Isolation and characterization of an intracellular aminopeptidase from the extreme thermophilic archaebacterium Sulfolobus solfataricus

148

Biochimica et Biophysica Acta, 1033 (1990) 148-153 Elsevier

BBAGEN 23251

Isolation and characterization of an intracellular aminopeptidase from the extreme thermophilic archaebacterium

Sulfolobus solfataricus Markus Hanner, Bernhard Redl and Georg Sttfffler Institute of Microbiology, Medical School, University of lnnsbruck (Austria)

(Received 28 June 1989)

Key words: Aminopeptidase; Thermostability; Archaebacterium; (S. solfataricus)

An intracellular aminopeptidase (EC 3.4.11.-) was purified from the extreme thermophilic archaebacterium, Sulfoiobus soifataricus. The molecular weight of the native enzyme was about 320 000, as calculated by gel-filtration studies, and a subunit M r of 80000 was estimated by SDS-polyacrylamide gel electrophoresis. The temperature optimum of the enzyme was at 75 °C and the pH optimum was found to be 6.5. The aminopeptidase was highly active against the chromogenic substrates L-Leu-p-NA and L-AIa-p-NA. The enzyme was inhibited by EDTA, but the activity could be partially restored by removal of the EDTA and incubation with Co 2+ or Mn 2+. Bestatin, a typical inhibitor of aminopeptidase, fully inhibited the enzyme activity, but inhibitors of serine proteinases had no effect. Beside a high thermostability, the enzyme showed a remarkable stability against 6 M urea, organic solvents and acetonitrile.

Introduction Hydrolysis of peptide bonds is a prerequisite for utilization of proteins as sources of amino acids for bacterial growth and represents an essential mechanism of modification of proteins at the posttranscriptional level. Understanding of this mechanism depends on knowledge of the intracellular proteinases, their characteristics and the cellular processes which they catalyze. A number of intracellular proteinases have been isolated and characterized in eukaryotic and eubacterial cells [1]. From archaebacteria, organisms which represent a third line of evolution [2] proteinases have been purified from a strain of Desulfurococcus [3] and from Halobacterium halobium [4]. However, these are extracellular enzymes, and so far no report describing the isolation of an intracellular proteinase from archaebacteria is known to us.

Abbreviations: PMSF, phenylmethylsulfonyl fluoride; TLCK, tosytL-lysine-chloromethylketone; Hepes, 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid; p-NA, p-nitroanilide; Ac-Leu-p-NA, acetyl-Leup-NA; Bz-Tyr-p-NA, benzoyl-Tyr-p-NA; Suc-Phe-Ala-p-NA, succinyl-Phe-Ala-p-NA; Z-GIy-Gly-p-NA, benzyloxycarbonyl-Gly-Glyp-NA Correspondence: B. Redl, Institute of Microbiology, Medical School, University of Innsbruck, Fritz-Pregl-Strasse 3, A-6020 Innsbruck, Austria.

In this study we report the isolation and terization of an aminopeptidase from the bacterium Sulfolobus solfataricus, an extremely philic sulfur-oxidizing organism that has been from hot acid habitats [5].

characarchaethermoisolated

Materials and Methods Materials. The chromogenic substrates Z-Gly-GlyLeu-p-NA, Ac-Leu-p-NA, Ac-Phe-p-Na, Phe-p-NA, Arg-p-NA, Bz-Tyr-p-NA, Bz-Arg-p-NA, GIy-p-NA, Suc-Phe-Ala-p-NA, S-benzyl-Cys-p-NA were from Bachem, Swiss. L-Leu-p-NA and L-Lys-p-NA were from Sigma, Munich. L-Ala-p-NA was from Serva, Heidelberg. Trisacryl®M (DEAE) was obtained from LKB, Bromma. Iodoacetamide, protamine sulfate from salmon, phenylmethylsulfonyl fluoride, N-(1-naphthyl)ethylenediamine, G r a d e III, azocasein and the standard proteins used in the SDS-polyacrylamide gel electrophoresis and M r studies were from Sigma. Nitrocellulose m e m b r a n e (BA 83; 0.2 /~m) was from Schleicher & Schtill, Dassel. All of the other reagents were of analytical grade and obtained from Merck, Darmstadt. Culture. The archaebacterium S. solfataricus, strain DSM 1616, was grown aerobically at 85 ° C as described by De Rosa et al. [6]. Purification of an intracellular proteinase. All steps were carried out at 4 °C. The cells were harvested by

0304-4165/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)

149 centrifugation at 1 0 0 0 0 X g for 15 min at 4 ° C and stored at - 8 0 ° C . For some preparations 0.05 m M Co 2÷ was added to all buffers. Step 1. Crude extract. Wet frozen cells (17 g) were thawed in 35 ml of buffer A (50 mM Tris-HC1 buffer (pH 7.5)) and disrupted by passing them twice through a French pressure cell at 14000 lb/in. The suspension was centrifuged at 40 000 x g for 45 rain at 4 ° C. Step 2. Precipitation with protamine sulfate. Precipitation of nucleic acids was carried o u t by adding protamine sulfate (2 m g / m l ) to the crude extract [7]. After 60 min at 4 ° C with slow stirring, the precipitated material was removed by centrifugation at 40000 x g for 30 min at 4 ° C and the supernatant was used for step 3. Step 3. Fractionation with ammonium sulfate. Solid ammonium sulfate was added to the supernatant. The proteins precipitated between 25 and 55% saturation were used for further purification. The precipitate was dissolved in buffer A and dialyzed against several changes of buffer B (25 mM potassium phosphate buffer (pH 7.0)) at 4 ° C overnight. Step 4. Ion-exchange chromatography using DEAETrisacryl M. The protein solution was applied to a DEAE-Trisacryl column (2.5 x 8 cm) previously equilibrated with buffer B. Proteins were eluted from the column by increasing the salt concentration stepwise (0.13 mM and 0.5 mM KC1). Fractions containing proteolytic activity were pooled, dialyzed against 10 mM Tris buffer (pH 7.5) and lyophilized. Step 5. Gel-filtration chromatography on Bio-Gel A-1.5. The lyophilized proteins were dissolved in buffer A and were applied to a column of Bio-Gel A-1.5 (1 x 60 cm) equilibrated in buffer A. Fractions containing proteolytic activity were pooled and stored at - 8 0 ° C. Assay for proteinase activity. The enzyme activity was determined spectrophotometrically by use of chromogenic substrates. L-Leu-p-NA was used as substrate in the standard assays. Chromogenic substrates were generally dissolved in 50% dimethylsulfoxide at a concentration of 10 mM and stored in the dark at - 2 0 ° C. Under these conditions the stock solutions were stable for at least 2 months. The incubation mixture contained 5/~1 to 40/~1 enzyme solution, 0.9 ml 50 mM Tris-HC1 (pH 7.0) and 0.1 ml of chromogenic substrate to a final concentration of 1 mM. After 5 to 20 min of incubation at 70 ° C, the reaction was stopped by chilling on ice and the amount of p-nitroaniline released was measured at 410 nm. One unit of activity was equal to the amount of enzyme that hydrolysis 1 #mol of p-nitroanilide per min under the standard conditions. A molar absorption coefficient of 8900 M - 1 . cm i for p-nitroaniline at 410 nm was taken for calculation of activity [8]. Protein determination. Protein was determined according to the method of Bradford [9] using crystalline bovine serum albumin as standard.

Polyacrylamide gel electrophoresis. SDS-polyacrylamide gel electrophoresis was performed in slab gels containing 10% acrylamide according to Laemmli [10] using the Bio-Rad Mini-Protean II gel electrophoresis system. Proteins of SDS-200 Kit from Sigma were used as molecular weight markers. Polyacrylamide gel electrophoresis under native conditions was done using an acidic or basic buffer system according to Davis [11], and isoelectrofoccusing in slab gels containing Servalyte ampholines pH 2-11 was carried out as described [12]. Enzymoblotting. Protein fractions were separated by electrophoresis under native conditions in polyacrylamide gels containing 7.5% acrylamide using a basic buffer system according to Davis [11]. The gels were equilibrated in transfer buffer (25 mM Tris + 20 mM glycine) for 15 min and electrotransfer of proteins to nitrocellulose membrane was performed in an LKBTransphor electroblotting unit at 4 ° C and 60 V for 120 rain. After electroblotting the membrane was incubated in 50 mM Tris-HC1 (pH 7.0) containing 2 mM L-Leu-pNA for 45 min at 60 ° C. p-Nitroaniline, the product obtained by proteolytic activity, was diazotized with N-(1-naphthyl)ethylenediamine to a red azo dye [13]. Molecular weight estimation of native enzyme. The purified aminopeptidase was chromatographed by gelfiltration on a Bio-Gel A-1.5 column (0.8 x 60 cm), calibrated with chymotrypsinogen ( M r 25 000), ovalbumin (45000), bovine albumin (67000), aldolase (160000), catalase (240000), and ferritin (450000). The eluting buffer was 50 mM Tris-HCl buffer (pH 7.0), fractions of 2 ml were collected at a flow rate of 12 ml/h. Effect of temperature on actiuity and stability of the enzyme. The activity of the purified enzyme at temperatures ranging from 25 ° C to 95 ° C was determined in 50 mM Tris-HC1 buffer (pH 6.5) containing 0.9 fig enzyme per test. The buffer was adjusted to pH 6.5 at each temperature employed. The rate of reaction assays were corrected for loss of enzyme activity caused by thermal inactivation throughout the incubation. For testing of thermal stability, the purified enzyme was preincubated at temperatures varying from 6 0 ° C to 95 ° C for 15 min in sealed vessels containing 50 mM Tris-HC1 (pH 6.5)with or without addition of 0.05 mM Co 2÷. After chilling the samples on ice, the remaining activity was determined immediately using the standard assay procedure.

Effect of pH on actiuity and stability of the enzyme. The enzymatic activity as a function of pH was tested using either 50 mM potassium phosphate buffer (pH 5-6.3), or 50 mM Hepes (pH 6.3-7) or 50 mM Tris-HC1 (pH 7-9). To each buffer, NaC1 was added to bring the ionic strength to the same value. The effect of the pH on the stability of the aminopeptidase was investigated by preincubation of the purified enzyme in 50 mM citrate/HC1 buffer (pH 3.0),

150 50 mM citrate/HC1 buffer (pH 5.0), 50 mM Tris-HC1 buffer (pH 7.0) and 50 mM boric acid/50 mM K C 1 / N a O H buffer (pH 9.0) for 12 h at 4 ° C, followed by determination of the residual activity using the standard test.

b

0

c

Results

Enzyme purification A typical purification procedure of the aminopeptidase from S. solfataricus is summarized in Table I. After treatment with ammonium sulfate, proteolytic activity was detected in fractions, which precipitated between 25 and 55% ammonium sulfate saturation. These fractions were further analyzed by a modified enzymoblotting procedure [13]. After electrophoretic separation by polyacrylamide gel electrophoresis under native conditions using a basic or acidic buffer system proteins were electroblotted to nitrocellulose and incubated with L-Leu-p-NA. Proteolytic activity on the enzymoblot was detected as a red band only when proteins were separated in gels using a basic buffer system (Fig. 2, lane a). Therefore it was decided to use a cation-exchange chromatography for further purification. The enzyme was purified to homogeneity by ion-exchange chromatography on Trisacryl M ® (DEAE) and gel-filtration on Bio-Gel A-1.5 as shown by SDS-polyacrylamide-gel electrophoresis (Fig. 1, lane c). The specific activity of the intracellular proteinase was increased about 200-fold with respect to the crude extract. Addition of Co 2÷, which is known to stimulate aminopeptidase activity [14], to all buffers used for the purification procedure, did not increased the specific activity of the purified enzyme.

Fig. 1. Electropherograms obtained after SDS-polyacrylamide gel electrophoresis at different stages of purification; lane a, 38/zg crude extract; lane b, 15 /zg of proteins from 25-55% ammonium sulfate fraction; lane c, 1.5/~g proteinase after purification by Bio-Gel A 1.5. Proteins were stained with Coomassie brilliant blue.

80 000. The native aminopeptidase would therefore appear to consist of four subunits of equal size. The isoelectric point of the purified aminopeptidase was at pH 4.4 as calculated from isoelectric focusing on slab gels. o

t9

Molecular weight, subunit structure and isoelectric point The molecular weight of the native enzyme was estimated by gel-filtration on a Bio-Gel A 1.5 column and exhibited an M r of 320000. The subunit M r was estimated by SDS-polyacylamide gel electrophoresis yielding a single protein band corresponding to a M r of

TABLE I

Summary of the purification of aminopeptidase from S. solfataricus Purification step

Protein (mg)

Activity (U)

Specific activity (mU/mg)

Purification (-fold)

Crude extract Protamine sulfate Ammonium sulfate fractionation DEAE-pool Gel-filtration on Bio-Gel A-1.5

1260 826

1.496 1.485

1.187 1.798

1 1.5

286 5.4

1.240 0.993

4.336 184

3.6 155

4.0

0.900

225

190

Fig. 2. Aminopeptidase activity visualized on nitrocellulose using enzymoblots from native polyacrylamide gels. Lane a, 16 /zg of proteins from 25-55% ammonium sulfate fraction; lane b, 2 ~g of pure enzyme.

151 ,00

100

80'

8O

60'

ea

40'

40'

~

20'

20'

0

0 20

40

60

80

100

temperature

Fig. 3. Effect of temperature on activity of the aminopeptidase. Assays were performed in 0.1 M Tris-HCl containing 1 mM L°LeupNA and 0.9 ~tg of proteinase at the various temperatures indicated. The buffer was adjusted to pH 6.5 at each temperature used and the incubation time was 6 rain.

Effect of temperature on actiuity and stability W h e n the enzymatic activity was tested at various temperatures, the aminopeptidase exhibited m a x i m u m activity at about 75 o C. Little proteolytic activity was observed at or below 25 o C, the level of activity doubled for each 1 0 ° C temperature increment from 2 5 ° C to 60 ° C and the activity fell off sharply above 85 ° C (Fig. 3). Addition of 0.05 m M each of Co 2÷, M n 2÷, Mg 2+, Ca 2÷ and Zn 2÷ to the incubation mixture had no effect on the temperature optimum. At higher concentration of divalent metal ions a decrease of enzyme activity occurred. The thermal stability of the purified enzyme was significantly increased by the presence of 0.05 m M Co 2÷. After preincubation for 15 min at 7 5 ° C the enzyme activity decreased to 10% in the absence of Co 2÷, whereas in the presence of Co 2÷ the remaining activity was still 76%. A preincubation at 85 ° C in the absence of Co 2÷ resulted in a complete loss of enzymatic activity, whereas after preincubation in the presence of Co 2+ the remaining activity was still 60% (Fig. 4).

•

55

i

,

,

65

75

i

7

85

95

temperature Fig. 4, Effect of temperature on enzyme stability. 0.3 /zg of purified proteinase was preincubated in 50 mM Tris-HC1 (pH 6.5) (D D) or in 50 mM Tris-HC1 (pH 6.5) containing 0.05 mM Co2+ (O O) for 15 rain at the temperatures indicated and the remaining activity was determined using L-Leu-pNA as substrate.

p-Na, the aminopeptidase was highly active against L-Ala-p-NA. L - A r g - p - N A and L-Phe-p-NA were cleaved with lower efficiency (63% and 62%), and very low activity was f o u n d against L-Gly-p-NA and L-Lys-p-NA (15% and 11%). Neither N-terminal modified substrates nor azocasein were hydrolyzed. The Michaelis constants were determined for two chromogenic substrates, LL e u - p - N A and L-Ala-p-NA and were found to be 0.1 m M and 0.21 m M , respectively.

Enzyme inhibitors and effect of chemical reagents A n u m b e r of°inhibitors were tested for their ability to affect the enzymatic hydrolysis of L-Leu-p-NA (Table III). F o r this purpose, the aminopeptidase was preincubated with the inhibitors for 60 rain at room tempera-

15 e~ O r~

,0

Effect of pH on activity and stability The enzymatic activity was determined at different p H values as described in Materials and Methods. Proteolytic activity occurred from p H 5.0 to 8.0, with an o p t i m u m at p H 6.75 (Fig. 5). The purified enzyme proved to be stable between p H 3.0 and p H 9.0, with at least 70% residual activity when preincubated for 14 h in buffers of different p H values.

Substrate specificity The substrate specificity of the enzyme was investigated using 13 chromogenic substrates (Table II). A p a r t from its activity against the standard substrate, L-Leu-

0 4,0

i

i

5,0

6,0

J

i

7,0

8,0

l 9,0

pH

Fig. 5. Effect of pH on the activity of aminopeptidase. The incubation mixture contained 0.7/~g of proteinase and the buffers employed were either 50 mM potassium phosphate (@) or 50 mM Hepes (El) or 50 mM Tris-HC1 (o). The incubation time was 6 min.

152 TABLE II

Substrate specificity of the Sulfolobus aminopeptidase Activities were measured as described in Materials and Methods. Specific activity for L-Leu-p-NA was taken as 100%. p-NA used

Relative rate (%) of hydrolysis

L-Leu L-AIa L-Arg L-Phe L-Lys L-Gly Ac-Phe Ac-Leu Bz-Arg Bz-Tyr Suc-Phe Z-Gly-Gly-Leu S-Bz-Cys

100 118 63 62 15 11 0 0 0 0 0 0 0

the enzyme activity could be restored up to 80%. Reconstitution with Ca 2+, Mg 2+ and Zn 2+ had only a minor effect. The enzyme was not inhibited by PMSF or soybean trypsin inhibitor, thus indicating that serine is not part of its active center. Histidine also seems not to be part of the active center, since T L C K did not effect the enzymatic activity. No influence on enzyme activity was seen after treatment with iodoacetamide, an inhibitor of thiolproteases. Preincubation in 50% ( v / v ) ethanol, 5% ( v / v ) acetic acid and 0.1% SDS solution resulted in a complete loss of enzymatic activity. After preincubation in 10% ( v / v ) acetonitrile and 1% ( v / v ) mercaptoethanol the remaining activity was still 74% and 44%, respectively. Preincubation in 50% ( v / v ) methanol and 6 M urea up to 14 h did not cause any appreciable loss of enzymatic activity. Discussion

ture and the remaining activity was determined after addition of substrate in the absence of inhibitors. The enzyme was inhibited by bestatin, known as an inhibitor of aminopeptidases. A complete inhibition of enzymatic activity also occurred in the presence of EDTA, an inhibitor of metalloproteinases. By removal of E D T A and addition of 1 m M Co 2÷ or 1 m M Mn 2 +

TABLE III

Effect of various compounds on the activity of the aminopeptidase 0.7/~g purified enzyme was preincubated with the compounds indicated for 60 min at room temperature. An aliquot was removed and the remaining activity was determined in 50 mM Tris buffer (pH 7.0) at 70 ° C. All chemicals were dissolved in water except for PMSF, 8-hydroxychinoline and TLCK, which were dissolved in methanol. Compounds

Final concentration

Activity against L-Leu-p-NA (% of control)

None PMSF PMSF EDTA Iodoacetamide 8-Hydroxychinoline TLCK Bestatin

1 mM 2 mM 1 mM 1 mM 1 mM 1 mM 1 mM

100 102 92 0 118 126 93 3

Ethanol Ethanol Methanol Methanol Acetonitrile Acetonitrile Acetic acid Acetic acid Urea SDS Mercaptoethanol

10% 50% 10% 50% 5% 10% 1% 5% 6M 0.1% 1%

0 0 93 95 96 74 14 0 106 0 44

We have purified an intracellular aminopeptidase from the thermophilic sulfur-dependent archaebacteriurn, S. solfataricus, to homogeneity by a combination of a m m o n i u m sulfate fractionation, anion-exchange chromatography and gel-filtration, thus allowing the characterization of the structural and catalytic properties of this enzyme. From gel-filtration studies, a molecular weight of 320000 for the native enzyme was calculated. In this respect, the aminopeptidase from Sulfolobus does not differ very much from other aminopeptidases isolated from different sources [15,16]. A subunit molecular weight of 80 000 was calculated by SDS-polyacrylamide gel electrophoresis, indicating a tetrameric structure fo the Sulfolobus aminopeptidase. The enzyme activity exhibited a maximum at about 75 ° C and therefore did not correlate with the optimum growth temperature of the organism at 85 ° C. This fact indicates extrinsic factors necessary for enzyme activity. We have found that the purified enzyme was completely inhibited by E D T A and addition of Co 2+ or Mn 2+ to the EDTA-treated enzyme could restore the proteolytic activity. From human liver an aminopeptidase was isolated for which Co 2+ and other divalent metals behave quite similar [17]. The effect of various divalent metal ions on enzyme activity was tested. No effect on temperature optimum was found, but the temperature stability was significantly increased by addition of 0.05 m M Co 2+. The specificity of the proteinase was determined from its ability to cleave a number of synthetic substrates. These studies demonstrated that the proteinase hydrolyzed only peptide bonds at the N-terminal position but no N-terminal modified substrates. The broad substrate specificity of the aminopeptidase from

153

Sulfolobus is similar to that of other known aminopeptidases [18,19]. It preferentially hydrolyzed peptide bonds containing the non-polar amino acids L-Ala and L-Leu. L-Phe and polar amino acid residues like L-Gly or basic amino acid residues like L-Lys and L-Arg were hydrolyzed more slowly. The results of the studies using several inhibitors led us to the conclusion that the aminopeptidase was a metalloenzyme requiring divalent cations at its active site, since no other inhibitors of proteinases except EDTA and bestatin could significantly affect the enzyme activity. Beside its thermostability, the proteinase remained fairly stable against urea, methanol and acetonitrile. A high chemostability could also be found with a number of different enzymes isolated from extreme thermophilic organisms [20,21]. In Bacillus stearothermophilus a membrane-bound aminopeptidase (AP I) was described [19] with similar properties in respect to the high thermoand chemostability of the aminopeptidase from Sulfolobus. In addition, the thermostability of AP I was also markedly reduced in cobalt free buffer. The 200-fold enrichment for the purified aminopeptidase indicated a high expression of this enzyme under the growth conditions used. Whether the aminopeptidase from Sulfolobus has a general function in the utilization of peptides as some sources of amino acids, or a more specific function in the removal of amino acids at N-terminal ends of nascent proteins has to be proved by further studies. Acknowledgement We kindly thank our colleague M. Schinkinger for providing some of the ammonium sulfate fractions and for many useful suggestions. We thank Biochemie Kundl for their generous gift of chromogenic substrates used in

our experiments. This work was supported by the Legerlotz-Stiftung.

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