Activity-Stability Relationships in Extremophilic Enzymes*
Salvino
D'Amico,
Jean-Claude
Marx,
Charles
Gerday, and
Georges
Feller
From the Laboratory of Biochemistry, University of Liège,
Institute of Chemistry B6,
B-4000 Liège-Sart Tilman, Belgium
Received for publication, December 9, 2002
 |
ABSTRACT |
Psychrophilic, mesophilic, and thermophilic
-amylases have been studied as regards their conformational
stability, heat inactivation, irreversible unfolding, activation
parameters of the reaction, properties of the enzyme in complex with a
transition state analog, and structural permeability. These data
allowed us to propose an energy landscape for a family of extremophilic
enzymes based on the folding funnel model, integrating the main
differences in conformational energy, cooperativity of protein
unfolding, and temperature dependence of the activity. In particular,
the shape of the funnel bottom, which depicts the stability of the native state ensemble, also accounts for the thermodynamic parameters of activation that characterize these extremophilic enzymes, therefore providing a rational basis for stability-activity relationships in protein adaptation to extreme temperatures.
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INTRODUCTION |
Our planet harbors a huge number of harsh environments that are
considered as "extreme" from an anthropocentric point of view, as
far as temperature, pH, osmolarity, free water, or pressure are
concerned. Nevertheless, these peculiar biotopes have been successfully
colonized by numerous organisms, mainly extremophilic bacteria and
archaea. As the curiosity of scientists stimulates the exploration of
new environments, it seems that there is no "empty space" for life
on Earth and, for instance, even the supercooled cloud droplets contain
actively growing bacteria (1). Among the extremophilic microorganisms,
those living at extreme temperatures have attracted much attention.
Thermophiles have revealed the unsuspected upper temperature for life
at about 113 °C (2, 3). Their enzymes have also demonstrated a
considerable biotechnological potential such as the various
thermostable DNA polymerases used in PCR that have boosted many
laboratory techniques. At the other end of the temperature scale,
metabolically active psychrophilic bacteria have been detected in
liquid brine veins of sea ice at
20 °C (4). These cold-loving
microorganisms face the thermodynamic challenge to maintain
enzyme-catalyzed reactions and metabolic rates compatible with
sustained growth near or below the freezing point of pure water (5, 6).
Directed evolution experiments have highlighted that, in theory, cold
activity of enzymes can be gained by several subtle adjustments of the
protein structure (7). However, in natural cold environments, the
consensus for the adaptive strategy is to take advantage of the lack of
selective pressure for stable proteins for losing stability,
therefore making the enzyme more mobile or flexible at temperatures
that "freeze" molecular motions and reaction rates (8).
The crystal structures of extremophilic enzymes unambiguously indicate
a continuum in the molecular adaptations to temperature. There is
indeed a clear increase in the number and strength of all known weak
interactions and structural factors involved in protein stability from
psychrophiles to mesophiles (living at intermediate temperatures close
to 37 °C) and to thermophiles (2, 9-11). Therefore, the same
mechanism of molecular adaptation is involved in response to two
distinct selective pressures, i.e. the requirement for
stable protein structure and activity in thermophiles and the
requirement for high enzyme activity in psychrophiles. This of course
suggests intricate relationships between activity and stability in
naturally evolved enzymes from these extremophiles. To date, these
relationships have not been investigated in details mainly because most
extremophilic enzymes were not analyzed in rigorously identical
conditions. In the present work, we have investigated three
structurally homologous
-amylases. The
-amylase from
Pseudoalteromonas haloplanktis
(AHA),1 formerly
Alteromonas haloplanctis, is the best characterized psychrophilic enzyme isolated from an Antarctic bacterium (12-14). Interestingly, its closest structural homologue is the pig pancreatic
-amylase (PPA), which is taken here as the mesophilic reference (15,
16). Bacillus species are able to colonize moderately thermophilic habitats, and Bacillus amyloliquefaciens
secretes a thermostable
-amylase (BAA). This enzyme has been
selected as the thermophilic counterpart because its unfolding is
completed below 100 °C (17, 18), therefore allowing biophysical
investigations at atmospheric pressure. The three-dimensional
structures of these
-amylases can be superimposed, showing a strong
conservation of the active site architecture, of the main secondary
structures, and of the overall fold (14). However, the melting points
of these proteins are markedly different: 45 °C for AHA, 65 °C
for PPA, and 85 °C for BAA (12). Therefore, they constitute an
adequate series of homologous enzymes for temperature adaptation studies.
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EXPERIMENTAL PROCEDURES |
Sources--
The recombinant AHA was expressed in
Escherichia coli at 18 °C and purified by DEAE-agarose,
Sephadex G-100, and Ultrogel AcA54 column chromatography, as described
previously (13). PPA and BAA were from Roche Molecular Biochemicals and
Sigma, respectively.
Enzyme Assays--
-Amylase activity was recorded between 3 and 25 °C using 3.5 mM
4-nitrophenyl-
-D-maltoheptaoside-4,6-O-ethylidene
as substrate and excess
-glucosidase as coupling enzyme in 100 mM Hepes, 50 mM NaCl, 10 mM
MgCl2, pH 7.2. Activities were recorded in a thermostated Uvikon 860 spectrophotometer (Kontron) and calculated on the basis of
an absorption coefficient for 4-nitrophenol of 8,980 M
1 cm
1 at 405 nm; a
stoichiometric factor of 1.25 was applied (13). Amylolytic activity
recorded over a wide range of temperatures, up to 90 °C, was assayed
by the dinitrosalicylic acid method (19) using 1% soluble starch as
substrate in 100 mM Hepes, 50 mM NaCl, pH 7.2. Heat inactivation of enzymes incubated in 100 mM Hepes, 50 mM NaCl, pH 7.2, was recorded as a function of time at
various temperatures. The residual activity of timed aliquots stored in ice for 1 h was measured by the standard assay at 25 °C.
Activation Parameters--
Thermodynamic parameters of
activation were calculated as described (20) using Equations
1-3,
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(Eq. 1)
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(Eq. 2)
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(Eq. 3)
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where kB is the Bolzmann constant,
h the Planck constant, Ea is the
activation energy of the reaction, and R the gas constant.
Unfolding Recorded by Intrinsic Fluorescence--
Fluorescence
was recorded using an SML-AMINCO Model 8100 spectrofluorimeter
(Spectronic Instruments) at an excitation wavelength of 280 nm
(1-nm band pass) and at an emission wavelength of 350 nm (4-nm band
pass). Heat-induced unfolding was recorded in 100 mM Hepes,
50 mM NaCl, pH 7.2, at a scan rate of 1 °C/min using a
programmed water bath Lauda Ecoline RE306. GdmCl-induced unfolding was
monitored at 20 °C after a 4-h incubation of the samples at this
temperature in 30 mM MOPS, 50 mM NaCl, 1 mM CaCl2, pH 7.2. Data were normalized
using the pre- and post-transition baseline slopes as described
(21). Least squares analysis of
G values as a function of
GdmCl concentrations allowed the estimation of the conformational
stability in the absence of denaturant,
GH2O, according to Equation 4,
|
(Eq. 4)
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The biphasic transition of BAA was analyzed using a three-state
model (22).
Dynamic Quenching of Fluorescence--
The
acrylamide-dependent quenching of intrinsic protein
fluorescence was monitored in the presence of ~50 µg of enzyme
(A280 <0.1) in a 1-ml initial volume of 30 mM MOPS, 50 mM NaCl, 1 mM CaCl2, pH 7.2, at an excitation wavelength of 280 nm (1-nm
band pass) and at an emission wavelength of 350 nm (2-nm band pass) after consecutive additions of 4 µl of 1.2 M acrylamide
in the same buffer. The Stern-Volmer quenching constants
KSV were calculated according to the relation (23)
shown in Equation 5,
|
(Eq. 5)
|
where F and F0 are the
fluorescence intensity in the presence and absence of molar
concentration of the quencher Q, respectively. The intrinsic protein
fluorescence F was corrected for the acrylamide inner filter
effect f, the latter being defined as shown in Equation 6,
|
(Eq. 6)
|
using an extinction coefficient
for acrylamide at 280 nm of
4.3 M
1 cm
1.
Differential Scanning Calorimetry (DSC)--
Measurements were
performed using a MicroCal MCS-DSC instrument as detailed (12). Samples
(~3 mg/ml) were dialyzed overnight against 30 mM MOPS, 50 mM NaCl, 1 mM CaCl2, pH 7.2. Thermograms of enzyme-acarbose complexes were recorded in the presence
of 1 mM acarbose (Bayer). Thermograms were analyzed
according to a non-two-state model in which the melting point
Tm, the calorimetric enthalpy
Hcal, and the van't Hoff enthalpy
Heff of individual transitions are fitted
independently using the MicroCal Origin software (version 2.9). The
magnitude and source of the errors in the
Tm and enthalpy values have been
discussed elsewhere (24). Fitting standard errors on a series of three DSC measurements made under the same conditions in the present study
were found to be ±0.05 K on Tm and
±1% on both enthalpies. Thermograms for equilibrium unfolding were
recorded at 2 K min
1 and in the presence of 0.5 M 3-(1-pyridinio)-1-propanesulfonate for PPA and BAA. The
thermodynamic parameters of unfolding were calculated using the
relations (25) shown in Equations 7-9,
|
(Eq. 7)
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(Eq. 8)
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(Eq. 9)
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with a heat capacity difference between the native and unfolded
state
Cp = 8.47 kcal mol
1
K
1 as determined experimentally (12). Kinetically driven
unfolding was recorded at low scan rates (0.1, 0.5, and 1 K
min
1), and the rate constant k was calculated
from the relation (26) shown in Equation 10,
|
(Eq. 10)
|
where v is the scan rate,
Cp is the excess heat capacity at a
temperature T,
cal is the total heat of the
process, and
(T) is the heat evolved at a given
temperature T.
 |
RESULTS AND DISCUSSION |
Conformational Stability--
The stability of AHA, PPA, and BAA
is illustrated in Fig. 1 by their
GdmCl-induced unfolding transitions. These enzymes unfold at distinct
denaturant concentrations (C1/2) and are
characterized by a decrease of unfolding cooperativity (m
value) and the appearance of intermediate states (BAA) as the stability
increases. These observations parallel the behavior of protein adapted
to different temperatures as recorded by thermal unfolding (12, 13).
Estimation of the conformational stability in the absence of denaturant
(
GH2O) at
20 °C using Equation 4 provides a ratio of 1/2/6 for AHA, PPA, and
BAA, respectively. The stability curves of the investigated enzymes
have been calculated from DSC data using the modified Gibbs-Helmholtz
equation (Equation 7). This function corresponds to the energy required
to disrupt the native state at any temperature (27) and by definition
is nil at the melting point (Fig. 2). The
psychrophilic AHA unfolds reversibly according to a two-state mechanism, and the validity of its stability curve has been
demonstrated by detection of the cold melting point predicted by the
function (12). The poorly reversible thermal unfolding of PPA
and BAA was analyzed according to the model of Lumry and Eyring, shown in Equation 11,

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Fig. 1.
Equilibrium unfolding of the psychrophilic
AHA ( ), the mesophilic PPA ( ), and the thermophilic BAA ( ) as
recorded by fluorescence emission. Thermodynamic parameters
of GdmCl-induced unfolding at 20 °C are as follows: for AHA,
C1/2 = 0.9 M, m = 4.3 kcal mol 1 M 1,
GH2O = 3.7 kcal mol 1; for PPA, C1/2 = 2.6 M, m = 2.7 kcal mol 1
M 1,
GH2O = 6.9 kcal mol 1; for BAA, C1/2 = 6.0 M,
GH2O = 23.8 kcal mol 1. Reversibility was 85% for the three
enzymes.
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Fig. 2.
Gibbs free energy of unfolding.
Stability curves are shown for the psychrophilic AHA, the mesophilic
PPA, and the thermophilic BAA, as calculated from microcalorimetric
data (Equation 7).
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(Eq. 11)
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using DSC data generated at fast scan rates (2 K/min) in the
presence of a non-detergent sulfobetaine to prevent aggregation (28).
In these conditions, it is assumed that the irreversible step has much
lower enthalpy contribution and rate constant than the reversible step.
As a result, the unfolding transition can be regarded to a first
approximation as an equilibrium transition and can be treated by the
equations of equilibrium thermodynamics (29, 30). Some general
conclusions concerning the stability of extremophilic enzymes can be
deduced from these curves. Despite large differences in melting point
Tm, the
Gmax
values (top of the curve) are centered around room temperature, as
anticipated from theoretical studies (31). Accordingly, the increasing
melting points of the three enzymes are predominantly reached by
lifting the stability curves. This is obtained by a corresponding
increase of
Hcal in Equation 7
(at constant
Cp), demonstrating the major involvement of enthalpic stabilization of the protein structure in
temperature adaptation (32, 33). Furthermore, the analysis of these
curves indicates that the heat-labile psychrophilic enzyme is also
cold-labile, contrary to what is intuitively expected, whereas the
heat-stable BAA is predicted to be the most cold-stable protein.
The enthalpic and entropic contributions to
G have been calculated according to Equations 8 and 9 (Table
I). The physiological temperatures for
the heat-stable PPA and BAA lie on the right side of the bell-shaped
G curves, and therefore, their structure is stabilized
enthalpically, whereas the thermal dissipative contribution of the
unfavorable entropic term provides the required mobility for the enzyme
function (25). In contrast, the physiological low temperatures for the
psychrophilic enzyme lie on the left side of its stability curve. It
follows that in environmental conditions, its structure is stabilized
entropically, whereas the enthalpic contribution becomes a
destabilizing factor. It is thought that hydration of polar and
nonpolar groups is responsible for the decrease of stability at low
temperatures, leading to cold unfolding (32, 34), and accordingly, this
factor should contribute to the required mobility for function at
environmental low temperatures.
Irreversible Unfolding and Heat Inactivation--
Heat
inactivation of activity was analyzed according to the kinetic model
shown in Equation 12,
|
(Eq. 12)
|
and the activation parameters were calculated from Arrhenius
plots. The irreversible conformational unfolding of PPA and BAA was
analyzed from DSC data generated at low scan rates (<1 K/min). Such
kinetic analysis is not possible for AHA (fully reversible unfolding),
and data were obtained from its mutant N12R. The latter displays the
same microcalorimetric properties as AHA but only 30% denaturation
reversibility (13), ensuring that unfolding is kinetically driven. As a
matter of fact, the melting point of the three enzymes is scan
rate-dependent below 1 K/min, and the rate constant
k was calculated from Equation 10. The corresponding activation parameters are provided in Table
II, which shows that both heat
inactivation of activity and irreversible unfolding of the structure
display the same trends. The increase in free energy of activation from
AHA to BAA reflects that the same denaturation rate constants
k are reached at increasing temperatures (44, 60, and
80 °C for heat inactivation of AHA, PPA, and BAA, respectively). The
small differences in
G*, however, arise from large
differences in the enthalpic and entropic contributions. Indeed,
the lowest
G* value for the psychrophilic enzyme
corresponds to the largest
H* and T
S*
contributions and conversely corresponds to the heat-stable BAA. The
decrease of
H* as enzyme stability increases mainly reflects the decrease in cooperativity of inactivation and of unfolding: for instance, the heat-labile AHA denatures in a shorter temperature range, leading to the sharp slope of the Arrhenius plots
and subsequently to high activation energy Ea and
H*. Such high cooperativity probably originates from the
lower number of interactions required to disrupt the active
conformation. In this formalism,
S* >0 suggests that the
randomness of the activated transition state increases before
irreversible inactivation or unfolding. Accordingly, the transition
state of the psychrophilic enzyme is reached through a larger entropy
variation than that of BAA, possibly reflecting an increased disorder
(of the active site or of the structure), which is the main driving
force of heat denaturation. In addition, the thermophilic enzyme
seems resistant to denaturation before the irreversible loss of
activity and conformation (low
S*). A thermophilic enzyme
can then be regarded as an intrinsically stable protein that
counteracts heat denaturation by a weak cooperativity of unfolding and
inactivation. The entropy loss associated with hydration of nonpolar
groups upon denaturation can also contribute to the differences in
entropic contribution reported in Table II. In the case of unfolding,
this is supported by the slight increase of hydrophobicity from AHA to
BAA noted for these enzymes (12).
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Table II
Thermodynamic parameters for the irreversible heat inactivation of
activity and for irreversible unfolding
As the three enzymes are denatured in different temperature ranges,
activation data are reported for an identical rate constant,
k = 0.05 s 1. Thermal unfolding data for AHA
were obtained with the mutant N12R.
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Conformational Flexibility and Protein Permeability--
It is
generally assumed that the increase in stability from psychrophilic to
thermophilic proteins arises from a decrease in conformational
flexibility (5, 25, 35-37). However, some experimental data do not
fully support this theory (38-40), but it is also recognized that the
time scale and motions possibly involved remain largely undefined (41).
The conformational flexibility of the three investigated enzymes was
probed by dynamic fluorescence quenching. Briefly, this technique
utilizes increasing concentrations of a small quencher molecule
(acrylamide in the present case) to probe the accessibility of
tryptophane within a protein. The decrease of fluorescence arising from
diffusive collisions between the quencher and the fluorophore reflects
the ability of the quencher to penetrate the structure and can be
viewed as an index of protein permeability (23). As illustrated in Fig.
3, this index significantly decreases
from AHA to BAA (at 10 and 37 °C) and is inversely correlated with
stability: the higher the stability, the lower the permeability. However, absolute values of the Stern-Volmer quenching constant (KSV) can be compared only if the
location of tryptophane residues in the structure is identical as well
as their environment. This condition cannot be met for most closely
related protein homologues (12, 19, and 17 Trp residues for AHA, PPA,
and BAA, respectively), and therefore, the variation of these constants with temperature becomes the relevant parameter. Again, Fig. 3 shows
that the variation of fluorescence quenching between 10 and
37 °C decreases in the order psychrophile
mesophile
thermophile. A similar variation was obtained between 10 and 25 °C
(data not shown). Thus, the increase in protein permeability in a
temperature range where the native state prevails is much larger for
AHA, intermediate for PPA, and low for BAA. It should be noted that this permeability is the result of all conformational opening processes, averaged in a large time scale, allowing the quencher to
diffuse into the protein. These data strongly support a correlation between stability and conformational flexibility.

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Fig. 3.
Stern-Volmer plots of
fluorescence quenching by acrylamide. Fluorescence quenching
values at 10 °C (upper panel) and at 37 °C
(middle panel) for AHA ( ), PPA ( ), and BAA ( ) are
shown. Lower panel, variation of fluorescence quenching
between 10 and 37 °C obtained by subtracting the regression lines of
Stern-Volmer plots at individual temperatures. The quenching constant
KSV values corresponding to the plot
slope (Equation 5) are 0.90, 0.68, and 0.32 mM 1·10 2 at 10 °C and 1.39, 1.02, and 0.56 mM 1·10 2 at
37 °C for AHA, PPA, and BAA, respectively.
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Activity-Stability Relationships--
It has been shown that the
high activity at low temperatures of cold-adapted enzymes
(corresponding to the low activation free energy
G* of
the reaction) mainly arises from low values of the activation enthalpy
that render enzymatic reactions less temperature-dependent
(42, 43). This is illustrated in Table III by the activation parameters of the
three investigated enzymes. It has been further proposed that this
property reflects the weak number of enthalpy-driven interactions that
have to be broken to reach the transition state, leading to a
heat-labile activity as these interactions also contribute to the
active site conformation (20). As a first experimental approach, the
thermodependence of the activity and the thermally induced unfolding
transition were recorded in the same conditions, as shown in Fig.
4, in the absence of added
Ca2+, which stabilizes the structure (12) but inhibits the
activity (44). In the case of the heat-stable PPA and BAA, the
temperature for maximal activity closely corresponds to the
fluorescence transition, showing that structural unfolding is a main
determinant for the loss of activity at high temperatures. In
contrast, the maximal activity of the psychrophilic enzyme is reached
at about 30 °C, below any significant conformational event (Fig. 4).
This shows that the active site of the psychrophilic enzyme (and the
catalytic intermediates) is even more heat-labile than its protein
structure. Such a result supports the concept of "localized
flexibility" (43), which assumes that the low stability of the active
site or of the structures bearing the active site is a main determinant of activity at low temperatures.

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Fig. 4.
Temperature dependence of activity
(upper panel) and of unfolding as recorded by
fluorescence emission (lower panel). Data for the
psychrophilic AHA ( ), the mesophilic PPA ( ), and the thermophilic
BAA ( ) are provided. Experiments were performed at similar protein
concentrations (5-40 µg/ml) in 100 mM Hepes, 50 mM NaCl, pH 7.2, in the absence of added Ca2+
(~1 µM residual Ca2+ as estimated by atomic
absorption). Note also the decrease in unfolding cooperativity as the
enzyme stability increases (lower panel).
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Table III also indicates that the magnitude of the entropic term of the
enzymatic reaction decreases as the enzyme stability increases. It has
been hypothesized that the flexible and mobile active site of
psychrophilic enzymes undergoes larger structural fluctuations between
the free and activated states than the more compact and rigid catalytic
center of stable enzymes (20, 43). If this is true, and on the basis of
the activation entropy (Table III), AHA trapped in the activated state
should display larger structural differences with the free enzyme, PPA
should display intermediate differences, and BAA should display minimal
structural differences. To check this hypothesis, the stability of the
three enzymes was recorded by DSC in the free state and in complex with acarbose, a large pseudosaccharide inhibitor acting as a transition state analog (16). Fig. 5 and Table IV
reveal that upon acarbose binding, the psychrophilic enzyme is indeed
strongly stabilized, as indicated by the large
increases of the melting point and of the
calorimetric enthalpy
Hcal. According to the
same criterion, PPA is less stabilized in the transition state, whereas
BAA is destabilized, as shown by the lower
Hcal value without melting point alteration.
It is worth mentioning that both the magnitude and sign of these
variations, as well as the macroscopic interpretation (assuming that a
stabilized transition state intermediate is more ordered), parallels
the structural fluctuations predicted by the activation entropy (Table
III). These data strongly suggest that the improved activity at low
temperatures of the psychrophilic enzyme is achieved by destabilizing
the active site domain to reduce the temperature dependence of the
activity (low
H*), which in turn implies large structural
motions upon substrate binding (high
S*). The low
activity of thermophilic enzymes at room temperature can be explained
in the same way. The catalytic domain designed to be active at high
temperature is stabilized by numerous interactions, resulting in a
higher temperature dependence (high
H*, and therefore, a
low activity as the temperature decreases), whereas the moderate conformational changes of the rigid active site result in weak activation entropy variations.

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Fig. 5.
DSC endotherms of
-amylases in the free state (thin
lines) and in complex with the transition state analog
acarbose (heavy lines).
Tmax corresponds to the top of the transition,
and Hcal corresponds to the surface below the
transition (Table IV). Base line subtracted data have been normalized
for protein concentration.
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Table IV
Microcalorimetric parameters of thermal unfolding for -amylases in
the free state and in complex with the pseudosaccharide inhibitor
acarbose
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Conclusion--
These results and available data on enzymes
adapted to extreme temperatures can be integrated in a model based on
the "new view" using the folding funnel to describe the
folding-unfolding reactions (45-48). Fig.
6 describes the energy landscape of
psychrophilic and thermophilic enzymes. The height of the funnel,
i.e. the free energy of folding, also corresponding to the
conformational stability, can be fixed in a 1/5 ratio according to the
stability curves (Fig. 1, Table I). The upper edge of the funnels is
occupied by the unfolded state in random coil conformations. It should be noted that psychrophilic enzymes tend to have a lower proline content than mesophilic and thermophilic enzymes, a lower number of
disulfide bonds, and a higher occurrence of glycine clusters (8-11).
Accordingly, the edge of the funnel for the psychrophilic protein is
slightly larger (broader distribution of the unfolded state) and is
located at a higher energy level. When the polypeptide is allowed to
fold, the free energy level decreases, as well as the conformational
ensemble. However, thermophilic proteins pass through intermediate
states corresponding to local minima of energy. These minima are
responsible for the ruggedness of the funnel slopes and for the reduced
cooperativity of the folding-unfolding reaction as demonstrated by
GdmCl and heat-induced unfolding. In contrast, the structural elements
of psychrophilic proteins generally unfold cooperatively without
intermediates, as a result of fewer stabilizing interactions and
stability domains (12, 13), and therefore, the funnel slopes are steep
and smooth. The bottom of the funnel, which depicts the stability of
the native state ensemble, also displays significant differences
between both extremozymes. The bottom for a very stable and rigid
thermophilic protein can be depicted as a single global minimum or as
having only a few minima with high energy barriers between them (49, 50), whereas the bottom for an unstable and flexible psychrophilic protein is rugged and depicts a large population of conformers with low
energy barriers to flip between them. Rigidity of the native state is
therefore a direct function of the energy barrier height (49, 50) and
is drawn here according to the results of fluorescence quenching. In
this context, the activity-stability relationships in these
extremozymes depend on the bottom properties. Indeed, it has been
argued that upon substrate binding to the association-competent
subpopulation, the equilibrium between all conformers is shifted toward
this subpopulation, leading to the active conformational ensemble
(49-51). In the case of the rugged bottom of psychrophilic enzymes,
this equilibrium shift only requires a modest free energy change (low
energy barriers), a low enthalpy change for interconversion of the
conformations, but is accompanied by a large entropy change for
fluctuations between the wide conformer ensemble. The converse picture
holds for thermophilic enzymes, in agreement with the activation
parameters (Table III) and with the proposed macroscopic
interpretation. Such energy landscapes integrate nearly all biochemical
and biophysical data from extremophilic enzymes, but they will
certainly be refined when other series of homologous proteins from
psychrophiles, mesophiles, and thermophiles will become available.

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Fig. 6.
Proposed model of folding funnels for
psychrophilic and thermophilic enzymes. In these schematic energy
landscapes, the free energy of folding or unfolding
(E) is represented as a function of the conformational
diversity, as detailed under "Conclusion."
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 |
ACKNOWLEDGEMENTS |
We thank N. Gérardin and R. Marchand
for skillful technical assistance and H. Bichoff (Bayer AG, Wuppertal,
Germany) for the kind gift of acarbose.
 |
FOOTNOTES |
*
This work was supported by the European Union (Grant
CT970131), the Région Wallonne (Grants Bioval 981/3860, Bioval
981/3848, Initiative 114705), the FNRS Belgium (Grant 2.4515.00), and
the Institut Polaire Français.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed. Tel.:
32-4-366-33-43; Fax: 32-4-366-33-64; E-mail: gfeller@ulg.ac.be.
Published, JBC Papers in Press, January 2, 2003, DOI 10.1074/jbc.M212508200
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ABBREVIATIONS |
The abbreviations used are:
AHA,
-amylase from P. haloplanktis;
PPA,
-amylase from pig
pancreas;
BAA,
-amylase from B. amyloliquefaciens;
GdmCl, guanidinium chloride;
DSC, differential scanning calorimetry;
MOPS, 3-(N-morpholino)propanesulfonic acid.
 |
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