Btochmuca et 8iophvswa Acta. 1073(1991) 142-148 © 1991 Elsevierbk:iencePublishersB.V.(BiomedncalDivision)0304-4165/91/$03.50 ADONIS 030441659100087F
142
Purification and uroperties of an extreme thermostable glutamate dehydrogenase from the archaebacterium Sulfolobus solfataricus M a n f r e d F. S c h i n k i n g e r 1, B e r n h a r d R e d l ~ a n d G e o r g StiSffler 1.2 I Institut fiir M~kro~iologie. Medlzinisehe Fakultiit der Universttlt~ lnnsbruck (Austria) and 2 Max-Planck-lnstitut ]'fir Molekulare Genetik. Abteilung Wittmann, Berlin (F.R.G.)
(Received 2;. January 1990) (Revised manuscriptreceived23 July 1990)
Key words: Glutamate dehydrogenasc;Thermophile;Archaebacterium;(S. sol/atancus) Glutamate dehydrogenase (L-glutamate: NAD(P)* oxidoreductase, deaminating, EC !.4.1.3.) of the extreme tberme-philic archaebacterium S u l f o l o b u s solfataricus was purified to homogeneity by (NH4)zSO 4 fractionation, anion-exchange chromatography and affinity chromatography on 5'-AMP-Sepharose. The purified native enzyme had a M, of abc,ut 270000 and was shown to be a hexamer of subunit M, of 44000. It was active from 30 to 95°C, with a maximum a,:tivity at 85°C. No significant loss of enzyme activity could be detected, either after incubation of the purified enzyme at 90°C for 60 min, or in the presence of 4 M urea or 0.1% SDS. The enzyme was catalytically active with both N A D H and N A D P H as eoenzyme and was specific for 2-oxoglutarate and L-glutamate as substrates. With respect to coenzyme utilization the S u l f o l o b u s solfatarlcus glutamate dehydrogenase resembled more closely the equivalent enzymes from eukaryotic organisms than those from eubacteria.
Introduction Sulfolobus solfataricus an extreme thermophilic sulfur
dependent archaebacterium with an optimal growth temperature of 85°C, has been isolated from hot acid habitats [1]. Since Sulfolobus grows at very high temperatures it should provide a source of enzymes with unusual physicochemical properties. In addition to its growth in extreme environments, the study of S u l f o l , bus, as a member of the third i:rimary kingdom of organisms [2] is interesting from a phylogenetic point of view. It has been shown that some biochemical features of Sulfolobus are more closely related to eukary~.'os than to eubacteria [3]. Glutamate dehydrogenase occupies a pivotal posi.t'on in the metabolism of most organisms because it Frovidzs a link between carbohydrate and nitrogen metabolism. It is a well-studied enzyme and has been purified from both prokaryotes [4-9] and eukaryotes [10-13]. Sequence analysis have shown that glutamate dehydrogenases belong to the most conserved enzymes [141.
Correspondence: B. Redl. Institut for Mikrobiologie. Medizinische Fakuh~it der Universit~it Innsbruck, Fritz-Pregl Str. 3. A-6020 lnnsbruck, Austria.
In this work we report the purification, the molecular and kinetic properties and the thermostability and resistance against dissociating reagents of a NAD(P)+-de pendent glutamate dehydrogenase from S u l f o l o b u s solfataricus.
Materials and Methods Materials
DE-32 cellulose was purchased from Whatman, Maidstone0 U.K. 5"-AMP-Sepharose was obtained by Pharmacia, Uppsala, Sweden. N A D H was of analytical grade and purchased from Scrva, Heidelberg, F.R.G. N A D P H Type V, pyridoxal 5-phosphate, iodoacetamide, protaminesulfate from Salmon, Grade llI and the standard proteins used in the SDS-polyacrylamide gel electrophoresis and M, studies were products from Sigma Chemicals, Munich, F.R.G. Rabbit antigoat-lgG alkaline phosphatase conjugate and 5-bromo4-chloro-3-indolyl phosphate were from Sigma Chemicals, Munich, F.R.G. The TSK-250 HPLC column, the standard proteins for the Mr studies and the protein assay kit were purchased from Bio-Rad Laboratories, Richmond, U.S.A. All other reagents were of analytical grade and obtained from Merck, Darmstadt. F.R.G.
143 Glutamate dehydrogenases from Proteus sp., Candida utilis and rat liver used for immunization were purchased from Sigma Chemicals (Munich, F.R.G.) and were further purified by gel permeation HPLC.
Culture o/cells The archaebacterium Sulfolobus solfataricus strain DSM 1616 was grown aerobically at 85°C as previously described [15].
of protein during the enzymatic amination of 2-oxoglutarate to L-glutamate. The enzymatic activity as a function of pH was tested in the standard assay mixture but with triethanolamine replaced by 60 m M K H 2 P O j / N a z H P O 4 (pH 5.0-8.0) or 50 mM diethanolamine (pH 8.0-10.0). All pH values were adjusted at the assay temperature (70°C). NaC1 was added to each buffer to bring the ionic strength to the same value.
Enzyme purificatton
Protein determination
Unless otherwise stated, all purification steps were carried out at 4°C. Wet cells (10 g) were resuspended in 2 volumes of buffer A (50 m M Tris-HCI (pH 7.6) containing 1 mM EDTA) and cell lysis was performed by passing the bacteria twice through a French pressure cell operated at a pres:~ure of 97 MPa. The extract was then clarified by centrifugat;on at 40000 x g for 45 min. Elimination of nucleic acids was carried out by adding 0.2 ml of a 2% (w/v) solution of protamine sulfate in buffer A per ml crude extract and stirring for 60 min at room temperature. After centrifugation at 40000 x g for 30 rain the supernatant was adjusted to 55% (w/v) a m m o n i u m sulfate and again stirred foJ 60 rain. The precipitate was collected by centrifugation at 40000 x g for 30 rain. The pellet was resuspended in buffer A and extensively dialysed against 200 volumes of the same buffer. The homogenate was then loaded onto a DE-32 (Whatman) anion-exchange column (1.5 x 12.5 cm) equilibrated with buffer A. After the column had been washed with equlibration buffer to remove unadsorbed material, elution was carried out with a linear gradient (140 ml) from 0 to 0.8 M NaCI in buffer A. Fractions (2 ml each) were collected at a flow rate of 60 m l / h . Those showing glutamate dehydrogenase activity were pooled and dialysed against buffer B (0.1 M sodium phosphate, pH 7.0). The solution was applied to a 5'-AMP-Sepharose column (0.8 x 8 cm) equilibrated with buffer B. The column was washed with buffer B until the adsorption at 280 n m returned to the baseline. The column was then washed with 20 ml buffer B containing 5 m M 2-o:toglutarate. Glutamate dehydrogenase was eluted specifically by using a solution of 0.5 m M N A D + and 5 m M 2-oxoglutarate in buffer B at a flow rate of 20 m l / h . The active fractions were pooled and stored at 80oc.
Protein concentration was determined according to Bradford [16] using the Bio-Rad Protein Assay Kit, with bovine serum albumin as standard.
-
Glutamate dehydrogenase assay The glutamate dehydrogenase activity was measured at 70"C by monitoring the NAD(P)H oxidation at 340 n m for 5 rain in a reaction mixture (1 ml) containing 50 m M triethanolamine-HCI (pH 7.6), 2.5 m M EDTA, 100 m M CH3COONH4, 0.2 m M NAD(P)H and 7 m M 2-oxoglutarate. One unit of dehydrogenase activity was equal to the release of 1 p,M NAD(P) + per rain and mg
M, determination of native enzyme Gel permeation chromatography by HPLC was used for determination of the M, of native glutamate dehydrogenase. Pure pi'eparatiuns ot" enzyme were applied to a TSK-250 column equilibrated and eluted with 20 mM phosphate buffer (pH 7.0) containing 0.15 M NaCI at a flow rate of 1 m l / m i n . The column was calibrated with the following standard proteins as mz,~kers: thyroglobulin (M, 669000), catalase (240000), y-globulin (180000), ovalbumin (45000) and myoglobin (17000). A computing integrator was used for monitoring peak area and retention time,
Polyacrylamide gel electrophores~.s SDS-polyacrylamide gel electrophoresis was performed using 10% (w/v) acrylamide slab gels according to Laemmli [171, run in a Bio-Rad Mini Protean vertical electrophoresis unit (Bio-Rad Laboratories, Richmond, U.S.A.). The subunit molecular weight was determined according to Weber and Osborn [18] by comparison with the migration of protein markers of known M, values (phosphorylase b, bovine serum albumin, ovalbumin and carbonic anhydrase). Isoelectric focusing was carried out in an LKB Multiphor apparatus using broad pH (3.5-9.51 thin-layer polyacrylamide gels [19]. A mixture of 11 standard proteins (Pharmacia calibration kit) with known p l values was run in parallel in the same gel. The proteins were stained with Coomassie brilliant blue R and densitometric gel scans were used to assess the pl.
Amino acid analysis Homogenous protein was subjected to acidic hydrolysis and the amino acid analysis was performed as described in Ref. 24. Amino acids were detected as their OPA (ortho-phtaldialdehyde/mercaptoethanol) derivates on a waters HPLC system equipped with a Shimazu RF 530 fluorescence detector. Cysteine residues were determined after performic acid oxidation as previously described [34]. Tryptophane was determined after hydrolysis in 6 M HCI in 7% (w/v)
144 kDa 2o5
thioglycolic acid according to Melzer et al. [35] and proline was determined as described by Bohlen and Mellet [36].
~116 0
hmnunological procedures Antisera were raised agair, st the glut.amate dehydrogenases from Sulfolobus solfataricus, Proteus sp., Candida utilis and rat liver, respectively, in sheep. The proteins were mixed with an equal volumn of Freund's complete adjuvant and subcutaneous injection of 250 to 500 I~g (Candida, Proteus, rat) and 50 to 100 /tg (Sulfolobus) were performed four times at 3 week intervals. The total amount of protein injected per sheep was 1.3 mg of Candida glutamate dehydrogenase, i.2 mg of Proteus glutamate dehydrogenase, 2 mg of rat glutamate dehydrogenase and 0.80 mg of Sulfolobus glutamate dehydrogenase, respectively. One week after the last booster, sera were collected and stored at - 20°C. Control sera were collected before the first injection. The immunological relatedness was tested by immunoblotting using a slight modification of the procedure of Towbin [20]. The modification included the use of a rabbit anti-goat-lgG alkaline phosphatase conjugate as secondary antibody and 5-bromo-4-ehloro-3indolyl phosphate as substrate for the phosphatase [21]. Immunoblots were made from SDS-polyacrylamide gels and from non-denaturating polyacrylamide gels using a gel system for acidic proteins according to Davis [22].
a
4 97.4 ~66
"~--
Fig. l. SDS-polyacrylamidegel electrophoresisof glutamate dehydrogenase from S. sol/ataricus(a) 3 Fg oI purified enz'ime.(b) molecular weight markers: myosin Mr = 205000 (subunit). /~-galactosidasc Mr = 116000 (subanit). phosphoryla~ b Mr = 97400 (subunit). albumin bovine Mr =66000. albumin egg M~= 45000. carbonic anhydras¢ M, = 29000. Protcins were stained with Coomassiebrilliant blue R.
the enzyme preparation appeared to be homogeneous (Fig. 1).
Molecular weight and isoelectric point The purified glutamate dehydrogenase haa a retention time on HPLC gel permeation column that corresponded to a M r of 270000 + 15000, based upon the regression equation for the retention time of the five standard proteins used. The subunit M r was shown to be 44000 5= 2000 by SDS-polyacrylam;de ",,el electrophoresis. The isoelectric point ( p l ) of the enzyme was 6.2 _+.0.2, as calculated from isoelectric focusing.
Results
Purification of glutamate dehydrogenase The results of a typical purification procedure are summarized in Table 1. The most effective purification step was represented by the affinity chromatography on 5'-AMP-Sepharose. The high efficiency was due to the complete adsorption of the enzyme on the affinity column and to its specific elution with 2-oxoglutarate and NAD+. The S. solfataricus glutamate dehydrogenase was purified about 604-fold over the crude extract, with a specific activity of 423 units per mg of protein at 70°C. As judged by SDS-polyacrylamide gel electrophoresis
Parameters affecting enzyme activity and stability Stability against thermal inactivation was tested after preincubation of the purified enzyme for 1 h at temperatures ranging from 70 to 100°C. No significant loss of enzyme activity could be deter.ted after preincubation at temperatures up to 90°C. Even boiling for 1 h did not inactivate the enzyme completely (Fig. 2).
TABLE I
Purifwationof Sulfolobu.~solfatarict~glutamatedehydrogenase
Crude extract
Step
Total protein (mg) 180
Protaminc sulfate precipitation Ammoniumsulfate fractionation DE-32 chromatography 5"-AMP-Sepharosechromatography
130 45 10 0.7
Total activity (units)
126 l 17 95 120 296
Specific acti,':w, (unlts/mg) 0,7
Purification (-fold) l
0.~ 2.1 12 423
1.3 3 17 604
145 to.!
I'ABLE 11
Stab,htl ,j S ~,;l[atart¢t~ glutantun, detrrdrogen~tte 80
.ACtlVltlC,, a t e
mean ranges of two determinations.
Treatment
60
40"
20"
70
80
90
I00
temperature (°C) Fig. 2. Effect of temperature on stability of glutamate dehyflrogena~e. Purified enzyme (70 ng) was preincubated in 50 mM tricthanolammeHCI (pH 7.5) for 1 h at the temperatures indicated and the remaining activity was determined using the standard as~,ay.
W h e n the e n z y m a t i c activity w a s tested at v a r i o u s t e m p e r a t u r e s , it w a s f o u n d that the g l u t a m a t e d e h y d r o g e n a s e exhibited m a x i m u m activity at 8 5 ° C . At 9 5 ° C the e n z y m a l i c activity w a s still 90% a n d 35% of the m a x i m u m activity were m e a s u r e d at 3 0 ° C ( d a t a not shown). T h e e n z y m a t i c ~c~i,,ity vs. p H w a s d e t e r m i n e d over a p H r a n g e o f 5 . 0 - 1 0 . 0 . In the r a n g e c ~ p H 7 . 0 - 8 . 0 , g l u t a m a t e d e h y d r o g e n a s e exhibited 9 0 - 1 0 0 % activity, with a m a x i m u m at p H 7.6, as s h o w n in Fig. 3. T h e p H stability o f S. solfataricus g l u t a m a t e d e h y d r o g e n a s e w a s e x a m i n e d b y p r e i n c u b a t i o n o f the p u r i fied e n z y m e in 50 m M c i t r a t e / H C I b u f f e r ( p H 3.0), 50 m M c i t r a t e / H C I b u f f e r ( p H 5,0) a n d 50 m M di16" 14" 12" I0 o 8" 6" 4~ 2" 0 5
6
7
8
9
tO
pH
Fig. 3. Effect of pH on activity of glutamate dehydrogenas¢. 70 ng of purified enzyme was incubated at 70°C for 5 rain in a reaction mixture containing 7 mM 2-oxoglutarate. 2.5 mM EDTA, 0.1 M CH COONH,=, 0.2 mM NADH and the buffers employed were 60 mM KH2PO,=/Na2HPO. = (I) or 50 mM diethanolamin¢ (A). NaCI was added to each buffer to bring the ionic strenght to the same value.
pH = 3,0. 14 h. 25~(" pH = 4.0. 14 h. 250(. pH =9(1, 14 h. 25~C Pyridoxal 5-pho,,phate 2 mM lodacetamtde 2 mM ,)-glutamate: 2 mM 3ram 4 mM 6 mM 4 M urea. 12 h. 25°C 6 ,M urea, 12 h, 25°C 75 M urea. 12 h, 25~(." 0.1~ SDS, 12 h. 25°C 3.5~ SDS, 12 h. 250( . 50~ (,./v)ethanol, 12 h, 250(.50"~ (,../v) methanol. 12 h, 25°C 50'~ (~,/~) t~propanol, 12 h, 25°C 90°( ". 1 h 100°C. I h
Actt','lty ("~ of control) 75 4-_7 64+5 83+3 0 le2 + 4 89 + 2 78+_3 71 ± 2 66 + 1 105 ± 7 88±2 45±6 98+_ 1 4±3 0 3±2 0 95 + 2 20+_.5
e t h a n o l a m i n e ( p H 9.0), respectively, for 14 h at r o o m t e m p e r a t u r e . T h e S. sol/atarwus e n z y m e w a s stable over a b r o a d p H r a n g e ( p H 3 - p H 9), with at least 65% residual activity a f t e r p r e i n c u b a t i o n for 14 h in buffers with different p H values ( T a b l e II). T h e effect o f d i s s o c i a t i n g r e a g e n t s a n d o r g a n i c solvents o n e n z y m e activity w a s studied b y p r e i n c u b a t ing the p u r i f i e d e n z y m e in these r e a g e n t s for 12 h at r o o m t e m p e r a t u r e . T h e residual activity w a s m e a s u r e d at 7 0 ° C as described, T h e e n z y m e w a s c o m p l e t e l y s t a b l e a f t e r 12 h preinc u b a t i o n in 4 M urea, a n d r e t a i n e d a l m o s t 90% o f the initial activity a f t e r 12 h i n c u b a t i o n in 6 M urea. It h a d a half-life of a b o u t 9 h in 7.5 M u r e a ( T a b l e II). N o loss o f activity w a s d e t e c t e d a f t e r 12 h o f p r e i n c u b a t i o n in 0.1% S D S (w/,.~: w h e r e a s the half-live o f the e n z y m e w a s 5 h in 0,5% S D S ( T a b l e I!). T h e influence of p y r i d o x a l p h o s p h a t e , a w e l l - k n o w n i n h i b i t o r o f a n u m b e r o f g l u t a m a t e d e h y d r o g e n a s e s 123] w a s tested. A d d i t i o n o f 2 m M p y r i d o x a l 5 - p h o s p h a t e to the a s s a y m i x t u r e resulted in a c o m p l e t e loss o f activity of the Sulfolobus enzyme. P r e i n c u b a t i o n with 2 m M i o d o a c e t a m i d e for u p to 10 h d i d not inhibit the e n z y m e activity. A c o n c e n t r a t i o n d e p e n d e n t inhibition o f enz y m e activity by D-glutamate, a s t r o n g c o m p e t i t i v e inhibitor o f both, the b o v i n e liver a n d Neurospora g l u t a m a t e d e h y d r o g e n a s e s [231 has been detected ('['able II). T r e a t m e n t with 50% ( v / v ) o f either e t h a n o l , m e t h a n o l or i s o p r o p a n o l resulted in a c o m p l e t e loss o f e n z y m e activity. W h e n the purified e n z y m e w a s stored at 4 ° C for m o r e t h a n 20 d a y s n o significant loss o f e n z y m e activity
146 TABLE 111 Apparent K,, t'aluesfor substrates and coencymes of glutamate dehydrogenasefrom Sulfolobtt~solfatarwus Substrale-coenzyme
2-Oxoglutarate-NADH 2-Oxoglutarate-NADPH t-Glutamate-NAD" t-Glutamate-NADP*
Km values substrate (raM)
coenzyme (/tM)
0.6 0.2 0.3 1.1
7 10 63 25
was seen. After 40 days at 4°C the activity decreased to 80%. For long-term storage a temperature of - 7 0 ° C was choosen, since no significant loss of activity was seen after a period of 6 month at this temperature. Kinetics The kinetic parameters of S. solfataricus glutamate dehydrogenase for various substrates and coenzymes were determined at 70°C using the standard assay. The purified enzyme exhibited Michaelis-Menten kinetics and Table 11I summarizes and compares the Michaelis constants (Kin) of the enzyme with respect to 2-oxoglutarate, L-glutamate (7 mM each) N A D +. NA D P* , N A D H and NA D P H (0.2 mM each), estimated by double-reciprocal plots. As shown in Table II1, the formation of L-glutamate by the S. solfataricus enzyme was not strictly N A D H TABLE IV ,4mino acid composition of hexameric glutamate deh)'drogena~esfrom S. solfatartcus (Ss), F~ colt (Ec). B. megatermm (Bin), N. crassa (Nc) and bovine liver (BI) Amino acid tool% Ss [da;a from thispaperl (res./mol J ) Cys Asp Glu His Set Gly Thr Arg Ala Tyr
Trp Met Val Phe lie Leu Lys Pro
1.0 (3.8) 10.5 (42.8) 9.4(38.3) 0.7 (2.9) 3.3 (13.4) 13.0(53.0) 4.2 (17.1) 4.4 (17.9) 10.1 (41.2) 2.8 (11.41 0.8 (3.6) 3.2 (13.2) 8.2 (33.4) 2.2 (8.9) 6.2 (25.7) 8.3 (34.6) 9.4 (38.1) 2.5 (10.1)
Ec [51
Bm [61
1.4 1.5 9.4 8.3 12.4 10.2 2.1 3.1 6.7 5.6 11.7 11.8 5.2 5.2 4.1 4.7 10.4 11.2 2.8 2.0 1.0 n.d. 2.3 3.0 7.8 7.1 3.4 4.6 3.5 4.2 7.1 9.3 5.1 4.1 3.7 3.9
A molecularweightof 45000 was assumed.
Nc [281
BI 1291
1.3 8.7 11.6 1.9 7.4 11.6 4.2 3.5 11.3 3.2 1.6 1.6 7.1 3.9 3.9 7.7 6.1 3.2
1.2 9.7 9.3 2.8 5.9 9.3 5.9 5.9 7.5 3.6 0.6 2.8 6.7 4.5 7.3 6.3 6.5 4.1
dependent. The enzyme was also able to use N A D P H as coenzyme. At the assay temperature (70°C) a V,,~ of 0.4 mM 2-oxoglutarate per rain and mg of protein was calculated. A m i n o acid analysis The amino acid composition of the glutamate dehydrogenase from S. solfataricus is given in Table IV together with the composition of glutamate dehydrogenases from Escherichia coil, Bacillus megaterium, of the hexameric NADP-dependent enzyme from Neurosporo crassa and of the glutamate dehydrogenase from bovine liver. For comparison of the amino acid compositions in Table IV we have used the composition divergence ( D ) of Harris and Teller [25]:
D = ~ f ~ t ( Xi.A- X,.~)-" were X,.^ and Xi.B are tL: values of amino acid i in proteins A and B, expressed as mol% x 10. The values of composition divergence between the S. solf~laricus enzyme and those of the eubacteria E. coli and B. megaterium were 76 and 79, respectively, and values of 74 and 79 were found between the S. solfataricus enzyme and those of N. crassa and bovine liver. All of these values are too high to deduce any relationship between these enzymes, since there is a lack of correlation between sequence difference and composition divergence above a value of 55 [261. However, it should be mentioned that the values of composition divergence between the enzyme of N. crussa and those of E. coli and B. megaterium were 28 and 45, respectively, thus suggesting a signifi,~ant homology between eubacterial glutamate dehydrogenases and the enzyme from the lower eukaryote N. crassa. Immunological relatedness o f the glutamate deh.vdrogenase antisera The immunological relatedness of the S. solfataricus glutamate dehydrogenase with enzymes from other organisms was studied by immunoblotting using antisera against the glutamate dehydrogenases from S. solfataricus, Proteus sp., Candida utilis and rat liver, respectively. As shown in Table V. the antiserum against the S. solfataricus enzyme reacted with the raised antigen, but with none of the ether antigens tested. When testing the three other antisera (against Proteus sp., C utilis and rat liver glutamate dehydrogenase) it was found that none of them reacted with the S. solfataricus enzyme. On the other hand, the antiserum against the glutamate dehydrogenase from the eubacterium Proteus sp.. as well as the antiserum against the glutamate dehydro-
147 TABLE V Immunological relatedness Antisen~magainst
S. solfatarwttr-GDH Proteus sp.-GDH C. utdis-GDH Rat liver-GDH
Antigen Sulfolobu.~ Proteus Candtda GDH GDH GDH + + + + + -
Rat GDH + ( +
Immunologicalcrossreactivityof the different antigens tested: +crossreactivity. (+) weak crossreactivity, - no crossreactivity. GDH = glutamate dehydrogenase.
genase from the lower eukaryote C utilis, reacted with the enzymes from Proteus sp., C utilis and rat liver. The antiserum against rat liver glutamate dehydrogenase, like that against the S. solfataricm" enzyme, only reacted with the own immunogen. Discussion
The present paper reports on the purification and subsequent characterization of a NAD(P)+-dependent glutamate dehydrogenase from the extreme thermophilic sulfur-dependent archaebacterium Sulfolobus solfataricus. The molecular weight of the native enzyme was about 270000, and the subunit M r was 44000, thus indicating the native glutamate dehydrogenase was a hexamer. A hexameric structure has also been described for most of the glutamate dehydrogenases isolated from different prokaryotes and eukaryotes [231. In addition to the hexameric structure, one of the main characteristics of glutamate dehydrogenases isolated from a large number of organisms is their difference in coenzyme specifity. The enzymes isolated from higher organisms, especially animals, can utilize both, N A D ' and NADP +, whereas the enzymes isolated from lower eukaryotes and bacteria are either specific for N A D + and not for NADP + or vice versa. T h e S. solfataricus glutamate dehydrogenase utilized both, N A D + and NADP +, and in this respect resembled more closely the homologous enzymes from eukaryotes than those from eubacteria. It should be noted, that dual cofactor specificity may be a common feature of dehydregenase enzymes from Sulfolobus [27]. The amino acid analysis, although different in certain respects, generally reflects the composition of known glutamate dehydrogenases [5-7,11,28,29]. However, significant lower amounts of phenylalanine and histidine, and a low arginine/lysine ratio of 0.47 was found within the S. solfataricus enzyme. A similar low arginine/lysine ratio was also found within some other enzymes from thermophilic archaebacteria [30,31]. A comparison of the amino acid composition of
different glutamate dehydrogenases by the composition-divergence method indicated no relationship between the S. sol[ataricus enzyme and glutamate dehydrogenases from eubacteria and eukaryotes. The results of our immunological studies support this conclusion. On the other hand. the immunological studies also support conclusions from previous work [32] indicating that glutamate dehydrogenases from eubacteria and lower eukaryotes are highly conserved. However. we cannot decide whether the fact, that no crossreaction of the antiserum against the S. sol[ataricus glutamate dehydrogenase with the antigens tested was observed, is a consequence of a different protein structure due to adaptation to high temperature, or a consequence of the evolutionary distance of S. solfamricus as a m,:mber of the archaebacterial branch. We have tested a number of substances commonly u:,ed as inhibitors of glutamate dehydrogenases. From ~nese, pyndoxal phosphate has been shown to be one of the most site-specific reagents in modification of ghitama:¢ dehydrogenases [33]. With a number of these ea-=ymes it has been demonstrated that pyridoxal phosphate reacts with a single lysine residue, which is suggested to be located at, or close to, the active site. Therefore the modified enzyme is unable to form camplexes with substrate [33] or cocnzyme [61, l'he complete inactivation of the S..~olfataricux enzyme by 2 mM pyridoxal phosphate indicates a similar mechanism. A remarkable property of the S. solfataricus glutamate dehydrogenase was its high thermostability. The enzyme had its temperature optimum at 85°C and was still active at temperatures up to 100°C, Therefore, to our knowledge it is one of ~he most thermostable enzymes isolated. In contrast to other enzymes from extreme thermophilic archaebacteria, most of which are known to be barely active at temperatures below 40°C, the S. solfataricus glutamate dehydrogenase was still active at tempcratures below 30°C. Acknowledgements The authors wish to thank Dr. B. Wittman-Liehold for performing the amino acid analysis, and K.H. Rak for the preparation of the antisera. The expert assistance of M. Hanner is gratefully acknowledged. This work was partially supported by the Legerlotz foundation. References I Brock, TD.. Brock, K.M., Belly, R.T. and Weiss. R.L. (1972)
Arch. M,croblol. 84. 55-68. 2 Woe.so,C.R. and Fox, G.E. 0977) Proc. Nat. Acad. Sci. USA 74, 5088-5090. 3 Woese.C.R. (1987) Microbiol. Roy.51,221-271. 4 Phibbs.P.V. and Bernlohr. R.W. 0971) J. Bactetiol. 106. 375-385.
148 5 Veronese. F.M.. Boccu. E and Conventi. L. (1975) Biochim Biophys. Acta 377. 217-228. 6 Hemmil~i. I.A. and M'~ntsafii. P.I. (1978) Biochem J. 173. 45-52. 7 Leicht. W.. Werber. M.M. and Eisenber8. H. (1978) Biochemistry 17, 4(X)4-4010. 8 Yarnam(,to. I.. Abe. A. and Ishimoto. M. (1986) J. Biochem. 101. 1391- 1397. 9 Bonete. M.J.. Camacho. M.L. and Cadenas. E. (1987) Int. J. Biochem. 19. 1149 - 1155. I0 Corman. L.. Prescott. L.M. and Kaplan. N.O. (1967) J. Biol. Chem. 242. 1383-1390. 11 Lehman. F.G. at;d Pncidcrcr. G. ~1969) Biol. Chem. Hoppe-Seyler 350. 609-616. 12 King. K.S. and Frieden. C. (1970) J. Biol. Chem. 245. 4391-4396. 13 McCarthy. A.D.. Walker..I.M. and Tipton. K.F. 0980) Biochem. J. 191. 605-611. 14 Mattaj. I.W.. McPher~on. M.J. and Wootton. J.C. (1982) FEBS Lett. 147.21-25. 15 De Rosa. M.. Gambacorta. A.. Nicolaus. B.. Giardina. P.. Poerio. E. and Buonocore. V. (1984) Biochem. J. 224. 407-414. 16 Bradford. M. (1976) Anal. Biochem. 72. 248-254. 17 Laemmli. U.K. (1970) Nature 277. 680-685. 18 Weber. K. and Osborn. J. (1969) J. Biol. Chem. 244. 4406-4412. 19 Radola. B.J. (1980) Electrophoresis I. 43-56. 20 Towbin. t4.. Staehlin. T. and Gordon. J. (1979) Proc. Nat. Acad. Sci. USA 78. 4350-4353.
21 Blake. M.S.. Johnston. K.H.. Russell-Jones. G.J. and Gntschlich. E.C. (1984) Anal. Biochem. 136. 175-179. 22 Davis. B.J. (1964) Ann. NY Acad. Sci. 121. 404-427. 23 Gore. M.G. (1981) Int. J. Biol. Chem. 13. 879-886. 24 Ashman. K. and Bosserhoff. A. (1985) in: Modern Methods in Protein Chemistry. (Tschesche. H.. ed.). Vol. 2. pp. 155-171. de Gruyter. Berlin. 25 Harris. C .E and Teller. D.C. (1973) J. Theor. Biol. 38. 347-362. 26 Blake. J.A. and Harkins. R.N. (1977) J. Theor. Biol. 66. 281-295. 27 Danson. M.J. (19881 Adv. Microb. Physiol. 29. 166-231. 28 Blumenthal. K.M. and Smith. E.L. 11973) J. Biol. Chem. 248. 6002-60. 29 Moon. K. and Smith. E.L. (1973) J. Biol. Chem. 248. 3082-3088. 30 Fabry. S.. Lang. J.. Niermann. T.. Vingron. M. and Hensel. R. (1989) Eur. J. Biochem. 179. 405-413. 31 Grossebilter. W.. Hartl. T.. Gt~risch. H. and Stezowski J. (1986) Biol. Chem. Hoppe-Seyler 367. 457-463. 32 Mountain. A.. MePherson. M.J.. Baron. A.J. and Wootton. J.C. (1985) Mol. Gen. Genet. 199. 141-145. 33 Brown. A.. Culver. J.M. and Fisher. H.F. (1973) Biochemistry 22. 4367-4373. 34 Hits. C.H.W. (1967) Methods Enzymol. I I. 197-199. 35 Melzer, N., Tous. G.. Gruber. S. and Stein. S. (19871 Anal. Biochem 160. 356-361. 36 Bohlen. P. and Mellet. M. (1979) Anal. Biochem. 94. 313-321.
142
Purification and uroperties of an extreme thermostable glutamate dehydrogenase from the archaebacterium Sulfolobus solfataricus M a n f r e d F. S c h i n k i n g e r 1, B e r n h a r d R e d l ~ a n d G e o r g StiSffler 1.2 I Institut fiir M~kro~iologie. Medlzinisehe Fakultiit der Universttlt~ lnnsbruck (Austria) and 2 Max-Planck-lnstitut ]'fir Molekulare Genetik. Abteilung Wittmann, Berlin (F.R.G.)
(Received 2;. January 1990) (Revised manuscriptreceived23 July 1990)
Key words: Glutamate dehydrogenasc;Thermophile;Archaebacterium;(S. sol/atancus) Glutamate dehydrogenase (L-glutamate: NAD(P)* oxidoreductase, deaminating, EC !.4.1.3.) of the extreme tberme-philic archaebacterium S u l f o l o b u s solfataricus was purified to homogeneity by (NH4)zSO 4 fractionation, anion-exchange chromatography and affinity chromatography on 5'-AMP-Sepharose. The purified native enzyme had a M, of abc,ut 270000 and was shown to be a hexamer of subunit M, of 44000. It was active from 30 to 95°C, with a maximum a,:tivity at 85°C. No significant loss of enzyme activity could be detected, either after incubation of the purified enzyme at 90°C for 60 min, or in the presence of 4 M urea or 0.1% SDS. The enzyme was catalytically active with both N A D H and N A D P H as eoenzyme and was specific for 2-oxoglutarate and L-glutamate as substrates. With respect to coenzyme utilization the S u l f o l o b u s solfatarlcus glutamate dehydrogenase resembled more closely the equivalent enzymes from eukaryotic organisms than those from eubacteria.
Introduction Sulfolobus solfataricus an extreme thermophilic sulfur
dependent archaebacterium with an optimal growth temperature of 85°C, has been isolated from hot acid habitats [1]. Since Sulfolobus grows at very high temperatures it should provide a source of enzymes with unusual physicochemical properties. In addition to its growth in extreme environments, the study of S u l f o l , bus, as a member of the third i:rimary kingdom of organisms [2] is interesting from a phylogenetic point of view. It has been shown that some biochemical features of Sulfolobus are more closely related to eukary~.'os than to eubacteria [3]. Glutamate dehydrogenase occupies a pivotal posi.t'on in the metabolism of most organisms because it Frovidzs a link between carbohydrate and nitrogen metabolism. It is a well-studied enzyme and has been purified from both prokaryotes [4-9] and eukaryotes [10-13]. Sequence analysis have shown that glutamate dehydrogenases belong to the most conserved enzymes [141.
Correspondence: B. Redl. Institut for Mikrobiologie. Medizinische Fakuh~it der Universit~it Innsbruck, Fritz-Pregl Str. 3. A-6020 lnnsbruck, Austria.
In this work we report the purification, the molecular and kinetic properties and the thermostability and resistance against dissociating reagents of a NAD(P)+-de pendent glutamate dehydrogenase from S u l f o l o b u s solfataricus.
Materials and Methods Materials
DE-32 cellulose was purchased from Whatman, Maidstone0 U.K. 5"-AMP-Sepharose was obtained by Pharmacia, Uppsala, Sweden. N A D H was of analytical grade and purchased from Scrva, Heidelberg, F.R.G. N A D P H Type V, pyridoxal 5-phosphate, iodoacetamide, protaminesulfate from Salmon, Grade llI and the standard proteins used in the SDS-polyacrylamide gel electrophoresis and M, studies were products from Sigma Chemicals, Munich, F.R.G. Rabbit antigoat-lgG alkaline phosphatase conjugate and 5-bromo4-chloro-3-indolyl phosphate were from Sigma Chemicals, Munich, F.R.G. The TSK-250 HPLC column, the standard proteins for the Mr studies and the protein assay kit were purchased from Bio-Rad Laboratories, Richmond, U.S.A. All other reagents were of analytical grade and obtained from Merck, Darmstadt. F.R.G.
143 Glutamate dehydrogenases from Proteus sp., Candida utilis and rat liver used for immunization were purchased from Sigma Chemicals (Munich, F.R.G.) and were further purified by gel permeation HPLC.
Culture o/cells The archaebacterium Sulfolobus solfataricus strain DSM 1616 was grown aerobically at 85°C as previously described [15].
of protein during the enzymatic amination of 2-oxoglutarate to L-glutamate. The enzymatic activity as a function of pH was tested in the standard assay mixture but with triethanolamine replaced by 60 m M K H 2 P O j / N a z H P O 4 (pH 5.0-8.0) or 50 mM diethanolamine (pH 8.0-10.0). All pH values were adjusted at the assay temperature (70°C). NaC1 was added to each buffer to bring the ionic strength to the same value.
Enzyme purificatton
Protein determination
Unless otherwise stated, all purification steps were carried out at 4°C. Wet cells (10 g) were resuspended in 2 volumes of buffer A (50 m M Tris-HCI (pH 7.6) containing 1 mM EDTA) and cell lysis was performed by passing the bacteria twice through a French pressure cell operated at a pres:~ure of 97 MPa. The extract was then clarified by centrifugat;on at 40000 x g for 45 min. Elimination of nucleic acids was carried out by adding 0.2 ml of a 2% (w/v) solution of protamine sulfate in buffer A per ml crude extract and stirring for 60 min at room temperature. After centrifugation at 40000 x g for 30 rain the supernatant was adjusted to 55% (w/v) a m m o n i u m sulfate and again stirred foJ 60 rain. The precipitate was collected by centrifugation at 40000 x g for 30 rain. The pellet was resuspended in buffer A and extensively dialysed against 200 volumes of the same buffer. The homogenate was then loaded onto a DE-32 (Whatman) anion-exchange column (1.5 x 12.5 cm) equilibrated with buffer A. After the column had been washed with equlibration buffer to remove unadsorbed material, elution was carried out with a linear gradient (140 ml) from 0 to 0.8 M NaCI in buffer A. Fractions (2 ml each) were collected at a flow rate of 60 m l / h . Those showing glutamate dehydrogenase activity were pooled and dialysed against buffer B (0.1 M sodium phosphate, pH 7.0). The solution was applied to a 5'-AMP-Sepharose column (0.8 x 8 cm) equilibrated with buffer B. The column was washed with buffer B until the adsorption at 280 n m returned to the baseline. The column was then washed with 20 ml buffer B containing 5 m M 2-o:toglutarate. Glutamate dehydrogenase was eluted specifically by using a solution of 0.5 m M N A D + and 5 m M 2-oxoglutarate in buffer B at a flow rate of 20 m l / h . The active fractions were pooled and stored at 80oc.
Protein concentration was determined according to Bradford [16] using the Bio-Rad Protein Assay Kit, with bovine serum albumin as standard.
-
Glutamate dehydrogenase assay The glutamate dehydrogenase activity was measured at 70"C by monitoring the NAD(P)H oxidation at 340 n m for 5 rain in a reaction mixture (1 ml) containing 50 m M triethanolamine-HCI (pH 7.6), 2.5 m M EDTA, 100 m M CH3COONH4, 0.2 m M NAD(P)H and 7 m M 2-oxoglutarate. One unit of dehydrogenase activity was equal to the release of 1 p,M NAD(P) + per rain and mg
M, determination of native enzyme Gel permeation chromatography by HPLC was used for determination of the M, of native glutamate dehydrogenase. Pure pi'eparatiuns ot" enzyme were applied to a TSK-250 column equilibrated and eluted with 20 mM phosphate buffer (pH 7.0) containing 0.15 M NaCI at a flow rate of 1 m l / m i n . The column was calibrated with the following standard proteins as mz,~kers: thyroglobulin (M, 669000), catalase (240000), y-globulin (180000), ovalbumin (45000) and myoglobin (17000). A computing integrator was used for monitoring peak area and retention time,
Polyacrylamide gel electrophores~.s SDS-polyacrylamide gel electrophoresis was performed using 10% (w/v) acrylamide slab gels according to Laemmli [171, run in a Bio-Rad Mini Protean vertical electrophoresis unit (Bio-Rad Laboratories, Richmond, U.S.A.). The subunit molecular weight was determined according to Weber and Osborn [18] by comparison with the migration of protein markers of known M, values (phosphorylase b, bovine serum albumin, ovalbumin and carbonic anhydrase). Isoelectric focusing was carried out in an LKB Multiphor apparatus using broad pH (3.5-9.51 thin-layer polyacrylamide gels [19]. A mixture of 11 standard proteins (Pharmacia calibration kit) with known p l values was run in parallel in the same gel. The proteins were stained with Coomassie brilliant blue R and densitometric gel scans were used to assess the pl.
Amino acid analysis Homogenous protein was subjected to acidic hydrolysis and the amino acid analysis was performed as described in Ref. 24. Amino acids were detected as their OPA (ortho-phtaldialdehyde/mercaptoethanol) derivates on a waters HPLC system equipped with a Shimazu RF 530 fluorescence detector. Cysteine residues were determined after performic acid oxidation as previously described [34]. Tryptophane was determined after hydrolysis in 6 M HCI in 7% (w/v)
144 kDa 2o5
thioglycolic acid according to Melzer et al. [35] and proline was determined as described by Bohlen and Mellet [36].
~116 0
hmnunological procedures Antisera were raised agair, st the glut.amate dehydrogenases from Sulfolobus solfataricus, Proteus sp., Candida utilis and rat liver, respectively, in sheep. The proteins were mixed with an equal volumn of Freund's complete adjuvant and subcutaneous injection of 250 to 500 I~g (Candida, Proteus, rat) and 50 to 100 /tg (Sulfolobus) were performed four times at 3 week intervals. The total amount of protein injected per sheep was 1.3 mg of Candida glutamate dehydrogenase, i.2 mg of Proteus glutamate dehydrogenase, 2 mg of rat glutamate dehydrogenase and 0.80 mg of Sulfolobus glutamate dehydrogenase, respectively. One week after the last booster, sera were collected and stored at - 20°C. Control sera were collected before the first injection. The immunological relatedness was tested by immunoblotting using a slight modification of the procedure of Towbin [20]. The modification included the use of a rabbit anti-goat-lgG alkaline phosphatase conjugate as secondary antibody and 5-bromo-4-ehloro-3indolyl phosphate as substrate for the phosphatase [21]. Immunoblots were made from SDS-polyacrylamide gels and from non-denaturating polyacrylamide gels using a gel system for acidic proteins according to Davis [22].
a
4 97.4 ~66
"~--
Fig. l. SDS-polyacrylamidegel electrophoresisof glutamate dehydrogenase from S. sol/ataricus(a) 3 Fg oI purified enz'ime.(b) molecular weight markers: myosin Mr = 205000 (subunit). /~-galactosidasc Mr = 116000 (subanit). phosphoryla~ b Mr = 97400 (subunit). albumin bovine Mr =66000. albumin egg M~= 45000. carbonic anhydras¢ M, = 29000. Protcins were stained with Coomassiebrilliant blue R.
the enzyme preparation appeared to be homogeneous (Fig. 1).
Molecular weight and isoelectric point The purified glutamate dehydrogenase haa a retention time on HPLC gel permeation column that corresponded to a M r of 270000 + 15000, based upon the regression equation for the retention time of the five standard proteins used. The subunit M r was shown to be 44000 5= 2000 by SDS-polyacrylam;de ",,el electrophoresis. The isoelectric point ( p l ) of the enzyme was 6.2 _+.0.2, as calculated from isoelectric focusing.
Results
Purification of glutamate dehydrogenase The results of a typical purification procedure are summarized in Table 1. The most effective purification step was represented by the affinity chromatography on 5'-AMP-Sepharose. The high efficiency was due to the complete adsorption of the enzyme on the affinity column and to its specific elution with 2-oxoglutarate and NAD+. The S. solfataricus glutamate dehydrogenase was purified about 604-fold over the crude extract, with a specific activity of 423 units per mg of protein at 70°C. As judged by SDS-polyacrylamide gel electrophoresis
Parameters affecting enzyme activity and stability Stability against thermal inactivation was tested after preincubation of the purified enzyme for 1 h at temperatures ranging from 70 to 100°C. No significant loss of enzyme activity could be deter.ted after preincubation at temperatures up to 90°C. Even boiling for 1 h did not inactivate the enzyme completely (Fig. 2).
TABLE I
Purifwationof Sulfolobu.~solfatarict~glutamatedehydrogenase
Crude extract
Step
Total protein (mg) 180
Protaminc sulfate precipitation Ammoniumsulfate fractionation DE-32 chromatography 5"-AMP-Sepharosechromatography
130 45 10 0.7
Total activity (units)
126 l 17 95 120 296
Specific acti,':w, (unlts/mg) 0,7
Purification (-fold) l
0.~ 2.1 12 423
1.3 3 17 604
145 to.!
I'ABLE 11
Stab,htl ,j S ~,;l[atart¢t~ glutantun, detrrdrogen~tte 80
.ACtlVltlC,, a t e
mean ranges of two determinations.
Treatment
60
40"
20"
70
80
90
I00
temperature (°C) Fig. 2. Effect of temperature on stability of glutamate dehyflrogena~e. Purified enzyme (70 ng) was preincubated in 50 mM tricthanolammeHCI (pH 7.5) for 1 h at the temperatures indicated and the remaining activity was determined using the standard as~,ay.
W h e n the e n z y m a t i c activity w a s tested at v a r i o u s t e m p e r a t u r e s , it w a s f o u n d that the g l u t a m a t e d e h y d r o g e n a s e exhibited m a x i m u m activity at 8 5 ° C . At 9 5 ° C the e n z y m a l i c activity w a s still 90% a n d 35% of the m a x i m u m activity were m e a s u r e d at 3 0 ° C ( d a t a not shown). T h e e n z y m a t i c ~c~i,,ity vs. p H w a s d e t e r m i n e d over a p H r a n g e o f 5 . 0 - 1 0 . 0 . In the r a n g e c ~ p H 7 . 0 - 8 . 0 , g l u t a m a t e d e h y d r o g e n a s e exhibited 9 0 - 1 0 0 % activity, with a m a x i m u m at p H 7.6, as s h o w n in Fig. 3. T h e p H stability o f S. solfataricus g l u t a m a t e d e h y d r o g e n a s e w a s e x a m i n e d b y p r e i n c u b a t i o n o f the p u r i fied e n z y m e in 50 m M c i t r a t e / H C I b u f f e r ( p H 3.0), 50 m M c i t r a t e / H C I b u f f e r ( p H 5,0) a n d 50 m M di16" 14" 12" I0 o 8" 6" 4~ 2" 0 5
6
7
8
9
tO
pH
Fig. 3. Effect of pH on activity of glutamate dehydrogenas¢. 70 ng of purified enzyme was incubated at 70°C for 5 rain in a reaction mixture containing 7 mM 2-oxoglutarate. 2.5 mM EDTA, 0.1 M CH COONH,=, 0.2 mM NADH and the buffers employed were 60 mM KH2PO,=/Na2HPO. = (I) or 50 mM diethanolamin¢ (A). NaCI was added to each buffer to bring the ionic strenght to the same value.
pH = 3,0. 14 h. 25~(" pH = 4.0. 14 h. 250(. pH =9(1, 14 h. 25~C Pyridoxal 5-pho,,phate 2 mM lodacetamtde 2 mM ,)-glutamate: 2 mM 3ram 4 mM 6 mM 4 M urea. 12 h. 25°C 6 ,M urea, 12 h, 25°C 75 M urea. 12 h, 25~(." 0.1~ SDS, 12 h. 25°C 3.5~ SDS, 12 h. 250( . 50~ (,./v)ethanol, 12 h, 250(.50"~ (,../v) methanol. 12 h, 25°C 50'~ (~,/~) t~propanol, 12 h, 25°C 90°( ". 1 h 100°C. I h
Actt','lty ("~ of control) 75 4-_7 64+5 83+3 0 le2 + 4 89 + 2 78+_3 71 ± 2 66 + 1 105 ± 7 88±2 45±6 98+_ 1 4±3 0 3±2 0 95 + 2 20+_.5
e t h a n o l a m i n e ( p H 9.0), respectively, for 14 h at r o o m t e m p e r a t u r e . T h e S. sol/atarwus e n z y m e w a s stable over a b r o a d p H r a n g e ( p H 3 - p H 9), with at least 65% residual activity a f t e r p r e i n c u b a t i o n for 14 h in buffers with different p H values ( T a b l e II). T h e effect o f d i s s o c i a t i n g r e a g e n t s a n d o r g a n i c solvents o n e n z y m e activity w a s studied b y p r e i n c u b a t ing the p u r i f i e d e n z y m e in these r e a g e n t s for 12 h at r o o m t e m p e r a t u r e . T h e residual activity w a s m e a s u r e d at 7 0 ° C as described, T h e e n z y m e w a s c o m p l e t e l y s t a b l e a f t e r 12 h preinc u b a t i o n in 4 M urea, a n d r e t a i n e d a l m o s t 90% o f the initial activity a f t e r 12 h i n c u b a t i o n in 6 M urea. It h a d a half-life of a b o u t 9 h in 7.5 M u r e a ( T a b l e II). N o loss o f activity w a s d e t e c t e d a f t e r 12 h o f p r e i n c u b a t i o n in 0.1% S D S (w/,.~: w h e r e a s the half-live o f the e n z y m e w a s 5 h in 0,5% S D S ( T a b l e I!). T h e influence of p y r i d o x a l p h o s p h a t e , a w e l l - k n o w n i n h i b i t o r o f a n u m b e r o f g l u t a m a t e d e h y d r o g e n a s e s 123] w a s tested. A d d i t i o n o f 2 m M p y r i d o x a l 5 - p h o s p h a t e to the a s s a y m i x t u r e resulted in a c o m p l e t e loss o f activity of the Sulfolobus enzyme. P r e i n c u b a t i o n with 2 m M i o d o a c e t a m i d e for u p to 10 h d i d not inhibit the e n z y m e activity. A c o n c e n t r a t i o n d e p e n d e n t inhibition o f enz y m e activity by D-glutamate, a s t r o n g c o m p e t i t i v e inhibitor o f both, the b o v i n e liver a n d Neurospora g l u t a m a t e d e h y d r o g e n a s e s [231 has been detected ('['able II). T r e a t m e n t with 50% ( v / v ) o f either e t h a n o l , m e t h a n o l or i s o p r o p a n o l resulted in a c o m p l e t e loss o f e n z y m e activity. W h e n the purified e n z y m e w a s stored at 4 ° C for m o r e t h a n 20 d a y s n o significant loss o f e n z y m e activity
146 TABLE 111 Apparent K,, t'aluesfor substrates and coencymes of glutamate dehydrogenasefrom Sulfolobtt~solfatarwus Substrale-coenzyme
2-Oxoglutarate-NADH 2-Oxoglutarate-NADPH t-Glutamate-NAD" t-Glutamate-NADP*
Km values substrate (raM)
coenzyme (/tM)
0.6 0.2 0.3 1.1
7 10 63 25
was seen. After 40 days at 4°C the activity decreased to 80%. For long-term storage a temperature of - 7 0 ° C was choosen, since no significant loss of activity was seen after a period of 6 month at this temperature. Kinetics The kinetic parameters of S. solfataricus glutamate dehydrogenase for various substrates and coenzymes were determined at 70°C using the standard assay. The purified enzyme exhibited Michaelis-Menten kinetics and Table 11I summarizes and compares the Michaelis constants (Kin) of the enzyme with respect to 2-oxoglutarate, L-glutamate (7 mM each) N A D +. NA D P* , N A D H and NA D P H (0.2 mM each), estimated by double-reciprocal plots. As shown in Table II1, the formation of L-glutamate by the S. solfataricus enzyme was not strictly N A D H TABLE IV ,4mino acid composition of hexameric glutamate deh)'drogena~esfrom S. solfatartcus (Ss), F~ colt (Ec). B. megatermm (Bin), N. crassa (Nc) and bovine liver (BI) Amino acid tool% Ss [da;a from thispaperl (res./mol J ) Cys Asp Glu His Set Gly Thr Arg Ala Tyr
Trp Met Val Phe lie Leu Lys Pro
1.0 (3.8) 10.5 (42.8) 9.4(38.3) 0.7 (2.9) 3.3 (13.4) 13.0(53.0) 4.2 (17.1) 4.4 (17.9) 10.1 (41.2) 2.8 (11.41 0.8 (3.6) 3.2 (13.2) 8.2 (33.4) 2.2 (8.9) 6.2 (25.7) 8.3 (34.6) 9.4 (38.1) 2.5 (10.1)
Ec [51
Bm [61
1.4 1.5 9.4 8.3 12.4 10.2 2.1 3.1 6.7 5.6 11.7 11.8 5.2 5.2 4.1 4.7 10.4 11.2 2.8 2.0 1.0 n.d. 2.3 3.0 7.8 7.1 3.4 4.6 3.5 4.2 7.1 9.3 5.1 4.1 3.7 3.9
A molecularweightof 45000 was assumed.
Nc [281
BI 1291
1.3 8.7 11.6 1.9 7.4 11.6 4.2 3.5 11.3 3.2 1.6 1.6 7.1 3.9 3.9 7.7 6.1 3.2
1.2 9.7 9.3 2.8 5.9 9.3 5.9 5.9 7.5 3.6 0.6 2.8 6.7 4.5 7.3 6.3 6.5 4.1
dependent. The enzyme was also able to use N A D P H as coenzyme. At the assay temperature (70°C) a V,,~ of 0.4 mM 2-oxoglutarate per rain and mg of protein was calculated. A m i n o acid analysis The amino acid composition of the glutamate dehydrogenase from S. solfataricus is given in Table IV together with the composition of glutamate dehydrogenases from Escherichia coil, Bacillus megaterium, of the hexameric NADP-dependent enzyme from Neurosporo crassa and of the glutamate dehydrogenase from bovine liver. For comparison of the amino acid compositions in Table IV we have used the composition divergence ( D ) of Harris and Teller [25]:
D = ~ f ~ t ( Xi.A- X,.~)-" were X,.^ and Xi.B are tL: values of amino acid i in proteins A and B, expressed as mol% x 10. The values of composition divergence between the S. solf~laricus enzyme and those of the eubacteria E. coli and B. megaterium were 76 and 79, respectively, and values of 74 and 79 were found between the S. solfataricus enzyme and those of N. crassa and bovine liver. All of these values are too high to deduce any relationship between these enzymes, since there is a lack of correlation between sequence difference and composition divergence above a value of 55 [261. However, it should be mentioned that the values of composition divergence between the enzyme of N. crussa and those of E. coli and B. megaterium were 28 and 45, respectively, thus suggesting a signifi,~ant homology between eubacterial glutamate dehydrogenases and the enzyme from the lower eukaryote N. crassa. Immunological relatedness o f the glutamate deh.vdrogenase antisera The immunological relatedness of the S. solfataricus glutamate dehydrogenase with enzymes from other organisms was studied by immunoblotting using antisera against the glutamate dehydrogenases from S. solfataricus, Proteus sp., Candida utilis and rat liver, respectively. As shown in Table V. the antiserum against the S. solfataricus enzyme reacted with the raised antigen, but with none of the ether antigens tested. When testing the three other antisera (against Proteus sp., C utilis and rat liver glutamate dehydrogenase) it was found that none of them reacted with the S. solfataricus enzyme. On the other hand, the antiserum against the glutamate dehydrogenase from the eubacterium Proteus sp.. as well as the antiserum against the glutamate dehydro-
147 TABLE V Immunological relatedness Antisen~magainst
S. solfatarwttr-GDH Proteus sp.-GDH C. utdis-GDH Rat liver-GDH
Antigen Sulfolobu.~ Proteus Candtda GDH GDH GDH + + + + + -
Rat GDH + ( +
Immunologicalcrossreactivityof the different antigens tested: +crossreactivity. (+) weak crossreactivity, - no crossreactivity. GDH = glutamate dehydrogenase.
genase from the lower eukaryote C utilis, reacted with the enzymes from Proteus sp., C utilis and rat liver. The antiserum against rat liver glutamate dehydrogenase, like that against the S. solfataricm" enzyme, only reacted with the own immunogen. Discussion
The present paper reports on the purification and subsequent characterization of a NAD(P)+-dependent glutamate dehydrogenase from the extreme thermophilic sulfur-dependent archaebacterium Sulfolobus solfataricus. The molecular weight of the native enzyme was about 270000, and the subunit M r was 44000, thus indicating the native glutamate dehydrogenase was a hexamer. A hexameric structure has also been described for most of the glutamate dehydrogenases isolated from different prokaryotes and eukaryotes [231. In addition to the hexameric structure, one of the main characteristics of glutamate dehydrogenases isolated from a large number of organisms is their difference in coenzyme specifity. The enzymes isolated from higher organisms, especially animals, can utilize both, N A D ' and NADP +, whereas the enzymes isolated from lower eukaryotes and bacteria are either specific for N A D + and not for NADP + or vice versa. T h e S. solfataricus glutamate dehydrogenase utilized both, N A D + and NADP +, and in this respect resembled more closely the homologous enzymes from eukaryotes than those from eubacteria. It should be noted, that dual cofactor specificity may be a common feature of dehydregenase enzymes from Sulfolobus [27]. The amino acid analysis, although different in certain respects, generally reflects the composition of known glutamate dehydrogenases [5-7,11,28,29]. However, significant lower amounts of phenylalanine and histidine, and a low arginine/lysine ratio of 0.47 was found within the S. solfataricus enzyme. A similar low arginine/lysine ratio was also found within some other enzymes from thermophilic archaebacteria [30,31]. A comparison of the amino acid composition of
different glutamate dehydrogenases by the composition-divergence method indicated no relationship between the S. sol[ataricus enzyme and glutamate dehydrogenases from eubacteria and eukaryotes. The results of our immunological studies support this conclusion. On the other hand. the immunological studies also support conclusions from previous work [32] indicating that glutamate dehydrogenases from eubacteria and lower eukaryotes are highly conserved. However. we cannot decide whether the fact, that no crossreaction of the antiserum against the S. sol[ataricus glutamate dehydrogenase with the antigens tested was observed, is a consequence of a different protein structure due to adaptation to high temperature, or a consequence of the evolutionary distance of S. solfamricus as a m,:mber of the archaebacterial branch. We have tested a number of substances commonly u:,ed as inhibitors of glutamate dehydrogenases. From ~nese, pyndoxal phosphate has been shown to be one of the most site-specific reagents in modification of ghitama:¢ dehydrogenases [33]. With a number of these ea-=ymes it has been demonstrated that pyridoxal phosphate reacts with a single lysine residue, which is suggested to be located at, or close to, the active site. Therefore the modified enzyme is unable to form camplexes with substrate [33] or cocnzyme [61, l'he complete inactivation of the S..~olfataricux enzyme by 2 mM pyridoxal phosphate indicates a similar mechanism. A remarkable property of the S. solfataricus glutamate dehydrogenase was its high thermostability. The enzyme had its temperature optimum at 85°C and was still active at temperatures up to 100°C, Therefore, to our knowledge it is one of ~he most thermostable enzymes isolated. In contrast to other enzymes from extreme thermophilic archaebacteria, most of which are known to be barely active at temperatures below 40°C, the S. solfataricus glutamate dehydrogenase was still active at tempcratures below 30°C. Acknowledgements The authors wish to thank Dr. B. Wittman-Liehold for performing the amino acid analysis, and K.H. Rak for the preparation of the antisera. The expert assistance of M. Hanner is gratefully acknowledged. This work was partially supported by the Legerlotz foundation. References I Brock, TD.. Brock, K.M., Belly, R.T. and Weiss. R.L. (1972)
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