(Received for publication, July 25, 1995; and in revised form, September 28, 1995)
From the
Pyrococcus furiosus uses a modified Embden-Meyerhof
pathway during growth on poly- or disaccharides. Instead of the usual
ATP-dependent glucokinase, this pathway involves a novel ADP-dependent
(AMP-forming) glucokinase. The level of this enzyme and some other
glycolytic enzymes appeared to be closely regulated by the substrate.
Growth on cellobiose resulted in a high specific activity of 0.96 units
mg, whereas on pyruvate a 10-fold lower activity was
found. The ADP-dependent glucokinase was purified 1350-fold to
homogeneity. The oxygen-stable enzyme had a molecular mass of 93 kDa
and was composed of two identical subunits (47 kDa). The glucokinase
was highly specific for ADP, which could not be replaced by ATP,
phosphoenolpyruvate, GDP, PP
, or polyphosphate. D-Glucose could be replaced only by
2-deoxy-D-glucose, albeit with a low efficiency. The K
values for D-glucose and ADP
were 0.73 and 0.033 mM, respectively. An optimum temperature
of 105 °C and a half-life of 220 min at 100 °C are in agreement
with the requirements of this hyperthermophilic organism. The
properties of the glucokinase are compared to those of less
thermoactive gluco/hexokinases.
During the past decade, an increasing number of microorganisms have been described that have their optimum growth temperature above 80 °C(1, 2) . Except for two bacterial genera, all of the more than 50 hyperthermophilic species isolated thus far are classified as Archaea (formerly Archaebacteria), the third domain of life(3) .
Because of its favorable culturing
conditions, Pyrococcus furiosus is the best studied anaerobic
hyperthermophile to date. Next to some polypeptides and
polysaccharides, P. furiosus is able to use maltose and
cellobiose as simple substrates(4, 5, 6) .
These disaccharides are transported into the cell, hydrolyzed to
glucose, and fermented to mainly acetate, alanine, H, and
CO
(7) . Initially, P. furiosus was
believed to use a novel non-phosphorylated type of Entner-Doudoroff
pathway, called pyroglycolysis(5, 8) . However, recent
C in vivo NMR data were not consistent with a
major role for the pyroglycolysis(9, 10) . The
C labeling pattern suggested that an Embden-Meyerhof-like
pathway was most likely to be involved(9) . Conventional
glucokinase and phosphofructokinase could, however, not be detected in
cell-free extracts(5) . Remarkably, two novel sugar kinases
were recently discovered that required ADP instead of ATP(9) .
In contrast to the key enzymes of the pyroglycolysis, the specific
activities of both kinases were sufficiently high to envisage a major
catabolic role. Instead of a classical NAD-dependent
glyceraldehyde-3-phosphate dehydrogenase, P. furiosus was
recently shown to harbor a novel tungsten-containing
glyceraldehyde-3-phosphate:ferredoxin oxidoreductase(11) . The
presence of an enzyme that converts glyceraldehyde-3-phosphate instead
of glyceraldehyde also substantiates the operation of a modified
Embden-Meyerhof pathway instead of the pyroglycolysis. The discovery of
the novel type of kinases and the tungsten proteins in P. furiosus supports the idea that life at elevated temperatures may involve
different metabolic steps as a result of an altered biochemistry or a
decreased stability of biomolecules.
In this paper, we describe the purification and characterization of the novel ADP-dependent glucokinase. The results show that ADP-dependent conversion of glucose is not only found in crude P. furiosus extracts but is catalyzed by a single enzyme that shows a characteristic specificity. The properties of the Pyrococcus enzyme are compared to those of glucokinases from less thermophilic sources.
Mass culturing (200 liters) was
performed on the same medium except that NaS was omitted,
the fermenter was sparged with N
, and potato starch was
used as substrate (5 g/liter).
For use in enzyme purifications,
cells and cell extracts were handled aerobically. Cells were suspended
in 50 mM Tris/Cl buffer (pH 7.8) (0.5 g of
cells/ml of buffer) containing DNase (10 µg/ml), and the suspension
was passed twice through a French press at 138 MPa. Cell debris were
removed by centrifugation for 1 h at 100,000
g. The
cell extract, containing 35-45 mg protein/ml, was stored at
-20 °C until use.
Protein was determined with Coomassie Brilliant Blue G250 as described by Bradford(12) , using bovine serum albumin as a standard. Occasionally, a modified more sensitive Coomassie Brilliant Blue G250 method was used as described by Löffler and Kunze(13) .
ADP-dependent glucokinase was determined by measuring the formation
of NADPH in a coupled assay with yeast glucose-6-phosphate
dehydrogenase. The assay was performed at 50 °C. At this
temperature, the yeast enzyme remained active, and the Pyrococcus enzyme was sufficiently active to measure its activity. The assay
mixture contained 100 mM Tris/Cl, pH 7.8, 10
mM MgCl
, 0.5 mM NADP, 15 mMD-glucose, 2 mM ADP, 0.35 units of D-glucose-6-phosphate dehydrogenase, and 5-50 µl of
enzyme preparation. The absorbance of NADPH was followed at 334 nm
(
= 6.18 mM
cm
). Care was taken that the activity of the
auxiliary enzyme was always in excess of the glucokinase activity.
Phosphoglucose isomerase (EC 5.3.1.9) and ADP-dependent phosphofructokinase were determined at 50 °C using auxiliary enzymes as described before(9) .
Purity of the enzyme was checked
by native and denaturing SDS-PAGE as described before(6) . For
determination of the subunit composition by SDS-PAGE, protein samples
were diluted in sample buffer, containing 2% SDS (w/v) and 5%
2-mercaptoethanol, and subsequently heated at 100 °C. In some
cases, 2-mercaptoethanol was omitted from the sample buffer, and the
sample was not heated. Silver staining was performed using the reagent
kit from E. Merck (Darmstadt, Germany). Activity staining was performed
on native PAGE gels by coupling the glucokinase activity to the
reduction of nitro blue tetrazolium. Therefore, the gel was incubated
at 37 °C in the dark for 30 min in a staining mix with the
following composition: 100 mM Tris/Cl, pH
7.8, 0.001% phenazin methosulfate, 0.035% nitro blue tetrazolium, 15
mM MgCl
, 0.5 mM NADP, 3 mM ADP,
15 mMD-glucose, 185 mM NaCl, and D-glucose-6-phosphate dehydrogenase (1.75 units).
Thermostability of the glucokinase was determined by incubating purified glucokinase in 200 mM Tris/maleate buffer (pH 8.5) at 100 °C in crimp-sealed vials, submerged in an oil bath. At certain time intervals, 50-µl aliquots were withdrawn and analyzed for activity in the standard assay.
Figure 1: Levels of glycolytic enzymes in cell-free extracts of P. furiosus grown on various substrates. The specific activities were determined as given under ``Experimental Procedures.'' For each substrate, the bars indicate the specific activity of the ADP-dependent glucokinase (left hatch), the phosphoglucose isomerase (empty), and the ADP-dependent phosphofructokinase (cross-hatch).
The use of affinity chromatographic techniques, like
ADP-agarose (either ribose-linked or N6-linked) or various
dyeligand-agaroses (Dyematrex screening kit, Amicon), was unsuccessful
because the enzyme did not bind to any of the ligands, even in the
presence of 10 mM MgCl. Therefore, a series of
seven sequential purification steps were required to obtain a
homogeneous preparation as judged by a silver-stained PAGE gel (Table 1). The colorless enzyme was 1346-fold purified with 2.1%
recovery and showed a specific activity of 307 units mg
at 50 °C. The identity of the band was confirmed by activity
staining.
Figure 2: SDS-polyacrylamide gel electrophoresis of the glucokinase from P. furiosus. Lane 1 shows a set of marker proteins with their molecular mass indicated. Lanes 2-5 contained the 980-fold purified protein (0.45 µg protein/lane). Lanes 2 and 3 contained glucokinase diluted in sample buffer without and with 2-mercaptoethanol, respectively, and which were not boiled. Lanes 4 and 5 contained glucokinase that was boiled in sample buffer for 2 and 45 min, respectively. Proteins were stained with Coomassie Brilliant Blue R250.
The temperature dependence of the activity is shown in Fig. 3. The optimum temperature was found at 105 °C (15-min
incubation period). Because of a rapid denaturation above this
temperature, this optimum value may increase or decrease depending on
the time of incubation (shorter or longer incubation time,
respectively). An Arrhenius plot of the data (Fig. 3, inset) showed a breakpoint at 60 °C, resulting in
activation energy values of 54.3 kJ mol between 30
and 60 °C and 37.4 kJ mol
between 60 and 105
°C.
Figure 3: Dependence of glucokinase activity on temperature. Activity was determined by measuring the amount of glucose-6-phosphate formed after incubation for an appropriate period of time at the desired temperature. Inset, Arrhenius plot of the data from 30 to 105 °C.
P. furiosus has recently been shown to utilize a modified Embden-Meyerhof pathway, which involves a glucokinase and a phosphofructokinase that are both ADP-dependent(9) . Here, the ADP-dependent glucokinase was purified and characterized. Cell-free extracts of P. furiosus contained high levels of this enzyme, especially when the organism was grown on cellobiose. This high level of the glucokinase and also of the phosphoglucose isomerase and the phosphofructokinase in cellobiose-grown cells as compared to maltose-grown cells clearly shows that at least the first steps of the glycolysis are closely regulated in this hyperthermophilic Archaeon. This also follows from the low activity of these enzymes on pyruvate- and peptone-grown cells.
The cytoplasmic and oxygen-stable
glucokinase was purified more than 1000-fold to homogeneity. Thus, the
glucokinase constitutes less than 0.1% of the total cellular protein.
This seems a rather low value for such a key enzyme. However, using the
experimentally determined relationship between activity and
temperature, the specific activity at 100 °C amounts to 2,233 units
mg (k
= 3,500
s
), which is the highest reported (Table 3).
Moreover, it has been calculated before that the specific activity in
cell-free extracts is more than satisfactory to sustain the glucose
flux(9) .
The ADP-dependent glucokinase had a native
molecular mass of approximately 93 kDa and consisted of two identical
subunits of approximately 47 kDa. This composition is
observed also for bacterial glucokinases and eukaryotic hexokinases,
but it differs from the eukaryotic glucokinases (bakers' yeast,
rat liver), which show a monomeric structure (Table 3).
Furthermore, the P. furiosus glucokinase differs from most
bacterial enzymes by its native molecular mass, which is about twice
the usual size of about 50 kDa. In this respect, it resembles more the
hexokinases from Eukarya. All the glucokinases described in
detail (with sequence information) can be grouped into three
evolutionary disconnected clusters, i.e. the mammalian
glucokinases, the yeast glucokinases, and the bacterial
glucokinases(25) . However, based on the partial N-terminal
sequence obtained for the P. furiosus enzyme, the latter does
not group within any of these. Also, no similarity was found within
other kinase families(25) . Nevertheless, many of these
kinases, if not all, exhibit a striking structural feature; each
subunit contains two lobes separated by a cleft(26) . Upon
binding of the substrate the two lobes come together. Whether the Pyrococcus enzyme also shows this substrate-induced cleft
closing remains to be elucidated.
A comparison with respect to the
substrate specificity is difficult since for many enzymes these data
are incomplete. Nevertheless, the glucokinase from P. furiosus showed a high specificity for the type of sugar as well as the
phosphoryl group donor. From previous NMR experiments, it can be
concluded also that the glucokinase is able to use both the - and
-anomer of D-glucose (9) . Thus, the enzyme is a
true glucokinase and highly specific for ADP. The specificity for ADP
is also reflected in the high affinity found for this compound, i.e. the K
value of 0.033 mM is
the among the lowest
reported(15, 16, 17, 18, 19, 20, 21, 22, 23, 24) .
The reason for the ADP dependence may lie in the ability to activate
sugars at conditions of low energy charge, e.g. after a period
of starvation. The ATP level is then probably very low, and the
relatively high ADP-level may still enable the phosphorylation of
sugars.
The dependence of the activity on temperature showed an optimum at 105 °C, which is in accordance with the requirements of the organism. The breakpoint in the Arrhenius plot at 60 °C has been observed before for other thermophilic enzymes and may reflect a conformational change of the protein(27, 28) .
The purified glucokinase showed a high thermostability (half-life of 220 min) at the physiological growth optimum of 100 °C. As has been found for most other enzymes from (hyper)thermophiles(29) , this thermostability is apparently not determined by extrinsic factors, like high salt concentrations or specific compatible solutes.
It is evident that P. furiosus harbors an exceptional ADP-specific glucokinase, perfectly fit for catalyzing the phosphorylation of glucose at elevated temperatures. Up to now, the ADP dependence is unique for P. furiosus. Other sugar-converting thermophiles examined thus far use either the classical Embden-Meyerhof pathway (30, 31) or a non-phosphorylated version of the Entner-Doudoroff pathway(32, 33) . Further research is focussing on the distribution of this type of enzyme among other (hyper)thermophiles and on the determination of their primary structure, which both will shed light on the evolutionary position of the ADP-dependent glucokinase. Moreover, the function of the ADP-dependent enzymes in relation to the bioenergetics of the organism is under present investigation.