Molecular and Cellular Biochemistry 216: 93–101, 2001.

© 2001 Kluwer Academic Publishers. Printed in the Netherlands.

93

L-asparaginase of Thermus thermophilus:

Purification, properties and identification of
essential amino acids for its catalytic activity
Agathi A. Pritsa and Dimitrios A. Kyriakidis
Laboratory of Biochemistry, Department of Chemistry, Aristotle University of Thessaloniki, Thessaloniki, Greece

Received 30 May 2000; accepted 20 September 2000

Abstract
L-asparaginase EC 3.5.1.1 was purified to homogeneity from Thermus thermophilus. The apparent molecular mass of L-as-
paraginase by SDS-PAGE was found to be 33 kDa, whereas by its mobility on Sephacryl S-300 superfine column was around
200 kDa, indicating that the enzyme at the native stage acts as hexamer. The purified enzyme showed a single band on acrylamide
gel electrophoresis with pI = 6.0. The optimum pH was 9.2 and the Km for L-asparagine was 2.8 mM. It is a thermostable
enzyme and it follows linear kinetics even at 77°C. Chemical modification experiments implied the existence of histidyl, arginyl
and a carboxylic residues located at or near active site while serine and mainly cysteine seems to be necessary for active form.
(Mol Cell Biochem 216: 93–101, 2001)

Key words: L-asparaginase, Thermus thermophilus, purification, renaturation, thermostable, active site

Introduction triolein and oleyl acetate [9]. These results suggest that the
reconstruction of a physiological hydrophobic environment
L-asparaginase (L-asparagine-amidohydrolase, EC 3.5.1.1) is necessary for maximal L-asparaginase activity. Previous
the enzyme which converts L-asparagine to L-aspartic acid reports to entrap L-asparaginase in liposomes have yielded
and ammonia has been used as a chemotherapeutic agent [1, rather unsatisfactory results, since the entrapped L-aspara-
2]. The clinical action of this enzyme is attributed to reduc- ginase displayed low catalytic [10] and antigenic activities
tion of L-asparagine since tumor cells, unable to synthesize [11]. We have shown that L-asparaginase of T. pyriformis
this amino acid, are selectively killed by L-asparagine dep- is regulated by phosphorylation-dephosphoryation reaction
rivation [3]. The literature on L-asparagine metabolism and resulting in the inactivation-activation of its catalytic activ-
L-asparaginase therapy has been extensively reviewed [4–7]. ity [12]. L-asparaginase of T. pyriformis also possesses in-
We have recently shown that L-asparaginase of Tetrahy- trinsic kinase activity and upon incubation with [γ-32P] ATP
mena pyriformis is associated with membranes [8]. The na- it is autophosphorylated predominantly on tyrosine residues
tive enzyme, a multimeric protein, has a relative molecular [13].
weight of 200 kDa, with a subunit size of 39 kDa. It is a Enzymes that work either at extreme pH values or at high
glutaminase-free enzyme and its activity is inhibited competi- temperatures are now of biotechnological interest. Therefore,
tively by D-aspartic acid and D-asparagine, as well as by L- purification of enzymes with these properties presents genu-
asparagine analogues with substituents at the β position. The ine commercial opportunities and a valuable contribution to
purified enzyme is a lipoprotein, since it is inactivated by the field of biotechnology.
phospholipase C and its activity is restored by the addition The present study provides evidence that L-asparaginase
of naturally occurring lipids such as phosphatidylcholine, of the thermophillic bacterium Thermus thermophilus is a

Address for offprints: D.A. Kyriakidis, Laboratory of Biochemistry, Department of Chemistry, Aristotle University of Thessaloniki, Thessaloniki, 54006,
Greece
94

thermostable, glutaminase-free enzyme and its activity is aspartic acid in one minute incubated at 60°C at the above
inhibited competitively by L-aspartic acid and D-asparagine. specific conditions. Specific activity of L-asparaginase is
L-asparaginase acts as an hexamer and requires the existence defined as the units per mg protein.
of histidine, arginine or an acidic amino acid residue at or near
the active site.
Purification of Thermus thermophilus L-asparaginase

Materials and methods Thermus thermophilus from the stationary phase (50 g) were
suspended in buffer A (50 mM Tris/HCl pH 8.5, 1 mM β-
Materials mercaptoethanol, 0.3 mM PMSF (phenylmethylsulfonyl
fluoride)) (3 ml/g cells). The cells were disrupted by sonic
Tryptone and yeast extract were purchased from Difco (De- vibration in a oscillator (UP200S dr. hielscher) and ultra-
troit, MI, USA). Phenyl sepharose CL-4B and Cibacron blue centrifuged at 105,000 × g for 1 h.
sepharose CL-6B were from Pharmacia (Upsala, Sweden). In the 105,000 × g supernatant solid ammonium sulfate
DE-52 was obtained from Whatmann (Kent, UK). Reactive was added and the pellet was (20–40% saturation) dissolved
red agarose and Heparin sepharose were obtained from Sigma in 25 ml buffer A and dialyzed overnight at 4°C against the
(St. Louis, MO, USA). Hydroxylapatite was product of Bio- same buffer. The dialysed sample was diluted 1:10 with buffer
Rad (CA, USA). All others chemicals were purchased from 50 mM Tris/HCl pH 8.5 and passed through a DE-52 column
Sigma (St. Louis, MO, USA). (11 × 2.5 cm) equilibrated with the same buffer. After wash-
ing the column with 600 ml of buffer 50 mM Tris/HCl pH
8.5, the bound proteins were eluted with a 500 ml 0–0.2 M
Bacterial strains and growth conditions NaCl gradient. Fractions were collected and assayed for L-
asparaginase activity.
The T. thermophilus strain HB8 used in all experiments. Active fractions were pooled and applied to a Phenyl
Micro-organisms were grown at 70°C in a medium contain- Sepharose CL-4B column (11 × 1.8 cm) equilibrated with
ing 0.5% (w/v) tryptone, 0.3% (w/v) yeast extract, 0.2% buffer 50 mM Tris/HCl pH 8.5, 50 mM NaCl. After washing
(w/v) NaCl, 0.1% (w/v) glucose, 2 µM FeCl3, 0.2 mM CaCl2 the column with 250 ml of the same buffer, the bound pro-
and 1 mM MgCl2. The pH was adjusted to 7.0 with NaOH. teins were eluted with 100 ml 20% ethylenglycol and 20–70%
Growth was monitored by measuring the turbidity at 600 nm. ethylene glycol gradient. Fractions were collected and as-
The bacteria were harvested in the static phase by centrifu- sayed for L-asparaginase activity. Active fractions were
gation at 6,000 g for 10 min. Cells were washed twice with pooled and diluted 1:1 with buffer 50 mM Tris/HCl pH 8.5.
0.9% (w/v) NaCl. The final yield was about 5 g of wet cells The enzyme solution was applied to a Cibacron Blue Seph-
per liter of culture medium. arose CL-6B column (10 × 1.6 cm) equilibrated with buffer
50 mM Tris/HCl pH 8.5 and 25% ethylenglycol. After wash-
ing the column with 250 ml of the same buffer, the bound
Assay for L-asparaginase proteins were eluted with a 200 ml 0–0.3 M NaCl gradient.
Active fractions were combined and applied on the next
The enzyme activity was measured by direct nesslerization column.
according to the method of Bergmeyer [14] in the assay Active fractions were diluted 1:3 with buffer 50 mM Tris/
buffer (50 mM Tris/HCl (Tris-(hydroxymethyl) amino- HCl pH 8.5 and applied to a Reactive Red Agarose column
methane) pH 10.0) and 10 mM L-asparagine or indirectly (7 × 1.4 cm) equilibrated with buffer 50 mM Tris/HCl pH 8.5
following the formation of NAD+ in a system where L-as- and 50 mM NaCl. The column was washed with 150 ml of
paraginase reaction was coupled to the reaction of aspartate the same buffer and the bound proteins were eluted with
aminotransferase and malate dehydrogenase [15]. All assays 100 ml 0.05–0.8 M NaCl gradient. Active fractions were
were carried out at 60°C (pH is about 9.2 at 60°C). pooled, dialysed against of 5 mM Na2HPO4-NaH2PO4 pH 8.0
The pH values for all buffers were measured at 25°C. The (buffer B) and passed through a Hydroxylapatite column (6
effect of temperature on pH for each buffer was taken into × 1.2 cm) equilibrated with buffer B. The enzyme was ap-
account and all pH values were corrected to L-asparaginase peared in the flow through of the column.
assay temperature to 60°C by the –dpH/dt (unit/degree) co- The enzyme solution was applied to a Heparin sepharose
efficient, specific for each buffer. column (5.5 × 1.1 cm) equilibrated with buffer B. After wash-
One international unit (IU) of L-asparaginase activity is ing the column with 100 ml of the same buffer, the bound
defined as the amount of enzyme liberating 1 µmol NH3 or proteins were eluted with a 50 ml 0–0.5 M NaCl gradient.
 95

Protein determination Superfine column (1.3 × 100 cm) equilibrated with Buffer B
and 0.05 M NaCl. Partially purified enzyme (1 ml) was ap-
Protein was determined by the method of Bradford [16] as plied to gel filtration and the column was washed with the
modified by Bearden [17], using bovine serum albumin as a same buffer. Fractions of 2 ml were collected and assayed for
standard. L-asparaginase activity.

Treatment of L-asparaginase with alkaline and acidic In situ renaturation of the enzyme
phosphatase
SDS electrophoresis was followed by in situ renaturation of
L-asparaginase is mixed with alkaline phosphatase from bo-
the proteins according to the method of Kameshita et al. [22],
vine and acidic phosphatase from T. pyriformis, in the opti-
except the change of the pH of the buffer used at the rena-
mal conditions for each phosphatase which are buffer 50 mM
turation stage. The method includes removal of SDS with
Tris-HCl pH 9.5, 100 mM NaCl, 5 mM MgCl2 for alkaline
20% isopropanol in buffer 50 mM Tris-HCl pH 8.0 (at room
phosphatase and buffer 50 mM Mes-NaOH pH 6.0, 5 mM
temperature), removal of isopropanol by washing the gel with
MgCl2 for acidic phosphatase. The mixtures are incubated at
buffer 50 mM Tris-HCl pH 8.0 and 5 mM β-MSH, denatura-
37°C for 30 min. The reaction for L-asparaginase is per-
tion with 6 M guanidine hydrochloride in buffer 50 mM
formed in the optimal conditions for L-asparaginase assay.
Tris-HCl pH 8.0 and 5 mM β-MSH and renaturation of the
proteins with buffer 50 mM Tris-HCl pH 7.0, 5 mM β-MSH
Con A-sepharose chromatography and 0.04% Tween-80 (4°C).

Con A-sepharose column that binds glycoproteins [18] was

equilibrated with buffer 50 mM Tris/HCl pH 8.5, 1 mM Chemical modification of L-asparaginase
CaCl2, 1 mM MnCl2 and 0.5 M NaCl.
Enzyme (0.1 IU) was incubated with 0–10 mM DEPC (di-
ethyl pyrocarbonate) (diluted in ethanol) in buffer 50 mM
SDS-polyacrylamide gel electrophoresis Na2HPO4-NaH2PO4 pH 7.0 at 37°C for 8 min at final volume
of 0.25 ml. At time intervals, 40 µl samples were removed,
L-asparaginase activity was assessed for purity by SDS-
the reaction was terminated by the addition of buffer 0.2 M
PAGE (polyacrylamide gel electrophoresis) according to the
imidazole-CH3COOH pH 7.0 (final concentration 25 mM)
method of Laemmli [19]. The SDS-PAGE gels were stained
and L-asparaginase activity was determined. The concentra-
with silver nitrate [20].
tion of ethanol in the mixture did not exceed 10% (v/v). To
test the reactivation by NH2OH, the enzyme solution was
Non-denaturing polyacrylamide gel electrophoresis preincubated with (0–10 mM) DEPC at 37°C for 10 min. The
reaction was terminated by the addition of buffer 0.2 M
The 7% nondenaturing polyacrylamide slab gel is made in imidazole-CH3COOH pH 7.0 (final concentration 25 mM),
the same manner as SDS-PAGE gels (buffer 375 mM Tris/HCl 100 mM (or 10, 20, 50 mM) NH2OH was added and the mix-
pH 8.9 but without SDS. Glucerol 10% w/v and bromophe- ture was incubated at 37°C for 60 min. The enzyme solution
nol blue 0.02% w/v are added to the samples. The electrode was dialysed against 500 ml buffer 50 mM Na2HPO4-NaH2PO4
buffer is 25 mM Tris/HCl and 192 mM glycine, pH 8.3. Elec- pH 7.0 and L-asparaginase activity was determined.
trophoresis is performed at 4°C. Other compounds used for modification were 1–50 mM
iodoacetamide at 37°C for 15 min (in buffer 50 mM Na2-
HPO4-NaH 2PO 4 pH 6.0 and 8.0), 0.1–10 mM NEM (N-
Isoelectric focusing ethylmaleimide) at 37°C for 15 min (in buffer 50 mM
Na2HPO4-NaH2PO4 pH 8.0), 0.05–1 mM p-HMB (p-mercury-
The isoelectric point of L-asparaginase was determined on
benzoate) at 37°C for 5 min (in buffer 50 mM Na2HPO4-
5.5% polyacrylamide gel following Laa’s procedure with
NaH2PO4 pH 8.0), 1–20 mM N-acetylo-imidazole at 37°C
ampholytes of pH range 3–10 [21].
for 30 min (in buffer 50 mM Na2HPO4-NaH2PO4 pH 7.0),
5–80 mM β-mercaptoethanol at 37°C for 30 min (in buffer
Molecular weight determination 50 mM Na2HPO4-NaH2PO4 pH 7.0), 0.5–10 mM PMSF at
37°C for 30 min (in buffer Tris-HCl pH 7.5), 1–25 mM EEDQ
The native molecular weight of L-asparaginase was deter- at 37°C for 20 min (2-ethoxy-1-ethoxycarbonyl-1,1-di-
mined by gel filtration chromatography on a Sephacryl S-300 hydro-quinoline) (in buffer 50 mM Mes-NaOH pH 6.0) and
96

0.5–10 mM phenyl-glyoxal at 37°C for 10 min (in buffer

50 mM Na2HPO4-NaH2PO4 pH 7.0).

Results
Purification of L-asparaginase to homogeneity from T. ther-
mophilus was achieved by 9 steps as indicated at Table 1, with
final yield 8.5 %, a purification factor 8842 fold and a spe-
cific activity of 840 IU/mg protein.
The specific activity of L-asparaginase rapidly increases
when the organisms enter the logarithmic phase and levels
off after reaching a maximum of 0.18 units per mg protein
in the stationary phase (Fig. 1). For all our purification ex-
periments cultures of T. thermophilus at late log phase were Fig. 1. L-asparaginase activity during growth of T. thermophilus. L-asparagi-
used. nase activity was measured at various times of growth in 1 liter cultures. Cells
(of 50 ml culture) were washed twice and suspended in 1 ml of buffer A (50
The molecular weight of L-asparaginase as estimated by
mM Tris-HCl pH 8.5, 1 mM β-mercaptoethanol and 0.3 mM PMSF) and L-
its mobility on Sephacryl S-300 superfine column, is around asparaginase activity was assayed as described under Materials and methods.
200 kDa (Fig. 2). By SDS electrophoresis the same prepara-
tion gave a single band of 33 kDa (Fig. 3). In situ renaturation
experiment on L-asparaginase (removal of SDS, as reported It was previously reported that L-asparaginase of L. michotii
in ‘Materials and methods’, with 20% isopropanol at pH 8.0, [28] and T. pyriformis is tremendously affected by a phos-
denaturation with 6 M guanidine hydrochloride at pH 8.0 and phorylation/dephosphorylation reaction [13]. When purified
renaturation at pH 7.0) and then run on SDS-polyacrylamide L-asparaginase (0.1 IU) from T. thermophilus was incubated
gel gave again a band of 33 kDa. This experiment shows that with increasing amounts of bovine alkaline phosphatase (0–
T. thermophilus L-asparaginase acts as hexamer and this is in 1 IU) no effect on the enzyme activity is observed. Similar
agreement with multimeric forms obtained so far from L-as- data were obtained when L-asparaginase was treated with an
paraginases of procaryotic and eucaryotic origin [23–27]. acidic protein phosphatase from T. pyriformis. The above
The purified L-asparaginase acts optimally at pH 9.2 (Fig. experiments show that either our purified enzyme does not
4) and presents an isoelectric point of 6.0 (Fig. 5). L-As- exist in a phosphorylated form or dephosphorylation by the
paraginase from T. thermophilus is quite stable and its ki- tested phosphatases do not affect L-asparaginase of T. ther-
netics are linear even at 70 or 77°C (Fig. 6). The divalent mophilus. Incubation of L-asparaginase with increasing
metals tested Mg2+, Mn2+, Ca2+ do not have any effect on L- concentration of phospholipase C resulted in no loss of its
asparaginase activity up to a concentration of 1 mM, while activity, indicating again the different catalytic properties of
Zn2+ at 0.5 or 1 mM inhibits L-asparaginase activity by 40 our enzyme and the lack of phospholipids dependence on its
or 60%, respectively. The Km for L-asparagine as deter- activity. When purified L-asparaginase was applied to a Con
mined from Lineweaver-Burk plot was found to be 2.8 mM A-Sepharose column, at the conditions that glycosylated
(Fig. 7). When D-asparagine or L-aspartic acid was included proteins are bound, all of its activity appeared in the flow
in the assay mixture L-asparaginase was inhibited competi- through indicating possibly that our protein is a non-N-
tively (Fig. 8). glycosylated molecule.

Table 1. Summary of the purification procedure of L-asparaginase

Purification steps Total protein (mg) Total activity (IU) Specific activity (IU/mg) Purification (fold) Yield (%)

Initial extract 4800 494 0.09 1 100

Supernate of 105,000 g 3511 378 0.11 1.2 78
(NH4)2SO4 (20–45%) 1017 320 0.31 3.2 65
DEAE-52 287 315 1.1 11.5 64
Phenyl sepharose 38 213 5.7 60 43
Cibacron blue sepharose 4 108 27 284 22
Reactive red agarose 0.2 84 420 4420 17
Hydroxylapatite 0.1 58 580 6105 11.7
Heparin sepharose 0.05 42 840 8842 8.5
 97

Fig. 2. Chromatography of L-asparaginase on Sephacryl S-300 superfine. Fig. 4. Plot of L-asparaginase activity vs. pH. The buffer used for pH 6.0–
Enzyme preparation (1 ml, 42 units) was applied on 100 × 1.3 cm column. 9.3 was 50 mM Tris-HCl, from 9.2–10.65 was 25 mM NaHCO3-NaOH and
The column was equilibrated and eluted with buffer 50 mM Tris-HCl pH from 10.3–11.3 was 25 mM NaHPO4-NaOH.
8.5 and 100 mM NaCl. The standard protein markers were: (1) Blue dex-
tran (2000 kDa); (2) β-Amylase (200 kDa); (3) Bovine albumin (67 kDa);
(4) Cytochrome c (12 kDa).
paraginase by DEPC was obtained when the substrate L-as-
paragine was added into the reaction mixture (Fig. 10). Af-
ter treatment of L-asparaginase with 10 mM DEPC in the
L-asparaginase was inhibited by the presence of DEPC
absence or in the presence of 10 mM L-asparagine for 7 min,
(0.5–10 mM). The kinetics of DEPC inactivation are shown
the residual activity was 30 and 65%, respectively. These
in Fig. 9. The slope of the replot at the inset of Fig. 9 presents
results suggest that one of the amino acid residues (serine,
a reaction rate of 0.8, indicating that reaction of one residue
histidine, lysine, cysteine or tyrosine) [29, 30] is located at
resulted in inactivation. Protection of inactivation of L-as-
or near active center of L-asparaginase. Modification of his-
tidine and serine is rapidly reversible by NH2OH, while ty-
rosine is more resistant and lysine or cysteine are irreversible
[31]. Unfortunately, in our case treatment with NH2OH at pH
7.0 of purified L-asparaginase was very sensitive and led to
the loss almost all of its activity. The effect of iodoacet-
amide, NEM, p-HMB [32], NAI (N-acetylo-imidazole), β-

Fig. 5. Isoelectric focusing of L-asparaginase. Approximately 30 µg of the

Fig. 3. Polyacrylamide gel electrophoresis of purified L-asparaginase. Puri- purified enzyme electrofocused in 5.5% polyacrylamide gel. The ampholytes
fied L-asparaginase (5 µg, lane 2) was subjected to SDS-PAGE. Protein was were at the pH range 3–10. The gel was cut in 5 mm sections and assayed
stained with silver nitrate method. Lane 1: molecular weight markers. for L-asparaginase activity.
98

Fig. 8. Double-reciprocal plots of L-asparaginase without inhibitor (–n–)

with 10 mM D-asparagine (–l–) and 7.5 mM L-aspartic acid (–s–).
Fig. 6. Kinetics of L-asparaginase at different temperatures.

mercaptoethanol, PMSF, EEDQ [33] and phenyl-glyoxal [34] T. thermophilus. The last few years a lot of attention was fo-
was also tested (Table 2). Treatment of purified L-asparagi- cused on stable enzymes derived from thermophilic and ex-
nase with the above mentioned compounds shows that the tra thermophilic bacteria. Thermostable enzymes isolated
amino acid residues which are modified and located at or near from thermophilic organisms, such as Taq-polymerase from
active site (protected by the substrate) are histidine, arginine Thermus aquaticus, Vent-polymerase from Thermococcus
and probably a carboxylic group of an acidic amino acid. A litoralis are in use now in most of the biochemical laborato-
serine and mainly a cysteine seems to play crucial role for ries.
the stability of the native enzyme or the formation of its ac- L-asparaginase of T. thermophilus was purified to homo-
tive sites, since modification of the mentioned amino acid geneity by ammonium sulfate precipitation and different
residues causes inactivation of the purified L-asparaginase, chromatographic columns. The enzyme was purified 8840 ×
with no protection by its substrate. fold and had a specific activity of 840 IU/mg protein.

Discussion
Here we describe for the first time the purification and char-
acterization of L-asparaginase from a thermophilic bacterium

Fig. 9. Kinetics of inactivation of L-asparaginase by DEPC. L-asparagi-

nase was incubated with different concentration of DEPC 0 mM (–n–),
0.5 mM (–l–), 1 mM (–s–), 2 mM (–t–), 3 mM (–u–), 5 mM (–+–), 7
Fig. 7. Determination of KmL-asn of purified L-asparaginase. Double-recip- mM (–x–) and 10 mM (–*–) at 37°C. At various times aliquots (40 µl) were
rocal plots of L-asparaginase versus concentration of substrate. The Km was removed and L-asparaginase activity was determined. Inset: Replot of the
determined by a Lineweaver-Burk plot. observed pseudofirst Kapp constants against log DEPC concentration.
 99

paraginases [35] and a Km for L-asparagine of 2.8 mM. Km

values of L-asparaginases vary from 0.01–7 mM. In the lit-
erature, L-asparaginases from S. marcescens, E. aroideae or
T. pyriformis with Km values close to that of T. thermophilus
L-asparaginase have been reported to possess antitumor ac-
tivity [14, 25, 36]. Our purified enzyme does not hydrolyse
D-asparagine or L-glutamine like asparaginases from several
other sources [37, 38].
Among the metals tested Mn2+, Mg2+ and Ca2+ do not ex-
ert any effect on the enzyme activity in contrast to Zn2+ which
partially inhibits L-asparaginase activity, similarly to other
L-asparaginases [39]. L-asparaginase of T. pyriformis and T.
thermophila were also inhibited by Zn2+ while activation was
observed only in the presence of Ca2+ [8, 27].
Different reagents were tested to find out which of the
amino acids are located at or near the active site of L-aspara-
Fig. 10. Protection of L-asparaginase from inactivation by DEPC. L-aspara-
ginase was incubated with 10 mM DEPC in the absence (–l–) or in the pres- ginase purified from T. thermophilus. Our results suggest that
ence (–s–) of 10 mM L-asparagine at 37°C. Control experiment without possibly histidine, arginine and a carboxyl amino acid are
treatment with DEPC (–n–) was also conducted. at or near the active site. Concerning the modification of
cysteines by pHMB or NEM or iodoacetamide, we can con-
clude that inactivation of L-asparaginase even by the presence
L-asparaginase of T. thermophilus is a multimeric protein of substrate, indicates that cysteine and less serine participate
of approximately 200 kDa and one subunit size of 33 kDa. in conformation or stabilization of the active enzyme.
Protein renaturation experiment on SDS-PAGE proved that Arginine was reported as possible amino acid at or adja-
the monomeric band of 33 kDa form the hexameric L-aspara- cent active site of L-asparaginase II from E. coli (EcA2) after
ginase. Similar to that we have shown that L-asparaginase of chemical modification of the enzyme with 2,3-butanodione.
T. pyriformis is a multimeric lipoprotein with relative molecu- Later, chemical modification of (EcA2) suggested that tyro-
lar weight approximately 200 kDa and one subunit size of sine and histidine are at or near active site [40, 41]. Site-di-
39 kDa [8]. This is in agreement with other reports, that L- rected mutagenesis indicated that a histidine (His-183) is
asparaginases occur in dimeric, tetrameric or hexameric form essential for assembly and stabilization of the native tetramer
in different biological systems [23–27]. of EcA2 [42]. Recent studies demonstrated that Thr-89, Asp-
In T. pyriformis the purified L-asparaginase exhibits a ki- 90 and Lys-162 are the amino acids residues that form the
nase activity as well and it is autophosphorylated on tyrosine surface of active site of EcA2 and these amino acids are con-
residues. Phosphorylation, or dephosphorylation, of L-as- nected by strong hydrogen bonds [43, 44]. Thr-12 is the ini-
paraginase resulted in a complete loss or activation by more tial nucleophile while Ser-58 and His-87 are involved in net
than 10-fold of its catalytic activity, respectively [12]. bond of substrate binding.
Most L-asparaginases act at pH range 7–9. The purified Crystallographic studies of EcA2 demonstrated that the
enzyme from T. thermophilus presents optimal activity at enzyme consists of four identical subunits A, B, C, D. EcA2
pH 9.2, an isoelectric point of 6.0 close to pI of other as- is active only in a tetrameric form. Firstly, two intimate pairs

Table 2. Chemical modification of L-asparaginase

Modifier Modified amino acid Effect Observations

DEPC Serine, histidine, lysine, Inactivation

cysteine, tyrosine
Iodoacetamide pH 6.0 Histidine Inactivation Protection by substrate
Iodoacetamide pH 8.0 Cysteine Inactivation No protection by substrate
pHMB Cysteine Inactivation No protection by substrate
NEM Cysteine Inactivation No protection by substrate
PMSF Serine Partial inactivation No protection by substrate
NAI Tysosine No effect
β-MSH Cystine No effect
N-EEDQ Aspartic acid, Glutamic acid Inactivation
Phenylglyoxal Arginine Inactivation Protection by substrate
100

of subunits are formed from extensive interactions between 12. Tsirka SAE, Kyriakidis DA: A model for the regulation of the activity
subunits A and B and between C and D. Tetramer is a dimer of L-asparaginase/kinase enzyme of Tetrahymena pyriformis. Biochem
Intern 19: 9–17, 1989
of identical intimate dimers AB and CD [45]. Each dimer has 13. Tsirka SAE, Kyriakidis DA: L-Asparaginase of Tetrahymena pyri-
two active sites but only the tetramer has been reported as formis is associated with a kinase activity. Mol Cell Biochem 95: 77–
the active. Each active site is made up primarily of residues 87, 1990
from the N-terminal domain, with contacts from the C-ter- 14. Wriston JC Jr: Asparaginase. In: L. Lorand, S.P. Colwick, N.O. Kaplan
minal domain of the intimately bound subunit. It has been (eds). Methods in Enzymology. Academic Press, New York, 1985, pp
608–618
proposed that most bacterial L-asparaginases are function- 15. Ho PKP, Milikin BE, Bobbit LJ, Grinnan LE, Burck JP, Frank HB,
ing as tetramers while L-asparaginases from plants are dimers Boech DLV, Squires RW: Crystalline L-asparaginase from Escherichia
in native form. coli. Purification and chemical characterization. J Biol Chem 245:
In the case of the purified L-asparaginase of T. thermophilus 3708–3715, 1970
we do not know where the active site might be. Therefore, 16. Bradford MM: A rapid and sensitive method for the quantitation of
microgram quantities of protein utilizing the principle of protein-dye
the experiments of site directed mutagenesis or reconstitu- binding. Anal Biochem 72: 248–254, 1976
tion experiments with X-rays analysis will elucidate this is- 17. Bearden JC: Quantitation of submicrogram quantities of protein by an
sue. Part of the sequence of our purified enzyme is already improved protein-dye binding assay. Biochim Biophys Acta 533: 525–
published [46]. Experiments are in progress now and will be 529, 1978
published shortly. 18. Goldstein IJ, Hollerman CE, Merrick JM: Protein-carbohydrate inter-
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19. Laemmli UK: Cleavage of structural proteins during the assembly of
the head of bacteriophage T4. Nature 277: 680–685, 1970
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