FEMS MicrobiologyLetters 94 (Iq92) 235-241) ~~ 1992 Federation of European MicrobiologicalSocieties1|37.~-11197/92/$1)5.111) Published by Elsevier

235

FEMSLE 04955

Purification and properties of pyruvate kinase from Thermoplasma acidophilum S i m o n P o t t e r and Linda A. F o t h c r g i l I - G i l m o r c Department o]"Biochemixtry, UnitersityOf I-dinhurgh. Ilugh Robson Buihlmg. Edinburgh. UK

Received 6 April Itlq2 Revision received 23 April Itlt~2 Accepted 27 April It~t~2

Key words: Pyruvate kinase; Arehaebacteria; Enzyme purification; Amino acid sequence; Thermoplasnla acidophihmz

1. S U M M A R Y

2. I N T R O D U C T I O N

Thermoplasma acidophilum is a thermoacidophi]ic archaebacterium occupying a paradoxical place in phylogenetic trees (phenotypically it is a thermoacidophile but phylogenetically it classifies with the methanogens). To better understand its phylogeny, the pyruvate kinase from this organism is being investigated as a molecular marker. The enzyme has been purified and has a native M r of 250000. It consists of four, apparently identical subunits each of M r 60011(I. No remarkable kinetic differences have been found between this thermophilic enzyme and its mesophilic counterparts other than its greater thermostability. Its amino acid composition has been determined and some partial sequencing has been done.

Pyruvate kinase (ATP-pyruvate 2-O-phosphotransferase. EC 2.7.1.40) catalyses the essentially irrcversible transphosphorylation from phosphoenolpyruvate (PEP) to ADP, a reaction that requires magnesium and potassium ions. The enzyme has been extensively studied because of its importance in controlling the flux through glycolysis from fructose 1,6-bisphosphate down to pyruvate. Amino acid sequences [1] are known for pvruvate kinases isolated from several sources, a r d a crystal structure is available for the cat muscle enzyme [2]. The apparent ubiquity of the enzyme, together with the maintenance of function in all of the organisms from which it has been isolated, suggested its use as a molecular marker for the construction of phylogenetic trees. It would thereby be complementary to the R N A or ribosome phylogenies proposed by Woese [3] and Lake [4]. Phenotypically, with regards to glycolysis, the archaebacteria fall into two groups: (a) the

Correspondence to: S. Potter, Department of Biochemistry.

University of Edinburgh, Hugh Robson Building, George Square, Edinburgh EH8 9XD, UK.

236 halophiles and methanogens, which possess an Entner-Doudoroff pathway and the enzymes in the lower part of the glycolytic pathway, and (b) the thermoacidophiles which utilise a non-phosphorylated pathway and possess only two of the common enzymes, enolase and pyruvate kinase [5]. These observations have led to the hypothesis that glycolysis evolved in the gluconeogenic direction (from the 'bottom up'). In order to investigate this hypothesis, the pyruvate kinasc of the thermoacidophilic archaebacterium Thermoplasma acidophilum has been purified to homogeneity, characterised and partially sequenced. This study provides the first kinetic and sequence information about a glycolytic enzyme from a thermoacidophilic archaebacterium.

3. METHODS

3.1. Cell culture Thermoplasma acidophilum was supplied as a freeze-dried culture by the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Braunschweig, FRG. Cells were cultured at 60°C and pH 2.0 (as recommended in the 1989 Catalogue of Strains provided by that organisation) in a temperature-controlled 2-1 glass vessel.

3.2. Purification of the pyrurate khlase The pyruvate kinase was purified from Thermoplasma crude extract by a series of chromatographic steps done at 45-50°C. Cells were collected by centrifugation and 200 mg were resuspended in 1.5 ml of lysis buffer (20 mM NaCi, 10 mM MgCI 2, 10 mM Tris. HCI (pH 7.5), containing 1,10-phenanthroline, E64C, 3,4-dichlorisocoumarin protease inhibitors; all 10 mM) and were homogenised with glass beads for 5 min at room temperature. The suspension was briefly centrifuged to remove cell debris, the crude extract was made up to 35% saturation with ammonium sulphate and left to stand at room temperature for 20 min with occasional mixing. The precipitate was removed by centrifugation for 2 min in a microcentrifuge and the supernatant was made up to 65% saturation with ammonium sul-

phate. After standing for 20 min, the mixture was centrifuged for 2 min and the supernatant was discarded. The pellet was resuspended in 1 ml of lysis buffer and this was dialysed overnight at 45°C against l I of lysis buffer (without the protease inhibitors). The dialysed sample was applied to a Mono-Q ion-exchange column (Pharmacia, :::~ mm z x 42 mm) and the column eluted with ~. linear gradient 0-300 mM NaCI in lysis buffer over 30 min at a flow rate of 0.5 ml/min. Fractions of l ml were collected, and those containing pyruvate kinase activity were pooled and applied to a Superose 6 gel filtration column (Pharmacia, 172 mm2× 285 mm). The column was eluted isocratically with lysis buffer at a flow rate of 0.5 ml/min. Active fractions of i ml were pooled and applied to a column of 5'-AMP agarose (Sigma, 79 mm2× 50 mm). The pure pyruvate kinase was eluted from the column with l0 mM phosphate buffer (pH 7.5) containing l0 mM ADP (Sigma).

3.3. Enzyme characterisation The pyruvate kinase was characterised by enzyme assays, SDS-polyacrylamide gel electrophoresis, amino-acid analysis and partial sequencing. Two assays for the enzyme were used. For qualitative detection of pyruvate kinase activity during the purification procedure, the coupled assay of Bucher and Pfleiderer [6] was used at 45°C (a compromise temperature to allow high activity of the pyruvate kinase without complete extensive denaturation of the coupling lactate dehydrogenase). For kinetic studies however, this coupled assay could not be used and so the direct, spectrophotometric assay of Pon and Bondar [7] was employed at 60°C. For each assay the cuvette contained in l ml: MgSO4, 7.2 mM; KCI, 7.2 mM; Tris, 0.05 M; pyruvate kinase, l /zg and ADP and PEP in invariant concentrations. The pH of the buffer was fixed at 7.5. Amino acid analysis was performed using an Applied Biosystems 420A Derivatizer with automated hydrolysis (Cronshaw, A.D., MacBeath, J.R.E., Shackleton, D.R., FothergilI-Gilmore, L.A. and Hulmes, D.J.S. unpublished results). PTC-amino acid derivatives were separated by reverse-phase HPLC and detected at 254 nm.

237 The N-terminus of the enzyme proved to be blocked and hence, prior to sequencing, the pyruvate kinase was proteolytieally cleaved by elostripain (Sigma). The pyruvate kinase was reduced and earboxymethylated prior to digestion, and the protease was pretreated with Ill mM DTE for 2 h at 4°C. Digestion was done at pH 7.5 for 2 h at 37°C and at a p r o t e i n / p r o t e a s e ratio of 125:1 and at 1.5 mg p r o t e i n / m l of 20 mM ammonium bicarbonate. Resultant peptides were separated and purified using an Applied Biosystems 130A microbore separation system with an Aquapore RP-300 column (7 v.m pore size; 2.1 mm x 30 mm). The purified peptides were then subjected to automated sequencing using an Applied Biosystems 477A instrument with a 120A on-iine phenylthiohydantoin analyser [8].

4. RESULTS AND DISCUSSION Pyruvate kinase purified by the above procedure was shown by S D S - P A G E to be homogeneous (Fig. 1). The increase in total activity noted

97.4

45

J

2g

Fig. 1. SDS-PAGE gel of the purified pyruvate kinase. The gel was stained with Coomassiebrilliant blue R (Sigma). The left lane contains the purified pyruvate kinase eluted from the 5'-ADP-agarosecolumn. The right t'~necontains high molecular mass markers (Sigma).The M~ valuesshown are × 10 3.

Tablc 1 Purification table Ibr Fhermoplasnla pyruvate kinase Step

Activity Protein Specific Purifications (mg) acti'~'ity (U/rag)

(U)

Cell-free extract Ammoniumsulphate precipitation Mono-O Superosc h 5'-AMP-agarosc

11.2

28.11

13.5 14.0 13.N 10.5

17.2 11.78 1.tJ6 I).56, 25.0 62.5 0.28 4tk5 124 11.1}5 2111.2 5113

(I.411

I

Units of specific activity arc #mol NADII converted min i (rag of protein) i. Final rec(wery (from maximal activity found) is 75r~.

during the procedure is possibly due to the presence of inhibitor molecules in the crude extract. The enzyme is a tetramer of subunits of M r 60000 (the subunit M r was determined from SDS-PAGE gels and the tetramer M r from the Superose 6 column), it has a specific activity of approximately 21111U / m g which is perhaps a little low for a pyruvate kinase (Table 2). This is not due to the assay conditions used (as the enzyme was incubated for Ill rain at 60°C before the assay was started) but may be explained by partial co!d denaturation of the enzyme - - activity is negligible below 40°C. The inactivation is reversible if the enzyme is incubated at room temperature but freezing denatures it irreversibly. A purification table for the enzyme is shown in Table !. The kinetic properties of the enzyme are listed in Table 2. They show that, whilst the enzyme exhibits fairly typical K0.5 values for its various substrates and is activated by AMP, it is much more stable at higher temperatures than any of its counterparts, with the possible exception of the enzyme from Bacillus stearothermophilus. Indeed, previous studies [9] have shown that the enzyme is stable for over 10 n:in at 90°C and half an hour at 70°C. It is hoped that primary structure information gleaned from cloning studies currently in progress may shed some light on this enhanced thermostability of the Thermoplasma enzyme. The amino acid composition of the pyruvate kinase has been determined and resembles that of other pyruvate kinases except that there are

238

elevated levels of proline residues. Proline is thought to stabilise a protein by reducing the number of conformations available to the unfolded state [12]. Further, asparagine levels are low, possibly because there would be a selective disadvantage in retaining this readily deamidated amino acid. The N-;Ierminus of the enzyme proved to be blocked. This appears to be common amongst pyruvate kinases from other sources and hence came as no surprise. The enzyme was, therefore, cleaved into peptides using the proteolytic enzyme clostripain, and selected peptides were sequenced. Two stretches of sequence were obtained. The first piece of sequence was A V A L D T K (initial yield 180 pmol; repetitive yield for alanine 82%). The initial aianine is in fact the fourth residue of the peptide as the first three cycles were contaminated with an unknown peptide. The sequence ~:an be identified with an

Table 2 Physical. chemical and kinetic properties of the pyruvate kinase Property

T. acido.

Specific activity (U/mg) 201 Temperature (°C) of assay 60 K,. 5 [PEP] (mM) 11.64 K,.5 [PEP]+ AMP (mM) (1.043 K,. 5 [PEP] + ATP (mM) 11.70 K,. 5 [ADP]+ AMP (mM) 0.1 K.. 5 [ADP] + ATP (mM) 11.68 Hill coefficient (PEP) 1.7 pH optimum (-AMP) 7,5 Native M r ( × 10- 3) 251) No. of subunits 4 Subunit M r ( x 10 -3) 60 Blocked N-terminus? yes

B. stearo.

Yeast

333

367

60 2.0 11.2

37 3.7 0.16 11.54

1.8 7.2

2.86 6.0-6.5

250 4

240 4

62 yes

59 yes

Units of enzyme activity ixmol/min. - indicates value not measured. K.. s is the dissociation constant fo~ the enzymesubstrate complex. K.. is not used as this would imply typical Miehaelis-Menten kinetics while the enzyme exhibits alIosteric control. B. stearo., Bacillns stearothennophihts; T. acido.. Thermoplasma acidophihml. Values for the Bacillus enzyme were from reference [lO] and those for the yeast (Saccharomyces cererisiae) enzyme from reference [l 1].

SEQUENCE1

AVALDTK

124 hummus2 ratmus2 ratmusl catmusl chimus humliv ratliv ratrbc potCy Anid Anig yea TbrCyl TbrCy2 Eco Bst

VAVALDTKGPEIRTGLIK VAVALDTKGPEIRTGLIK VAVALDTKGPEIRTGLIK VAVALDTKGPEIRTGLIK VAIALDTKGPEIRTGLIK VAIALDTKGPEIRTGILQ VAIALDTKGPEIRTGVLQ VAIALDTKGPEIRTGVLQ CAVMLDTKGPEIRTGFLT VAIALDTKGPEIRTGNTV LAIALDTKGPEIRTGNTP LAIALDTKGPEIRTGTTT IGIALDTKGFEIRTGLFK IGIALDTKGPEIRTGLFK AAILLDTKGPEIRTMKLE VAILLDTKGPEIRTHNME **********

Fig. 2. Alignment of the Thermoplasma sequence to known pyruvate kinase sequences. Sequences shown are all those so far obtained between positions 107 and 124 according to the numbering for the human muscle enzyme (top). * indicates residue completely conserved. Abbreviations: hummus, human muscle: ratmus, rill muscle; chimus, chicken muscle: humliv, human liver; ratliv, rat liver; ratrbc, rat erythrocyte; potCy, potato cytoplasm: Anid. Aspergillus niduhms" Anig, Aspergillus niger" yea. Saccharomyces cerecisiae; TbrCi, To'panosonm brucei cytoplasm: Eco, E. coli: Bst, Bacillus

slearolhermophihls.

active-site region of pyruvate kinase: AVALDTKGPEIRT. The last ten residues of this region are conserved in all known sequences (Fig. 2). It seems likely that the Thermoplasma enzyme also maintains this conserved sequence. A second stretch of sequence from a separate peptide was also obtained by Edman degradation: AGIYLPG A N (initial yield 150 pmol, repetitive yield for glycine 91%). This sequence could not be aligned with other pyruvate kinase sequences and hence its position in the sequence has yet to be determined. Oligonucleotide probes were designed for both of these sequences using codon usage tables derived from the sequences of the citrate synthase [13] and the glucose dehydrogenase [16] of Thermoplasma. Both probes are currently in use with the eventual aim of cloning the pyruvate kinase gene and expressing the pr,:~tein in sufficient quantities for further structural studies.

239 T h e similarity s h o w n b e t w e e n t h e partial seq u e n c e o b t a i n e d a n d t h e c o n s e r v e d r e g i o n o f all p y r u v a t e k i n a s e s (Fig. 2) is striking, s u g g e s t i n g t h a t t h e Thermoplasma p y r u v a t e kinase is o n e o f a family o f divergently evolved e n z y m e s . T h i s c o m p l e m e n t s w o r k on p h o s p h o g l y c e r a t e k i n a s e s which has shown that the archaebacterial phosp h o g l y c e r a t e kinase s e q u e n c e s a r e 3 0 - 3 6 % identical to t h e i r e u b a c t e r i a l a n d e u k a r y o t i c c o u n t e r p a r t s [14]. T a k e n t o g e t h e r , t h e s e s t u d i e s a n d t h e p r e s e n t o n e on t h e Thermoplasma p y r u v a t e kin a s e a d d weight to t h e s u g g e s t i o n that t h e archaebacterial glyceraldehyde-phosphate dehydrog e n a s e - - w h i c h s h o w s only 7 - 1 5 % identity with e u b a c t e r i a l a n d e u k a r y o t i c c o u n t e r p a r t s [15] - has been derived from a non-homologous enzyme w h i c h h a s c o n v e r g e d to f u n c t i o n as a glyceraldehyde-phosphate dehydrogenase.

ACKNOWLEDGEMENTS T h a n k s a r e d u e to A n d r e w C r o n s h a w , L i n d a K e r r a n d S t e p h e n P e a c o c k for t h e s e q u e n c i n g a n d a m i n o acid analysis w o r k a n d to Dr. A n d r e w Ryle for his h e l p f u l c o m m e n t s o n t h e m a n u s c r i p t . T h e w o r k w a s s u p p o r t e d by t h e Science a n d E n g i n e e r i n g R e s e a r c h Council.

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