The molecular basis of the effect of temperature on enzyme activity

Article abstract of DOI:10.1042/BJ20091254

Experimental data show that the effect of temperature on enzymes cannot be adequately explained in terms of a two-state model based on increases in activity and denaturation. The Equilibrium Model provides a quantitative explanation of enzyme thermal beha

Full text of DOI:10.1042/BJ20091254

Biochem. J. (2010) 425, 353–360 (Printed in Great Britain) doi:10.1042/BJ20091254
353
The molecular basis of the effect of temperature on enzyme activity
Roy M. DANIEL*, Michelle E. PETERSON*, Michael J. DANSON†, Nicholas C. PRICE‡, Sharon M. KELLY‡, Colin R. MONK*,
1
Cristina S. WEINBERG*, Matthew L. OUDSHOORN* and Charles K. LEE*
*
Department of Biological Sciences, University of Waikato, Private Bag 3105, Hamilton, New Zealand, †Centre for Extremophile Research, Department of Biology and Biochemistry,
University of Bath, Bath BA2 7AY, U.K., and ‡IBLS Division of Molecular and Cellular Biology, University of Glasgow, Glasgow G12 8QQ, U.K.
Experimental data show that the effect of temperature on enzymes
cannot be adequately explained in terms of a two-state model
based on increases in activity and denaturation. The Equilibrium
Model provides a quantitative explanation of enzyme thermal be-
haviour under reaction conditions by introducing an inactive (but
not denatured) intermediate in rapid equilibrium with the active
form. The temperature midpoint (Teq) of the rapid equilibration
between the two forms is related to the growth temperature of the
organism, and the enthalpy of the equilibrium (ꢀHeq) to its ability
to function over various temperature ranges. In the present study,
we show that the difference between the active and inactive forms
is at the enzyme active site. The results reveal an apparently uni-
versal mechanism, independent of enzyme reaction or structure,
based at or near the active site, by which enzymes lose activity
as temperature rises, as opposed to denaturation which is global.
Results show that activity losses below Teq may lead to significant
errors in the determination of ꢀG*cat made on the basis of the two-
state (‘Classical’) model, and the measured kcat will then not be a
true indication of an enzyme’s catalytic power. Overall, the results
provide a molecular rationale for observations that the active site
tends to be more flexible than the enzyme as a whole, and that
activity losses precede denaturation, and provide a general explan-
ation in molecular terms for the effect of temperature on enzyme
activity.
Key words: adaptation, enzyme, Equilibrium Model, evolution,
kinetics, temperature.
INTRODUCTION
of quaternary structure as well as the reaction concerned. The data
suggest that the Equilibrium Model is universally applicable to
all enzymes where Vmax data can be obtained.
Using the Equilibrium Model, the variation of enzyme activity
with temperature can be expressed by:
The way enzymes respond to temperature is fundamental to
many areas of biology. Until recently, the effect of temperature
on enzyme activity has been understood in terms of raised
temperature increasing activity and, at the same time, causing
activity to be lost by denaturation (e.g. [1–3]). However, it is now
clear that these two opposing effects are insufficient to explain
the effect of temperature, and that the effect of temperature on
enzymes over time cannot be predicted from the ꢀG*cat (activation
energy of the catalysed reaction) and ꢀG*inact (activation energy
of the thermal inactivation process) of the enzyme ([4], but see [5],
kinact Keqt
−
1
+Keq
k
cat
0
E e
V
max
=
1 + Keq
with
[
6], but see [7], [8]). The Equilibrium Model ([4], but see [5], [6],
ꢀ
Heq
1
1
(
− T
)
R
Teq
but see [7]) has provided a quantitative explanation of enzyme
thermal behaviour by introducing an intermediate inactive (but
not denatured) form that is in rapid equilibrium with the active
form.
Keq = e
,
∗
k
B
T
ꢀGcat
−
RT
k
cat
=
e
h
Keq
k
and
inact
E
act ꢀ Einact ꢀ X
∗
k
B
T
ꢀGinact
−
RT
k
inact
=
e
where Eact is the active form of the enzyme, which is in equilibrium
with the inactive form, Einact; Keq is the equilibrium constant
describing the ratio of Einact/Eact; kinact is the rate constant for the
h
where kcat is the enzyme catalytic rate constant, t is the assay
duration, E is the enzyme concentration, ꢀH is the change
in enthalpy associated with the E /E
E
inact to X reaction; and X is the irreversibly denatured form of the
enzyme.
Table 1 shows a variety of enzymes for which Equilibrium
0
eq
act
inact
equilibrium, Teq is
equilibrium, kB is
the temperature midpoint of the E /E
act
inact
Model parameters have been determined; all fitted the Model. The
enzymes cover most reaction classes and could all be measured
directly and continuously, ensuring rapid and accurate collection
of Vmax data. It is apparent from the range of structures, from
monomeric to hexameric, and including a citrate synthase where
the active site is at a subunit interface ([6], but see [7], [9–14]), that
conformity with the Equilibrium Model is apparently independent
Boltzmann’s constant, R is the gas constant, T is temperature,
and h is Planck’s constant.
The experimental data (Figure 1A) fit the Equilibrium Model
(Figure 1B), but not a simpler two-state model that only considers
ꢀG*cat and ꢀG*inact (the ‘Classical Model’, Figure 1C). The
new parameters associated with the model provide tools for
understanding and quantifying the temperature-dependence of
0
Abbreviations used: DTT, dithiothreitol; Eact, active enzyme; Einact, inactive enzyme; E , enzyme concentration; GDH, glutamate dehydrogenase; MDH,
malate dehydrogenase; Nle, norleucine; Teq, temperature at which the concentrations of Eact and Einact are equal.
1
To whom correspondence should be addressed (email cklee@waikato.ac.nz/charles.lee@gmail.com).
ꢀc The Authors Journal compilation ꢀc 2010 Biochemical Society

354
R.M. Daniel and others
Table 1 Thermodynamic parameters for 28 enzymes fitted to the Equilibrium Model
The parameters of all enzymes were derived by fitting assay data to the Equilibrium Model and thus relate to active enzymes in the presence of substrate and cofactor. The exceptions are the growth
temperature optima (Tgrowth) of the source organism, which are cited from various sources [9] and are included here only to give a broad approximation of the expected ‘working’ temperature of the
enzyme. AAA, aryl acylamidase; ACP, acid phosphatase; AKP, alkaline phosphatase; DHFR, dihydrofolate reductase; GCS, citrate (si)-synthase; GGT, γ -glutamyltransferase; IPMDH, isopropylmalate
dehydrogenase; PAL, phenylalanine ammonia lyase.
◦
ΔG*cat (kJ · mol−1
ΔG*inact (kJ · mol−1
ΔHeq (kJ · mol−1
◦
Organism
Enzyme
T
growth ( C)
)
)
)
T
eq ( C)
Number of subunits
Thermus sp. RT41a
Thermus aquaticus
Bacillus caldovelox
Caldocellulosiruptor sacchrolyticus
Bacillus sp. strain Ak1
Thermoplasma acidophilum
Geobacillus stearothermophilus
Geobacillus stearothermophilus
Bacillus licheniformis
Escherichia coli
Escherichia coli
Bos taurus
Bos taurus
Bos taurus
Bos taurus
Sus scrofa
Candida utilus
Bacillus subtilis
Bacillus cereus
Bacillus cereus
Saccharomyces cerevisiae
Pseudomonas fluorescens
Rhodotorula glutinis
Wheat germ
AKP
α-Glucosidase
IPMDH
β-Glucosidase
Protease
GCS
α-Glucosidase
DHFR
Subtilisin A
AKP
MDH
MDH
AKP
GDH (EC 1.4.1.3)
GGT
Fumarase
GDH (EC 1.4.1.4)
IPMDH
DHFR
MDH
α-Glucosidase
AAA
PAL
ACP
β-Glucosidase
IPMDH
75
70
70
70
65
60
55
55
50
40
40
39
39
39
39
39
31
30
30
30
28
25
24
20
20
20
15
2
72
88
76
78
72
77
64
67
61
68
55
53
57
63
63
60
57
73
66
51
71
74
80
79
63
72
55
67
99
103
101
105
102
103
96
97
98
99
96
85
97
93
98
92
94
94
90
86
90
92
97
95
95
100
79
93
305
149
573
119
134
164
225
96
106
121
619
826
86
264
109
378
409
255
261
243
272
115
181
142
100
123
139
104
90
2
1
2
1
1
2
1
1
1
2
4
2
2
6
2
4
6
2
1
4
1
1
4
1
1
2
1
1
74.4
63.9
72
75
87.8
67.8
53.9
62.6
70
70.7
67.4
59.6
53
52
59.1
59.5
52.8
58.5
40
39.4
35.8
56.5
63.9
55.9
56.7
29.3
54.6
Prunus dulcis
Bacillus psychrophilus
Antarctic bacterium HK47
Moritella profunda
AKP
DHFR
enzyme activity and the adaptation of enzymes and organisms to
temperature, and to ranges of temperature. Teq, the temperature of
the midpoint of the equilibrium between the active and reversibly
inactive forms of the enzyme, is an evolved property of enzymes
related to the organism’s growth temperature, being more closely
correlated with the environmental temperature of the enzyme
than is its stability [9]. ꢀHeq, the enthalpic change associated
with the equilibrium, governs the temperature range over which
the equilibrium occurs and thus the ability of the enzyme
to function at different temperatures and temperature ranges
EXPERIMENTAL
Continuous enzyme assays
Continuous spectrophotometric enzyme assays were carried
out as described previously [9,14,16] using a Thermo Spec-
tronic Helios γ -spectrophotometer equipped with a Thermo
Spectronic single-cell Peltier-effect cuvette holder. Assays were
performed across a suitable range of temperatures for each
enzyme, and spectrophotometric data were collected at 0.5
or 1 s intervals for 3–5 min using Vision32 (version 1.25;
Thermo Spectronic) on a Windows XP PC connected to the
spectrophotometer.
[
9].
The Equilibrium Model quantitatively explains the effect of
temperature on all enzymes for which Vmax can be measured over
a range of temperatures ([4], but see [5], [6], but see [7], [9,11–
All reactions were started by adding rapidly no more than 10 μl
◦
of enzyme solution (kept at 0 C) to ꢁ1000 μl of temperature-
1
3,15,16]) and has predicted and explained the counterintuitive
equilibrated reaction mixture (containing substrate/cofactor and
reaction buffer) in a quartz cuvette. Data collection started within
behaviour of enzyme reactors at some temperatures ([15], and
M.L. Oudshoorn and R.M. Daniel, unpublished work). We
cannot exclude the possibility that more complex models for the
dependence of enzyme activity will fit experimental data, but none
has been demonstrated.
3 s of enzyme addition, and the temperature of the reaction
◦
remained within + 1 C of the desired temperature. Where
−
possible, substrate concentrations were set to at least ten times
K
m
to approach Vmax; where it was not possible to do so (e.g.
The Equilibrium Model in itself does not offer an explanation
of the molecular basis of these effects, or the physical nature of
high K or poor substrate solubility), K values were determined
m
m
and used to compensate for the deviation from Vmax. Blank rates
were measured and used to correct reaction rates if necessary.
All criteria required for accurate and valid determination of
Equilibrium Model parameters [14] were met.
All enzymes shown in Table 1 have been assayed directly
and continuously to allow rapid and accurate data collection and
temperature control.
E
inact. In the present study, we provide evidence that the difference
between the active and inactive forms is at the enzyme active
site, and confirm that Keq and its components Teq and ꢀHeq are
independent of global stability. This enables a general explanation
in molecular terms for the effect of temperature on enzyme activity
and reveals a new structurally localized and apparently universal
mechanism for enzyme activity loss with increasing temperature,
additional to denaturation.
Bacillus subtilis subtilisin A (catalogue number P5380; Sigma–
Aldrich) was assayed in 50 mM phosphate buffer (pH 7.3) using
ꢀc The Authors Journal compilation ꢀc 2010 Biochemical Society

Molecular basis of temperature effects on enzyme activity
355
Figure 1 Comparison of experimental data with the predictions of the Equilibrium and Classical Models: the effect of temperature on β-glucosidase from
C. saccharolyticus with 30 mM p-nitrophenyl β-D-glucopyranoside as substrate
(
A) Experimental data. (B) Simulation of the effect of temperature using parameters derived from fitting the experimental data to the Equilibrium Model (ꢀG*cat = 78 kJ · mol−1; ꢀG*inact = 105
−
1
−1
◦
kJ · mol ; ΔHeq = 119 kJ · mol ; Teq = 72 C). (C) Simulation of the effect of temperature using only the values of ꢀG*cat and ꢀG*inact from fitting the experimental data to the Classical
Model.
the following substrates (purchased from Bachem AG) (where
is enzyme concentration): 10 mM succinyl-Ala-Ala-Pro-
Ala-p-nitroanilide (E
Bacillus sp. strain AK1 protease was cloned and expressed in
E. coli; cells were harvested and lysed by sonication, and the
enzyme was purified using phenyl-Sepharose [18]. The enzyme
E
0
0
= 118 nM); succinyl-Ala-Ala-Pro-Leu-p-
◦ ◦
nitroanilide (7 mM at 60–65 C, 10 mM at 70–75 C, 15 mM
was assayed in 100 mM Hepes/NaOH with 5 mM CaCl (pH 7.5),
2
◦
at 80–85 C; E
0
= 4.53 nM); 10 mM succinyl-Ala-Ala-Pro-Phe-
using 22.5 mM succinyl-Ala-Ala-Ala-p-nitroanilide and 50 mM
succinyl-Ala-Ala-Pro-Phe-p-nitroanilide. The assay volume was
400 μl in a quartz cuvette of 2 mm pathlength. The final enzyme
concentration was 30.4 nM. Enzyme activity was measured by
monitoring the production of p-nitroaniline at 410 nm (ε=7930
p-nitroanilide (E
p-nitroanilide (E
Ala-Pro-Val-p-nitroanilide (25 mM at 30–70 C, 50 mM at 75–
0
= 2.28 nM); 10 mM succinyl-Ala-Ala-Pro-Nle-
0
= 3.37 nM; Nle is norleucine); succinyl-Ala-
◦
◦
8
5 C; E
0
= 337 nM). The assay volume was 400 μl in a quartz
−
1
−1
cuvette of 2 mm pathlength. Enzyme activity was measured by
M
· cm ). The enzyme was pre-treated by incubation in buffer
monitoring the production of p-nitroaniline at 400 nm (ε=9290
in the absence and presence of 10 mM DTT (dithiothreitol) for
60 min at room temperature (20 C), then diluted 1:1 before adding
−
1
−1
◦
M
· cm ).
Caldicellulosiruptor saccharolyticus β-glucosidase, expressed
to the reaction mixture.
recombinantly in Escherichia coli, was purified using
chromatography following heat treatment [17]. It was assayed
as described previously [9] using various substrates (purchased
from Sigma–Aldrich), including 30 mM p-nitrophenyl β-D-
glucopyranoside, 20 mM p-nitrophenyl β-D-galactopyranoside,
Data processing
The processing of enzyme assay data was performed as described
in detail in [14] using the MATLAB-based Equilibrium Model
data-processing application on a CD-ROM (available on request
from R.M.D.). In short, absorbance data from Vision32 were
first converted into progress curves of product concentration
(M) against time (s) in Excel, which were then loaded
into the Equilibrium Model application, which performs the
facile derivation of Equilibrium Model parameters from the
experimental data. The application employs a fitting algorithm
based on a least squares approximation run over multiple iterations
to identify a set of thermodynamic parameters that best describe
the experimental data within the confines of the Equilibrium
Model. The resulting parameters were used to generate a
simulated three-dimensional plot of enzyme activity profile
(activity against temperature against time) that is then compared
with the corresponding three-dimensional plot generated from
smoothed raw assay data to ensure the validity of the final
2
0 mM p-nitrophenyl β-D-fucopyranoside and 52 mM p-
nitrophenyl β-D-xylopyranoside. For reaction buffer, 50 mM
sodium phosphate buffer (pH 6.3) was used, and the concentration
of enzyme in the assay was 0.586 μM for all substrates except
for p-nitrophenyl β-D-xylopyranoside, which was 2.93 μM. The
assay volume was 400 μl in a quartz cuvette of 2 mm pathlength.
Enzyme activity was measured by monitoring the production of
−
1
−1
p-nitrophenol at 410 nm (ε=7930 M · cm ).
Bos taurus GDH (glutamate dehydrogenase) (L-glutamic
dehydrogenase from bovine liver; catalogue number G7882;
Sigma–Aldrich) was assayed in 90 mM phosphate buffer (pH 7.3),
with 0.05 mM EDTA, 160 mM ammonium acetate, 10 mM
2
-oxoglutarate and 0.5 mM NADPH or NADH. The assay
volume was 1 ml in a 10 mm pathlength quartz cuvette; enzyme
activity was measured by monitoring absorbance at 340 nm (ε=
−
1
−1
6
220 M · cm ).
ꢀc The Authors Journal compilation ꢀc 2010 Biochemical Society

356
R.M. Daniel and others
Table 2 Effect of substrate on the Equilibrium Model parameters of three enzymes
Enzymes were assayed using different substrates and the Equilibrium Model parameters derived as outlined in Experimental. Data are presented to three significant figures. Values in parentheses are
the S.D. of the fit of the data to the Model.
Enzyme
Substrate
ꢀG*cat (kJ · mol−1)
ꢀG*inact (kJ · mol−1
)
ΔHeq (kJ · mol−1
)
Teq (K)
Subtilisin (Bacillus subtilis)
Succinyl-Ala-Ala-Pro-Leu-p-nitroanilide
Succinyl-Ala-Ala-Pro-Nle-p-nitroanilide
Succinyl-Ala-Ala-Pro-Phe-p-nitroanilide
Succinyl-Ala-Ala-Pro-Ala-p-nitroanilide
Succinyl-Ala-Ala-Pro-Val-p-nitroanilide
p-Nitrophenyl β-D-fucopyranoside
p-Nitrophenyl β-D-galactopyranoside
p-Nitrophenyl β-D-glucopyranoside
p-Nitrophenyl β-D-xylopyranoside
NADH
61.4 (0.1)
61.5 (0.1)
68.1 (0.1)
71.0 (0.1)
76.6 (0.1)
77.0 (0.1)
77.0 (0.1)
78.0 (0.1)
84.0 (0.1)
62.6 (0.1)
62.6 (0.1)
98.4 (0.1)
98.7 (0.1)
96.8 (0.3)
99.0 (0.6)
96.8 (0.1)
101 (1)
103 (1)
105 (1)
105 (1)
92.5 (0.1)
91.6 (0.1)
106 (2)
78.6 (0.6)
142 (3)
107 (3)
110 (1)
141 (2)
123 (2)
119 (1)
112 (1)
264 (2)
144 (2)
336 (1)
323 (1)
333 (1)
338 (1)
332 (1)
349 (1)
345 (1)
345 (1)
338 (1)
326 (1)
319 (1)
β-Glucosidase (Caldicellulosiruptor saccharolyticus)
GDH (Bos taurus)
NADPH
Equilibrium Model parameters. The values in parentheses in the
Tables are the S.D. of the fit of the data to the model [14].
RESULTS
act/Einact transition timescale
E
The timescale for the Eact/Einact equilibration is clearly rapid, since
all the variation of activity at zero time {e.g. Figure 1A; ([4],
but see [5], [6], but see [7])} occurs as a result of changes in
the Eact/Einact equilibrium, and is thus attained over timescales
shorter than the mixing process, probably <2 s. The implication
is that for every enzyme tested (Table 1) the Eact/Einact equilibration
timescale is of this order or shorter, whereas most proteins
take very much longer than this to unfold [19,20]. This is
particularly the case given the relatively low temperatures at
which some Eact/Einact equilibrations take place. The results shown
in Table 1 demonstrate that many enzymes are only partially
active at physiological temperatures, depending on the value of
Circular dichroism
Saccharomyces cerevisiae α-glucosidase (E-SUCR; Megazyme
International) was chosen for its prominent CD responses in
the near-UV spectrum, which in turn are caused by the distinct
characteristics of tryptophan residues, especially those near its
active site (results not shown); however, it should be noted that the
CD spectrum cannot be considered a quantitative measure of the
overall conformation of the enzyme. CD time-course observations
were performed at 292 nm, the wavelength at which the CD
signal is maximal. Time-course experiments and CD spectral
scans performed in the presence of substrate (i.e. under assay
conditions) were carried out using 0.5 mg · ml S. cerevisiae
α-glucosidase in the presence of 300 mM maltose (SigmaUltra,
catalogue number M9171; Sigma–Aldrich). A high concentration
of maltose was used to avoid significant substrate depletion
during measurement and thus to mirror the conditions used for
the collection of Equilibrium Model data. Neither maltose nor
its product glucose absorb significantly in the near UV range,
regardless of temperature (results not shown). The buffer used was
ꢀHeq. For enzymes with low ꢀHeq values, the Eact into Einact
conversion takes place over a broad temperature range, beginning
−
1
at relatively low temperatures. For example, 10% of the Prunus
−
1
dulcis β-glucosidase enzyme (ꢀHeq = 100 kJ · mol ) is already
◦
in the Einact form at 36 C. On the other hand, for E. coli MDH
−
1
(
malate dehydrogenase) (ꢀHeq = 619 kJ · mol ), 10% Einact is
◦
not reached until 67 C (see Figure 6).
2
0 mM sodium phosphate buffer (pH 7.5), and the total volume
Magnitude of ꢀHeq
for each reaction was 2 ml. All CD measurements were made
using a quartz cuvette of 10 mm pathlength. For CD spectral data,
approx. 60 s passed between enzyme addition and the completion
of the CD spectral scan. For CD time-course experiments, data
recording started immediately (i.e. within 6 s) after enzyme was
added to a pre-temperature-equilibrated reaction mixture.
S. cerevisiae α-glucosidase (4.84 nM) was assayed discontinu-
ously using 300 mM maltose as substrate in 18 mM phosphate
buffer (pH 7.0). Aliquots (100 μl) of the reaction were transferred
The enthalpic change associated with the Eact/Einact equilibrium,
ꢀH
eq, is much smaller than that associated with denaturation
(
ꢀHunfold) per amino acid residue. For example, the ꢀHeq for
◦
−1
subtilisin at 60 C (i.e. at Teq) is approx. 110 kJ · mol (Table 2),
whereas the ꢀHunfold at this temperature has been determined to be
−
1
approx. 1350 kJ · mol [21]. For ten single-subunit enzymes with
◦
T
eq values between 53 and 75 C, the average ꢀHeq is 0.47 + 0.40
−
−
1
−1
−1
−1
kJ · mol · residue (range 0.08–1.51 kJ · mol · residue ).
Similar values apply to multi-subunit enzymes. An estimate
for the average ꢀHunfold in this temperature range is 3.0–4.3
at desired intervals to screw-capped tubes containing 100 μl of
◦
5
0 mM phosphate buffer (pH 7.0) at 100 C. The tubes were
−
1
−1
◦
kJ · mol · residue [22,23]. Although this is, at best, an
capped and placed in a water bath at 100 C for 7 min to terminate
the α-glucosidase reaction, and then transferred to an ice-water
bath until samples for the full time course had been collected
approximation, it confirms that the expected ꢀHunfold for these
enzymes is about one order of magnitude greater than their ꢀHeq
.
(
8.5 min at 30 s intervals). To each 200 μl of stopped reaction
mixture, 3 units of glucose dehydrogenase (catalogue number
G5059; Sigma–Aldrich) and β-NADP to a final concentration
of 4.5 mM were added and incubated at 35 C for 35 min. The
+
Structural studies
◦
We have used CD to probe the extent and timing of the
structural changes associated with the Eact/Einact equilibrium and
with denaturation in the α-glucosidase from S. cerevisiae under
concentrations of NADPH were calculated from absorbance data
at 340 nm (5 mm pathlength cuvette).
ꢀc The Authors Journal compilation ꢀc 2010 Biochemical Society

Molecular basis of temperature effects on enzyme activity
357
Figure 2 CD spectra of S. cerevisiae α-glucosidase
◦
S. cerevisiae α-glucosidase was scanned at 15 C in the presence of 300 mM maltose
◦
(
continuous line) and, after a 10-min incubation at 43 C, in the absence of substrate (broken
line).
assay conditions. Near-UV CD spectral features specific to the
◦
native (non-denatured) enzyme were observed at 15 C in the
presence of 300 mM maltose (Figure 2, continuous line), whereas
◦
at 43 C in the absence of substrate (i.e. non-Equilibrium Model
Figure 4 An experimental time–temperature–activity plot of the S.
condition), where the enzyme is denatured, no distinct features
can be seen (Figure 2, broken line). Similar observations were
made from CD time-course experiments monitoring CD signals
cerevisiae α-glucosidase
ꢀ
G*cat = 63.5 kJ · mol−1; ꢀG*inact = 96.9 kJ · mol−1
;
ꢀHeq = 215 kJ · mol−1
;
T
eq = 316.5K.
◦
at 292 nm (Figure 3). At 15 C in the presence of substrate, the
initial CD signal of the enzyme at 292 nm is very similar to
the value seen in Figure 2 (Figure 3, dashed line) and changes
only slightly over the duration of the experiment (25 min); this is
expected since under such conditions the enzyme exists mostly
in its active form ([Eact]>99.95%) and undergoes very limited
over the course of the experiment is comparable with the rate of
enzyme denaturation in the assay (see Figure 4).
CD time-course experiments suggest that the changes in tertiary
structure occur in at least two stages, over different timescales. The
first stage (the Eact/Einact transition) can be seen in the difference
◦
denaturation. In contrast, at 43 C in the absence of substrate, the
◦
between the initial CD signals at 15 and 43 C in the presence
CD signal at 292 nm decreases quickly (Figure 3, dotted line) and
irreversibly (results not shown), reflecting both the absence of
any stabilizing effect of substrate, and the buffering effect of the
of substrate (Figure 3, dashed and continuous lines respectively),
◦
which corresponds to a shift from ∼100% Eact (15 C) to approx.
◦
5
0% Eact and 50% Einact (43 C); this shift is rapid since the time
E
act/Einact equilibrium against denaturation ([4], but see [5], [13]).
course was started at near zero time, i.e. within 6 s of addition
of the enzyme to substrates and buffer. The second and time-
dependent change is thermal denaturation, which occurs at a rate
comparable with that of the conversion from Einact into X (Figure 3,
continuous and dashed-dotted lines, and Figure 4, changes along
The initial CD signal under this condition is also considerably
◦
lower than that at 43 C in the presence of substrate (Figure 3,
continuous line), where approx. 50% of the enzyme exists as
E
inact and where the initial CD signal is itself significantly reduced
◦
compared with that at 15 C (Figure 3, dashed line). Furthermore,
the decrease in CD signal at 43 C in the presence of substrate
◦
the rate/time axes at 43 C) and is lower than the rate of decrease
◦
in the absence of substrate (Figure 3, dotted line). Furthermore,
◦
one enzyme/substrate mixture was incubated at 43 C for an
additional 30 min after the time-course experiment and monitored
again for another 25 min (Figure 3, dashed-dotted line), after
which its CD signal is essentially identical with that of the fully
denatured enzyme (Figure 3, dotted line). It is likely that the
enzyme denatured fully during the extended incubation and exists
solely as the denatured form.
Substrate effects
T
eq and ꢀHeq are substrate-specific, i.e. differentenzyme/substrate
combinations have their own characteristic values of Teq and ꢀHeq
Table 2). These are often markedly different from one another,
(
with some relationship between the size of the differences in Teq
and ꢀHeq and the extent of the structural differences.
In the case of subtilisin, the effect is evident at the S1 site,
since the only difference in the substrates is at the P1 position.
The change from leucine to norleucine at the P1 position of the
substrate has relatively little effect on ΔG*cat or ΔG*inact, but
Figure 3 Time-dependent changes in CD signals at 292 nm of S. cerevisiae
α-glucosidase
◦
S. cerevisiae α-glucosidase CD time-course experiments were performed at 15 C in the
◦
presence of 300 mM maltose (dashed line), at 43 C in the presence of 300 mM maltose
continuous line), at 43 C in the absence of substrate (dotted line), and after a 30-min
◦
(
◦
◦
marked changes can be seen for Teq and ꢀH ; the change from
incubation at 43 C following the time-course experiment at 43 C in the presence of 300 mM
maltose (dashed-dotted line).
eq
norleucine to alanine does not significantly affect stability, but
ꢀc The Authors Journal compilation ꢀc 2010 Biochemical Society

358
R.M. Daniel and others
Table 3 Effect of disulfide bond cleavage at the active site on the Equilibrium Model parameters of AK1 protease with two different peptide substrates
Data are presented to three significant figures. Values in parentheses are the S.D. of the fit of the data to the Model.
Substrate
ꢀG*cat (kJ · mol−1)
ꢀG*inact (kJ · mol−1
)
ΔHeq (kJ · mol−1
)
Teq (K)
Succinyl-Ala-Ala-Ala-p-nitroanilide
76.0 (0.1)
76.2 (0.2)
71.5 (0.1)
71.5 (0.1)
105 (1)
106 (1)
102 (1)
100 (1)
94.0 (0.6)
115 (2)
134 (2)
123 (2)
337 (1)
342 (1)
348 (1)
342 (1)
Succinyl-Ala-Ala-Ala-p-nitroanilide+DTT
Succinyl-Ala-Ala-Pro-Phe-p-nitroanilide
Succinyl-Ala-Ala-Pro-Phe-p-nitroanilide+DTT
Figure 5 Effects of temperature on the Eact/Einact equilibrium and K
m
The continuous lines represent the initial (zero time) activity of the enzyme at various temperatures based on its Equilibrium Model parameters derived from experimental data. The percentages refer
to the proportion of Einact relative to the total enzyme population at temperatures indicated by arrows. The broken lines represent the effect of temperature on the K
A) E. coli MDH; K values are for oxaloacetate. (B) Alvinella pompejana epibionts GDH; K values are for NADPH.
m
of the enzyme (means + S.E.M.).
−
(
m
m
there are major differences in Teq and ꢀHeq as well as in ꢀG*cat
.
change at a well-defined position in the active site of an enzyme
has an effect on the Equilibrium Model parameters describing the
For phenylalanine, the stability and the Teq are the same as for
valine, but the ꢀHeq is higher than for any of the other substrates.
For β-glucosidase, the effect of the 6-deoxyhexose fucose is
to lower stability and raise Teq and ꢀHeq compared with the
aldohexose substrates galactose and glucose. These two have
the same Teq and similar stabilities. The pentose substrate xylose
substantially lowers Teq, while maintaining high stability.
E
act/Einact equilibrium.
Effect of ꢀHeq on the determination of ꢀG*cat and kcat
In addition to the known effect of ꢀHeq on the temperature range
over which enzymes operate [9], Figure 6 shows that, for enzymes
with a low ꢀHeq, such as β-glucosidase, a significant proportion of
the enzyme can be in the inactive form at temperatures well below
In the case of GDH, cofactors affect Teq and ꢀHeq in the same
way as substrates, with substantial differences in the absence of
any significant effect on ΔG*cat or ΔG*inact
changes often coincide with the Eact/Einact transition. There
have been a number of general observations that enzyme K
.
T
eq. This leads to a relatively straighter shape of the ascending limb
K
m
of the temperature–activity curve for the P. dulcis β-glucosidase,
because the effect of Teq is imposed upon that of ꢀG*cat. In this
case, attempts to determine ꢀG*cat from a simple plot of the
increase in activity with temperature for this enzyme will be open
to significant errors, as will measurements of kcat if made over the
affected temperature range.
m
values often increase with temperature [24]. We find that these
increases are relatively common and often coincide with the shift
from Eact to Einact. For example, Figure 5 shows the sharp increase
in the K
m
of MDH for oxaloacetate coincident with a sudden
of
increase in the proportion of Einact, and a slower increase in K
m
GDH for NADPH coincident with an increase in Einact spread over
a larger temperature range.
DISCUSSION
A variety of evidence from the present study indicates that
the Eact/Einact transition involves relatively little unfolding,
and that Einact is not significantly denatured, i.e. the results show
that the Einact form of an enzyme is relatively similar to the
An active-site change affects Teq
The AK1 protease is unusual in that it has a disulfide bond
in the active site, between Cys and Cys . Cleavage of this
bond has a significant impact on the active site, since residues
137
139
Eact form, rather than to the denatured/unfolded form, or to a
molten globule form. The speed of the Eact/Einact equilibration
is much greater than that of denaturation and consistent
with the conformational switching time between the sub-states
of the myoglobin-binding site [26]. The ꢀHeq values are much
lower than that expected for denaturation and are consistent with
conformational changes, very limited unfolding and/or changes
1
33–136 form a promontory that projects between the S1 and S4
sites, and the chain displacement arising from cleavage affects the
and kcat values of the enzyme, depending upon the temperature
K
m
and substrate [18,25]. As shown in Table 3, reductive cleavage of
this disulfide bond using DTT results in significant changes in
T
eq and ꢀHeq, without significant changes in ΔG*cat or ΔG*inact
.
in solvent interactions. However, the range of values for ꢀHeq,
from less than 90 kJ · mol to more than 800 kJ · mol , and
−
1
−1
This is found with both substrates used, showing that a point
ꢀc The Authors Journal compilation ꢀc 2010 Biochemical Society

Molecular basis of temperature effects on enzyme activity
359
are often associated with shifts in the Eact/Einact equilibrium,
are both consistent with changes at the active site from an
optimum configuration for substrate binding to a less optimum
one, coincident with a shift in the Eact/Einact equilibrium towards
the Einact form. We would not expect this to be invariably the case,
since, in some enzymes, shifts in the Eact/Einact equilibrium might
depend on active-site residues that do not affect K . Overall, the
m
results show that the active sites of enzymes dictate the effect of
temperature on enzyme activity. This is entirely consistent with,
and may provide part of the rationale for, observations that the
active site tends to be more flexible than the enzyme as a whole
[
20,27–34]. The exact nature of the physical changes involved
are not clear and, given the range of reactions and structures
covered by the Equilibrium Model, seem likely to be different in
different enzymes, but it would be very surprising if some type of
conformational change was not involved.
Figure 6 Effect of ꢀHeq on the temperature-sensitivity of enzymes via the
E
act/Einact equilibrium
The continuous line shows the zero time activity of E. coli MDH (ꢀHeq = 619 kJ · mol−1), and
The inference from Figure 6 is that, for enzymes with a low
−
1
the broken line shows that of P. dulcis β-glucosidase (ꢀHeq = 100 kJ · mol ). The vertical
ꢀH , such as the β-glucosidase shown, attempts to determine
eq
arrows show the percentage of the enzyme existing as the inactive form.
ꢀ
G*cat graphically, without taking account of Teq, are likely to
have significant errors. Any determination of kcat will only be
a true measure of the enzyme’s catalytic power if it is made
at temperatures where none of the enzyme is in the inactive
form. Since the average ꢀHeq of the enzymes for which it has
been determined is 226 kJ · mol , and 150 kJ · mol for single-
subunit enzymes (Table 1), this may often be at surprisingly low
temperatures.
−
1
−1
less than 0.1 kJ · mol · residue (e.g., B. taurus alkaline
phosphatase and P. dulcis β-glucosidase) to more than 1.2
−
1
−1
kJ · mol · residue (B. cereus dihydrofolate reductase and
B. taurus MDH) is large, and it seems likely that different events
are involved in different enzymes. Earlier work has shown that a
low concentration of a denaturing agent or stabilizing agent can
affect temperature stability without affecting Teq and ꢀHeq, also
indicating that these parameters are independent of enzyme global
stability [15]. A statistical analysis of a number of enzymes [9] has
already shown a stronger correlation of the growth temperature
of the source organism with Teq than with ꢀG*inact (stability);
the correlation of ꢀG*inact with Teq is weaker and predictable
given that both correlate with growth temperature. The CD
data support the notion that the Eact/Einact transition occurs very
rapidly compared with denaturation, is temperature-dependent
and is physically distinct from the slower denaturation process.
Overall, these findings provide an explanation for the results of
Tsou [20,27] showing that thermally induced loss of enzyme
activity occurs at lower temperatures and much more rapidly than
denaturation [20,27–29]. It may be that the change from Eact to
−
1
−1
As discussed elsewhere [15], the Equilibrium Model has
significant applications for the effects of temperature on enzyme
evolution and adaptation [9,15,16]. The several-fold lower initial
rate that is observed for the Equilibrium Model compared with
the Classical Model (Figure 1) is a direct consequence of the
values assigned to the thermodynamic parameters in the former,
particularly the value of Teq being lower than the temperature
bringing about significant irreversible thermal inactivation. The
initial rate observed thus increases as the value of Teq is increased.
This may help to explain why thermophilic enzymes only achieve
catalytic rates equivalent to those found with mesophilic enzymes,
despite the higher temperature of assay. That is, a thermophilic
enzyme may have a high global stability that results in low rates
of thermal inactivation, but the active-site structure may not be
optimized for thermal stability but for effective catalysis, and thus
of necessity may comprise a more flexible portion of the protein.
As the temperature of the enzyme is raised, the flexible active-site
region may be deformed and Eact thus converted into Einact with a
concomitant reduction in catalytic activity. However, as the major
portion of the protein molecule is optimized for stability and can
act as a stable scaffold, the temperature-induced conformation
change at the active site can be prevented from leading to total
unfolding and the conversion of Eact into Einact is consequently
reversible at potentially destabilizing temperatures. However, this
effect does lead to lower catalytic rates and a lower Teq than
would be expected from the optimum growth temperature of the
organism.
E
inact is the first, but very limited, step in a pathway leading to
complete unfolding, but this is not a necessary consequence of
these results.
Other evidence indicates that changes at the active site are
responsible for the Eact/Einact equilibration. The parameters
defining the transition, Teq and ꢀHeq, are substrate-specific,
with similar substrates often giving similar parameters for
a given enzyme, and structurally different substrates giving
rise to significant differences in the parameters, often without
significant changes in ꢀG*cat (succinyl-Ala-Ala-Pro-Leu-
p-nitroanilide/succinyl-Ala-Ala-Pro-Nle-p-nitroanilide; p-nitro-
phenyl β-D-fucopyranoside/p-nitrophenyl β-D-galactopyrano-
side; NADH/NADPH) or ꢀG*inact (succinyl-Ala-Ala-Pro-Leu-p-
nitroanilide/succinyl-Ala-Ala-Pro-Nle-p-nitroanilide; succinyl-
Ala-Ala-Pro-Nle-p-nitroanilide/succinyl-Ala-Ala-Pro-Ala-p-nit-
The results imply that evolution of the enzyme active site
is likely to be constrained by its temperature-dependence.
Manipulation of stability by mutation, whether naturally or
by directed mutagenesis, may not allow activity at higher
temperatures unless Teq is also raised; since the basis of Eact/Einact
roanilide;
succinyl-Ala-Ala-Pro-Phe-p-ni-troanilide/succinyl-
Ala-Ala-Pro-Val-p-nitroanilide; p-nitrophenyl β-D-glucopyrano-
side/p-nitrophenyl β-D-xylopyranoside) (Table 2).
A point change at the active site, in this case cleavage of a
disulfide bond that produces insignificant changes in the global
structure [25], but that gives rise to a structural change at the
active site, can also produce changes in the parameters Teq and
equilibration is at the active site, this may lead to changes in K
and/or kcat. This may explain the difficulty of engineering enzymes
to operate at higher temperatures.
Of particular interest is the discovery of a localized and
apparently universal mechanism by which enzymes lose activity
as temperature rises, as opposed to denaturation which is global
[35]. The Einact state of the Equilibrium Model is clearly different
m
ꢀH
eq, providing direct evidence for involvement of the active site
in Eact/Einact equilibration (Table 3). The general observation that
tends to rise with temperature [24], and that these changes
K
m
ꢀc The Authors Journal compilation ꢀc 2010 Biochemical Society

360
R.M. Daniel and others
from a fully unfolded or molten globule state and is thus
distinguishable from the Lumry–Eyring model [36] and other
models [15]. The molecular basis of any specific Eact/Einact
equilibration seems likely to be as diverse as the enzymes
themselves. The precise details of the local changes occurring in
any specific enzyme as the equilibrium shifts from Eact to Einact have
yet to be determined. This determination may not be simple since
the structural difference between the two forms is evidently small,
and the temperature required to yield a dominating proportion of
12 Daniel, R. M., Danson, M. J., Eisenthal, R., Lee, C. K. and Peterson, M. E. (2008) The
effect of temperature on enzyme activity: new insights and their implications.
Extremophiles 12, 51–59
1
3
Daniel, R. M., Danson, M. J., Hough, D. W., Lee, C. K., Peterson, M. E. and Cowan, D. A.
2008) Enzyme stability and activity at high temperatures. In Protein Adaptation in
Extremophiles (Siddiqui, K. S. and Thomas, T., eds), Nova Publishers, New York
(
14 Peterson, M. E., Daniel, R. M., Danson, M. J. and Eisenthal, R. (2007) The dependence of
enzyme activity on temperature: determination and validation of parameters. Biochem. J.
4
02, 331–337
15
16
17
18
Eisenthal, R., Peterson, M. E., Daniel, R. M. and Danson, M. J. (2006) The thermal
behaviour of enzyme activity: implications for biotechnology. Trends Biotechnol. 24,
E
inact will cause rapid denaturation of most enzymes.
The applicability across such a wide range of enzyme reactions
2
89–292
Lee, C. K., Cary, S. C., Murray, A. E. and Daniel, R. M. (2008) Enzymic approach to
eurythermalism of Alvinella pompejana and its episymbionts. Appl. Environ. Microbiol.
74, 774–782
Plant, A. R., Oliver, J. E., Patchett, M. L., Daniel, R. M. and Morgan, H. W. (1988) Stability
and substrate specificity of a β-glucosidase from the thermophilic bacterium Tp8 cloned
into Escherichia coli. Arch. Biochem. Biophys. 262, 181–188
Toogood, H. S., Smith, C. A., Baker, E. N. and Daniel, R. M. (2000) Purification and
characterization of Ak.1 protease, a thermostable subtilisin with a disulphide bond in the
substrate-binding cleft. Biochem. J. 350, 321–328
and structures, the active-site location, and the association with
growth temperature [9] all strongly indicate that the Equilibrium
Model describes an important natural phenomenon.
AUTHOR CONTRIBUTION
Roy Daniel and Michael Danson conceived the general hypothesis and, with Charles Lee,
wrote the paper. Roy Daniel planned the experiments and interpreted the data. Michelle
Petersen, Charles Lee, Cristina Weinberg, Matthew Oudshoorn and Colin Monk planned
and carried out the experiments to determine the Equilibrium Model parameters and
analysed the data. Colin Monk carried out much of the data processing and prepared the
Figures. Nicholas Price and Charles Lee planned the CD experiments and interpreted the
data. Sharon Kelly and Charles Lee carried out the CD experiments. All of the authors
contributed to a critical review of the paper and approved the final version.
19 Fulton, K. F., Devlin, G. L., Jodun, R. A., Silvestri, L., Bottomley, S. P., Fersht, A. R. and
Buckle, A. M. (2005) PFD: a database for the investigation of protein folding kinetics and
stability. Nucleic Acids Res. 33, D279–D283
20 Tsou, C. L. (1995) Inactivation precedes overall molecular conformation changes during
enzyme denaturation. Biochim. Biophys. Acta 1253, 151–162
21 Arroyo-Reyna, A., Tello-Solis, S. R. and Rojo-Dominguez, A. (2004) Stability parameters
for one-step mechanism of irreversible protein denaturation: a method based on nonlinear
regression of calorimetric peaks with nonzero ꢀCp. Anal. Biochem. 328, 123–130
2
2
2
3
Creighton, T. E. (1993) Proteins, W.H. Freeman, New York
Privalov, P. L. and Khechinashvili, N. N. (1974) A thermodynamic approach to the
problem of stabilization of globular protein structure: a calorimetric study. J. Mol. Biol.
ACKNOWLEDGEMENTS
8
6, 665–684
We thank Alan Cooper for helpful discussions, and Martin Seefeld and Andreas Pickl for
technical assistance.
2
2
4
5
Daniel, R. M. and Danson, M. J. (2001) Assaying activity and assessing thermostability of
hyperthermophilic enzymes. Methods Enzymol. 334, 283–293
Smith, C. A., Toogood, H. S., Baker, H. M., Daniel, R. M. and Baker, E. N. (1999)
Calcium-mediated thermostability in the subtilisin superfamily: the crystal structure of
FUNDING
Bacillus Ak.1 protease at 1.8 A˚ resolution. J. Mol. Biol. 294, 1027–1040
2
2
2
6
7
8
Ishikawa, H., Kwak, K., Chung, J. K., Kim, S. and Fayer, M. D. (2008) Direct observation
of fast protein conformational switching. Proc. Natl. Acad. Sci. U.S.A. 105, 8619–8624
Tsou, C. L. (1993) Conformational flexibility of enzyme active sites. Science 262,
We thank the Royal Society of New Zealand Marsden Fund for financial support [grant
number UOW0501].
3
80–381
Lin, Y. Z., Liang, S. J., Zhou, J. M., Tsou, C. L., Wu, P. Q. and Zhou, Z. K. (1990)
Comparison of inactivation and conformational changes of D-glyceraldehyde-3-phosphate
dehydrogenase during thermal denaturation. Biochim. Biophys. Acta 1038, 247–252
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Received 17 August 2009/12 October 2009; accepted 22 October 2009
Published as BJ Immediate Publication 22 October 2009, doi:10.1042/BJ20091254
ꢀc The Authors Journal compilation ꢀc 2010 Biochemical Society