid pPYK20. Plasmid pPYK20 had been constructed from plasmid pUC18 and the 7-kbp Hind111 restriction fragment containing the Pykl gene derived from plasmid pAYE4(34) (see McNally et al., 1989). Plasmid pPYK20 was then linearised by digestion with Hind111 and used to transform yeast strain SF747 by the method of Ito et al. (1983). The ends of the linear DNA molecule promote homologous recombination. Transformants were obtained by selection on uracil-deficient medium containing 3% (massivol.) glycerol and 3 % (by vol.) ethanol as carbon sources. The desired pyruvate-kinase-disrupted strain would be able to grow in the absence of uracil, would survive on glycerol/ethanol but would be unable to grow on glucose. 14 transformants were obtained after 12 days growth, and five of these were discarded because they proved able to grow on glucose. Presumably the Ura3 gene had integrated at another site in the yeast genome, perhaps the Ura3 locus itself. Southern blotting of the remaining transformants showed that they contained a 1.2-kbp insert within the genomic pyruvate kinase gene. It was found that growth of the Pykl : : Ura3 strains was very slow on glycerol/ethanol, with colonies taking approximately 5 days to form on solid media. Two alternative carbon sources were tested: ethylpyruvate and pyruvate (both over the concentration range 0.1 -4%, mass/vol.). The former compound was unable to sustain cell growth, but pyruvate supported growth, with 2% being the optimum concentration. There is apparently no specific carrier for pyruvate into yeast cells, but the pyruvate can enter via the lactate/proton symporter (Cassio et al., 1987). For the purposes of expression of yeast pyruvate kinase mutants, it would have been preferable to produce a deleted rather than a disrupted pyruvate-kinase-deficient yeast. However, repeated attempts at producing such a mutant (A. J. P. Brown, personal communication) failed and so we were forced to resort to a disrupted mutant. Mutagenesis of yeast pyruvate kinase Mutagenesis of the yeast pyruvate kinase gene was performed according to Zoller and Smith (1982). Yeast pyruvate kinase assay Pyruvate kinase was assayed spectrophotometrically after the method of Bucher and Pfleiderer (1955) with the following modifications. Assays were performed in 1-cm-path-length silica or quartz cuvettes in a total volume of 1 ml at 30°C. Unless otherwise stated the cuvette contained 6 pmol ADP, 6 pmol phosphoenolpyruvate, 0.25 pmol NADH, 1 unit lactate dehydrogenase, 15 pmol magnesium sulphate, 100 pmol KCl and 50 pmol Mes, pH 6.2. The reaction was started by the addition of pyruvate kinase and its progress recorded by
51 5 monitoring the change in absorbance at 340 nm in a PerkinElmer i 2 spectrophotometer. 1 unit pyruvate kinase is defined as the amount of enzyme which produces 1 pmol pyruvate and ATP . min-' from phosphoenolpyruvate and ADP. Protein concentration determination
The concentration of pure yeast pyruvate kinase was determined spectrophotometrically using an absorbance of 0.51 at 280 nm for a 1 mg . ml-I solution, as determined by Yun et al. (1976). Protein concentration during purification was measured by the dye-binding assay of Bradford (1976) with bovine serum albumin as the standard. Preparation of yeast polypeptide protease inhibitors
Pringle, in his review (1974), suggests utilising the naturally occurring yeast polypeptide protease inhibitors and this approach was adopted here as follows. An extract of yeast rich in the naturally occurring heat-stable polypeptide protease inhibitors was prepared from commercial bakers yeast. Approximately 50 g fresh bakers yeast was lysed as described below and the cell-free extract boiled for 10 min. After cooling to room temperature, the boiled lysate was centrifuged for 30min at 9000rpm in the GS3 rotor of a Sorvall RC-5C centrifuge and the pellet discarded. Solid trichloroacetic acid was added to the remaining supernatant to 5% (massivol.) to precipitate the soluble polypeptides, which were then collected by centrifugation as before. The pellet was redissolved in 10 ml 1 M Tris/HCl, pH 8.0, and this preparation was used as described. Growth and harvest of yeast expressing high levels ojpyruvate kinase
On day 1, 10-ml aliquots of appropriate selection media (one for each litre of yeast culture required) were inoculated with a single colony of transformed yeast and grown for 24 h at 30°C in a shaking incubator. After growth they were added to 1-1 vol. of the same medium and growth continued until the A600,was 1.1 1.4. There is strong evidence that yeast pyruvate kinase is cold-labile and so all purification procedures were performed at room temperature (Kuczenski and Suelter, 1970). The yeast cells were harvested by centrifugation at 5000 rpm in a Sorvall RC-3C centrifuge for 30 min. The cells were pooled and washed once by resuspension and centrifugation as before in 100 mM Tris/HCl, pH 7.5, 100 mM KCl, 5 mM EDTA, 20% (by vol.) glycerol. The harvested cells were then used immediately or snap-frozen in liquid nitrogen and stored at - 70°C until use. No differences in yield or properties of the purified enzyme were found between frozen and unfrozen cells.
the level of the meniscus. The slurry was stirred for 1 h at 2000 rpm from overhead with a glass paddle mounted in a Bosch CSB 420E variable-speed power drill. The glass paddle was a circle of glass 20 mm in diameter and 3 mm thick, fused at the edge to an 8-mm-diameter glass rod. After lysis the slurry was filtered through sintered glass and the glass beads washed by further addition of 200 ml lysis buffer. This filtrate was the cell-free extract. Ammonium sulphate fractionation
Finely divided solid ammonium sulphate was added, with stirring, to the cell-free extract to a final saturation of 40%. The solution was left to stir for a further 30 min and then centrifuged at 13 500 rpm in the GSA rotor of a Sorvall RC5C centrifuge. The resulting pellet (P40) was discarded. Further solid ammonium sulphate was added to the supernatant (S40) as before to a final saturation of 60% and the solution stirred and centrifuged as before. The resulting pellet (P40 - 60) contained approximately 80% of the pyruvate kinase activity. Q-Sepharose chromatography
P40 - 60 was dissolved in the minimum volume of 50 mM Tris/HCl, pH 8.3, 50 mM KCl, 5 mM EDTA, 20% (by vol.) glycerol, 12 mM 2-mercaptoethanol, 0.05 mM phenylmethylsulphonyl fluoride and 0.1 mM benzamidine. The protein was then desalted though a Sephadex G-25 column (3.0 cm x 25 cm) equilibrated in the same buffer. It was then passed through a Q-Sepharose column (4.5 cm x 10 cm) equilibrated in the same buffer. Over 85% of the applied pyruvate kinase activity eluted in the break-through volume of the column and was greater than 90% pure, as determined by SDSjPAGE (see Fig. 1). The pyruvate-kinase-containing fractions were pooled and the protein precipitated by the addition of solid ammonium sulphate to 75% saturation. This was known as the post-Q protein. The Q-Sepharose column was regenerated by washing with the same buffer containing 1 M KCl.
~
Gel filtration
The post-Q protein was dissolved in the minimum volume of 100 mM Tris/HCl, pH 7.5, 200 mM KCl, 20% (by vol.) glycerol, 5 mM EDTA and 12 mM 2-mercaptoethanol. It was then passed through a Sephacryl S-300 (3.5 cm x 80 cm) column equilibrated in the same buffer. The pyruvate kinase activity eluted as a single peak and was pooled and stored as a suspension in 75% saturated ammonium sulphate. RESULTS AND DISCUSSION
Yeast cell lysis
Expression of yeast pyruvate kinase
The harvested yeast cells were resuspended in 10ml 100 mM Tris/HCl, 100 mM KCl, 5 mM EDTA, 12 mM 2mercaptoethanol, 20% (by vol.) glycerol, 0.5% (by vol.) yeast polypeptide protease inhibitors, 0.1 mM benzamidine and 0.05 mM phenylmethylsulphonyl fluoride. An alternative protease inhibitor cocktail containing 0.1 mM 1,lO-phenanthroline, 0.1 mM 3,4-dichloroisocoumarin and 0.05 mM E-64 was also found to be effective throughout. The suspension was transferred to a 50-ml thick-walled round-bottomed test tube, and acid-washed 0.2-mm glass beads were added to
Plasmid pMA91-PYK is a yeast Escherichia coli shuttle vector bearing the yeast pyruvate kinase gene under the control of the yeast phosphoglycerate kinase promoter (Rathjen, 1989).Yeast strain Pykl ::Ura3 was transformed with plasmid pMA91 -PYK and the resulting transformants expressed yeast pyruvate kinase to very high levels (see Fig. 1). The transformants also recovered the ability to grow with glucose or dextrose as the carbon source, demonstrating that the highly expressed pyruvate kinase was active in vivo. A number of sitespecific mutants of yeast pyruvate kinase have been subcloned
516 Table 1. The stages of purification of yeast pyruvate kinase The table shows the degree of purification and yield achieved at each stage in the preparation of yeast pyruvate kinase Step
Cell lysate S40 P40 - 60 Post-Q Post S-300
Volume
ml 190 210 40 54 45
Amount of enzyme
protein
units 52907 37758 30608 25 575 21 720
mg 540 386 195 93 61
into plasmid pMA91-PYK and expressed to similarly high levels in yeast Pykl ::Ura3. Mutants that displayed very little or no activity were not able to restore the ability of the disrupted strain to grow with glucose or dextrose as the carbon source. There is considerable evidence that the intracellular concentration of pyruvate kinase is regulated at both transcriptional and translational levels in yeast (Moore et al. 1990a, b, c). Yeast transformed with a high-copy-number plasmid bearing the yeast pyruvate kinase gene and its promoter, transiently overexpresses active enzyme. This overexpression is characterised by slow growth, and within tens of generations expression reverts to near constitutive levels. It has been shown here that yeast cells will continue to overexpress active yeast pyruvate kinase to very high levels under the control of the yeast phosphoglycerate kinase promoter. The cells do not appear to suffer any deleterious effects and grow reasonably well in selection media.
1
Specific activity
Yield
units/mg
%
98 98 157 214 355
100 71 58 48 41
2
3
4
5
6
7
6
9
W
Purification of yeast pyruvate kinase The method of yeast cell lysis used was chosen after a number of trials of other methods. Both ammonia lysis and toluene lysis (Yun et al., 1976) were tested on 2 0 3 portions of fresh bakers yeast but neither produced greater than 20% of the pyruvate kinase activity predicted from the literature. The glass-bead mill described here reproducibly produced 100% or more of the pyruvate kinase activity/cells obtained by Yun et al. (1976). During lysis the lysis vessel was immersed in a water bath maintained at room temperature to dissipate the small amount of heat produced during the process. Table 1 presents a typical purification of wild-type yeast pyruvate kinase from 3 1 cultured overexpressing yeast. Fig. 1 is of a 10% SDSjPAGE gel showing the protein composition at each stage of the purification. The purification procedure described here is simple, rapid and yields a stable, protease-free preparation of greater than 95% pure yeast pyruvate kinase. This procedure was developed with a strong emphasis on minimising proteolytic artefacts. The strategy adopted was twofold. Firstly, conditions were manipulated to inhibit as many proteases as possible by including a cocktail of protease inhibitors and working above pH 6.0 where the acid proteases of yeast are inactive. Secondly, the protein was held in dilute solution for as short a time as possible by the use of a desalting column rather than dialysis and by storing the protein as an ammonium sulphate precipitate as soon as possible after each purification step. It is apparent that the anti-proteolysis strategy employed was a success, as proteolysis was not a significant problem during purification.
Fig. 1. SDSjPAGE gel showing the stages of purification of yeust pyruvate kinase. This is a photograph of a 10% SDSjPAGE gel (Laemmli, 1970). Tracks 1 and 9, molecular mass standards (phosphorylase b, M , 94000; bovine serum albumin, M , 67000; ovalbumin, M , 45000; carbonic anhydrase, M , 30000); tracks 2 and 8, blank; track 3, yeast cell-free extract; track 4, the S40 ammonium sulphate fraction; track 5, the P40-60 ammonium sulphate fraction; track 6, the post-Q-Sepharose fraction; track 7 pure yeast pyruvate kinase
Yun et al. (1976) report the partial purification of yeast pyruvate kinase by passage over DEAE-cellulose at pH 7.5 (further steps were required to complete purification). The purification described here employs a similar principle of adsorbing contaminating proteins to an anion-exchange column while the pyruvate kinase elutes in the break-through volume. However, the use of higher pH, a stronger anion exchanger and starting material highly enriched in pyruvate kinase, results in a much greater degree of purification. Gel filtration removed a significant amount of material with an A 2 6 0 / A 2 8 0ratio typical of nucleic acid, whilst the protein composition of the material was unchanged, as determined by SDSjPAGE (see Fig. 1). Table 1 shows that the specific activity of the preparation increases after gel filtration and it is proposed that the nucleic-acid-like material interfered with the determination of the protein concentration. Yun et al. (1976) report the need for a final gel-filtration step to remove a contaminating protease. The gel filtration therefore fulfils two functions: the removal of extraneous nucleic-acid-
51 7 Table 2. A comparison of recombinant with constitutively expressed yeast pyruvate kinase Subunit M , was determined by SDS/PAGE, tetramer M , by gel filtration. Where Fru(1,6)P2 is shown in parenthesis, it was present at 1 mM. PPyr, phosphoenolpyruvate Rcfcrence
Hill constant
Km
PPyr
PPyr [Fru(l,6)P21
ADP
Specific activity
Subunit pH M , x 1 0 - ~ optimum
Tetramer M,X
10-~
ADP [Fru(l,W',I units . mg-'
mM This work
3.7
0.16
1.15
0.54
2.86
367
57-59
6.0-6.5
210-240
Yun et al., 1976
1.8
0.10
0.34
0.16
2.3
350-400
50-52
6.2
208
Johannes and Hess, 1973
2.9
0.21
-
-
2.46
200
-
-
-
Weiker and Hess, 1971
2.91
0.21
-
-
2.10
200
6.3
-
Hunsley and Suelter, 1969
1.78
0.13
0.40
0.20
2.85
21 9
-
-
166
Haeckal et al., 1968
4.5
0.46
0.47
0.18
2.80
200
-
6.0-6.5
150
like material and the putative removal of a contaminating protease. Characterisation of wild-type yeast pyruvate kinase Table 2 lists a number of parameters determined for the pure recombinant enzyme in comparison with a number of previously reported preparations of yeast pyruvate kinase. As can be seen, pyruvate kinase expressed and purified as described in this report exhibits very similar behaviour to enzyme prepared from constitutively expressing yeast. The parameters that differ significantly are the specific activity, the molecular mass and the K, values determined when the ADP concentration is varied in the presence and absence of Fru( 1,6)P2. The reason for the high K , values is as yet unclear. The ratio of the values in the presence and absence of Fru(1,6)P2 is constant for all the preparations, suggesting that the effect of Fru(l,6)P2 is constant. It was observed here that ADP produced a significant apparent substrate inhibition when present at a concentration higher that 6 mM (see Fig. 2). As a result, the K, values were calculated from initial rates measured at or below 6 mM ADP. Many previous preparations report the routine assaying of yeast pyruvate kinase at 10 mM ADP, suggesting that either a substrate inhibition affect was not observed or was not taken into account when calculating kinetic parameters. The reason for this substrate inhibition effect is not apparent but has been reproduced with at least three separate preparations of this enzyme. Examination of Table 2 shows a correlation between a specific activity of approximately 200 units . mg protein-' and a tetramer M , of approximately 150000. The more recent preparations, including the current one, show a specific activity of approximately 350 units . mg protein-' and a tetramer M , of 210000-240000. The tetramer M , of yeast pyruvate kinase is 218 180 as calculated from the DNA-derived amino acid sequence (McNally et al., 1989). Therefore it appears that the enzyme was truncated in some way in the earlier preparations which produced a smaller enzyme with a lower specific activity. It is proposed that this is due to proteolysis which has now been avoided with the current emphasis on prevention.
+
1.0
e
.
0.2
0
2
1
6 [ADPI mM
a
10
Fig. 2. Yeast pyruvate kinase activity versus ADP concentration. Yeast pyruvate kinase activity is plotted as a function of ADP concentration in the presence (+) and absence (0)of 1 mM Fru(1,6)P2 showing the observed substrate inhibition effect. The curves have been fitted to the data collected a t ADP concentrations of 6 mM and lower. The rates have been normalised as fractions of the calculated Vmar.The assays were performed in a 1-cm-path-length cuvette in a total volume of 1 ml at 30°C. The cuvette contained 6 pmol phosphoenolpyruvate, 0.25 pmol NADH, 1 unit rabbit muscle lactate dehydrogenase. 15 pmol magnesium sulphate, 100 pmol KCI and 50 pmol Mes, p H 6.2. The reaction was started by addition of pyruvate kinase and monitored by recording the change in absorbance at 340 nm
Mutagenesis of yeast pyruvate kinase Two active-site mutants of yeast pyruvate kinase were expressed and purified in the manner described above. The mutations were Glu271 +Gin and Arg293jLys. The residue numbering is standardised to the cat muscle enzyme sequence (Muirhead et al., 1986). Steady-state kinetic measurements determined that mutant Glu271 +Gin exhibits a k,,, 18 500fold lower than the wild type. The K, for phosphoenolpyruvate in the presence of 1 mM Fru(l,6)P2 was determined as 0.32 mM. Mutant Arg293jLys displayed no pyruvate kinase activity under the wide range of conditions tested including very high enzyme, substrate and metal ion concentration, pH range 5.0 -8.0 and different bivalent cations.
518
IPPyrI (mM)
Fig. 3. Fluorometric titration of' wild-type and mutant yeust pyruvate kinase with phosphoenolpyriivate. The steady-state protein fluorescence quenching plotted as a function of phosphoenolpyruvate (PPyr) concentration. Wild type (+) mutant Glu271+Lys ( A ) ; Arg293+Lys (0).The protein was dialysed beforehand against one change of 50 mM triethanolamine/acetate, pH 6.2, 100 mM KCI, 20% (by vol.) glycerol containing approximately 0.2 g activated charcoal to remove contaminating fluorophores. The final protein concentration was 0.05 mg . ml-', the fluorescence was recorded with a SLM instruments SLM8000 fluorescence spectrophotometer with the excitation wavelength at 295 nm and the emission recorded at 334 nm
The bivalent cation specificity was determined by substituting the sulphate salt of the relevant cation for magnesium sulphate in the standard assay buffer. Both wild-type and mutant Glu271 +Gln exhibited the same specificity; MgZt >Co2'>Mn2' > > N i Z + >>Zn2'. The addition of phosphoenolpyruvate to yeast pyruvate kinase produces a significant protein fluorescence quenching when excited at a wavelength of 295 nm and the emission is recorded at 334 nm. This quench was used to determine the phosphoenolpyruvate-binding constant to the wild-type and mutant enzymes, see Fig. 3. The Kd and maximum fluorescence quenches for the wild type, Glu271 +Gln and Arg293 +Lys were 0.15 mM, 0.20 mM and 0.27 mM, and 22.8%, 23.8% and 2.38%, respectively. Circular dichroism spectra (Fig. 4) show that the proportion of secondary structure elements is very similar for wild-type enzyme and mutant Glu271 +Gln. Mutant Arg293 +Lys appears to have lost a proportion of a-helix and gained a proportion of random chain. The three-dimensional crystal structure of the cat muscle enzyme implicates Glu271 as a ligand for binding the enzymeassociated bivalent cation. It was predicted that substitution of Glu271 for a Gln would reduce or abolish bivalent-cation binding and hence activity. The preliminary data presented here supports but does not prove this hypothesis. The k,,, is profoundly reduced whereas the K, for phosphoenolpyruvate is not greatly altered. In addition the binding of phosphoenolpyruvate seems unaltered when measured by protein fluorescence quenching. Rose and Kuo (1989) suggest that Glu271 is involved in a proton-relay system in rabbit muscle pyruvate kinase. Changing this residue to a Gln would disrupt such a proton relay and would reduce the k,,, of the mutant, which is what is seen in the case of the yeast mutant Glu271 +Gln. However further work will be required to determine whether Glu271 is involved in bivalent-cation binding, a proton relay, a combination of the two or some other function. NMR may be used
1
190
I
210
I
I
230 Wavelength nm
I
I
250
Fig. 4. Peptide CD spectra of wild-type and mutant yeast pyruvate kinases. The peptide CD spectra of wild-type (WT) and mutants Glu271 +Gln (E271Q) and A r g 2 9 3 - L ~ ~(R293K) of yeast pyruvate kinase in the presence of 50 mM triethanolamine/acetate, pH 6.2, 100 mM KCI and 20% (by vol.) glycerol. Protein concentration was 1-2 mg ml-' and the spectra were corrected for protein concentration after collection. Each spectrum is an average of five scans with an instrument time constant of 1 s
to measure the Mn2' binding directly and repetition of the isotope-trapping experiments of Rose and Kuo will determine if a putative proton relay has been disrupted. Arg 293 has not been assigned a specific binding or catalytic role in pyruvate kinase, but appears from the X-ray data to be involved in a complex network of salt bridges at the active site. The data presented above show that mutant Arg293+Lys binds phosphoenolpyruvate poorly or that phosphoenolpyruvate binding does not induce the same degree of the tryptophan fluorescence quenching observed for the wild type. The CD spectra, and the fact that it has proved impossible to purify this mutant without some degree of proteolysis, suggest that it is not folded correctly. It is a common observation that incorrectly folded proteins are more susceptible to proteolysis (Pringle, 1974). The lack of activity and poor phosphoenolpyruvate binding are consistent with an incorrectly folded active site. It appears therefore, that Arg293 plays a key role in stabilising the conformation of the enzyme and, in particular, the active site. The conclusion is that yeast pyruvate kinase expressed and purified in the manner described in this report is similar if not identical to the same enzyme purified previously from constitutively expressing yeast. Constitutively expressed enzyme has been purified in this laboratory to the order of 50% pure, but the enzyme was of poor quality and unstable and so a detailed comparison with the highly expressed enzyme was not possible. The preparations of both wild-type and mutant enzymes remain stable for at least four months as precipitates
519 in 75% saturated ammonium sulphate. Furthermore, the protein remains stable at room temperature for up to a week in a suitable buffer (pH 6.0-8.0) in the presence of 20% glycerol. This is significantly more stable than many previous preparations and the same degree of stability as the enzyme purified from constitutively expressing yeast by Yun et al. (1976). The high level of expression does not appear to have affected its properties under the conditions tested. This high level of expression coupled to the modified purification protocol means that large amounts (20 mg from 1 1 culture) of this enzyme can be purified easily and rapidly. Furthermore the production of the pyruvate-kinase-deficient yeast permits sitespecific mutants to be expressed and purified free from wildtype enzyme contamination. This purification procedure removes the obstacle of enzyme availability and means that methods of investigation that require large amounts of the enzyme, such as crystallization for X-ray crystallography, can be readily attempted. The high-resolution crystal structure of cat muscle M I pyruvate kinase has been solved in conjunction with the protein sequence (Muirhead et al., 1986) and will greatly aid future work on the yeast enzyme in two ways. Firstly, the X-ray structure studies, should crystals be obtained, will be performed using the techniques of molecular replacement based on the known cat muscle M I tertiary structure. Secondly, the interpretation of the activities of site-directed mutants at a molecular level will require correlation with the three-dimensional structure of the enzyme. \
I
We thank Dr Jane Mellor and Joy Rathjen for the gift of plasmid pMA91-PYK and D r Stephen R. Martin for help recording and interpreting the C D spectra. We are also grateful to A. J. P. Brown and to our colleagues at the University of Bristol Molecular Recognition Centre for helpful discussions. T. H. L. M., T. M. and S. C. A. were funded by the Science and Engineering Research Council.
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