|| Issue 1, July 2002
Biological & Biomedical Sciences
A Comparison of the Thermostability of Glyceraldehyde 3-phosphate Dehydrogenase From Thermophiles and Mesophiles in Different Ionic Salt Solutions
Western Washington University
Advisor: Salvatore F. Russo, Ph.D.
Western Washington University
The thermostability of D-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) from two mesophilic sources
was compared to that of GAPDH isolated from two thermophilic microorganisms. Based on published
crystal structures of GAPDH from the thermophiles Thermus aquaticus, Thermatoga maritime,
and Bacillus stearothermophilus, it has been proposed that the primary source of
thermostabilization in these enzymes is the presence of extra ion pairs that are not present in
mesophilic GAPDH. To test this hypothesis, the rate of irreversible thermal inactivation of mesophilic
and thermophilic GAPDH dissolved in solvents of varying ionic strength and composition was measured.
Surprisingly, the thermophilic enzymes were not significantly destabilized by any of the salts tested,
while mesophilic GAPDHs were destabilized by NaCl, KCl, and NH4Cl. Both thermophilic and mesophilic
GAPDH showed stabilization against irreversible inactivation with (NH4)2SO4
and KF. The salts KCl and NH4Cl had opposite effects on the two classes of enzymes: stabilizing
for the thermophilic versus destabilizing for
the mesophilic enzymes. Taken together, these results
show no evidence for the importance of solvent-accessible ion pairs in the thermostabilization of the
GAPDH from T. aquaticus or B. stearothermophilus.
The diverse organisms found in nature have been divided into domains: Eukaryotes, Eubacteria, and Archaebacteria. The defining characteristic
of eukaryotes is the presence of a well-defined nucleus within each cell. Prokaryotes lack a nucleus and have been classified as either
Eubacteria or Archaebacteria based on their biochemical structure. In addition, organisms have evolved to survive at different temperatures.
Mesophiles exhibit optimum growth in the 20-37° C range, whereas thermophiles grow optimally in the
50-70° C range, and extreme thermophiles at > 65° C.
The ability of an organism to produce macromolecules with an appropriate degree of structural and functional stability is a prerequisite for
evolutionary success. As life has evolved to meet the exceptional demands imposed by extreme environments, the fundamental cellular
constituents - proteins, nucleic acids, and lipids - have been forced to undergo simultaneous chemical adaptations to facilitate adaptation to
selective pressures. This phenomenon can be illustrated by considering Archaebacteria. Members of this domain include organisms that live in
extreme environments: Thermophiles, halophiles, acidophiles, and methanogens. Archaebacteria exhibit unique macromolecular traits, such as
ether-linked membrane lipids, modified cell walls, and proteins with unsurpassed thermostability. These special molecular traits allow them to
endure extreme conditions.
There are several practical reasons for attempting to examine the mechanisms of enhanced thermostability of proteins from thermophilic microorganisms.
First, thermostable enzymes are useful as industrial catalysts. For example, the glucose isomerase-catalyzed conversion of glucose to high-fructose
syrup is carried out at 65° C. Clearly, an enzyme that is rapidly inactivated at this temperature would not be an efficient choice of
catalyst. Second, thermophilic enzymes provide enhanced research applications. The most obvious example of this is the use of
T. aquaticus DNA polymerase for the polymerase chain reaction.
The thermodynamic stability of a protein is a balance between large stabilizing forces derived from non-covalent intramolecular interactions and large
destabilizing forces, primarily chain conformational entropy. The free energy difference between folded and unfolded states of a small, monomeric,
globular protein is on the order of -30 to -60 kJ mol-1 (Jaenicke 1991). Typical intramolecular interactions (hydrogen bonds,
salt bridges, and hydrophobic interactions) contribute between -2 and -20 kJ mol-1. Thus, the stability of the folded state can be
affected by relatively minor changes in primary structure.
From a biochemical perspective, the molecular adaptations found in thermophiles provide keys to understanding the mechanisms responsible for
macromolecular stabilization and regulation. This is especially true when a model system is available to compare specific traits of a mesophile
with those of a thermophile. One such model system is the enzyme D-glyceraldehyde 3-phosphate dehydrogenase (GAPDH).
GAPDH provides four major advantages as a model system for the study of enzyme thermostabilization. First, GAPDH has been purified and
characterized from several sources. Crystal structures, conformational studies, and kinetic analyses are available from multiple mesophilic and
thermophilic sources, including the facultative thermophile Bacillus coagulans (Crabb et al. 1977; Crabb et al. 1981; Lee et al. 1982;
McLinden et al. 1984), the thermophile Bacillus stearothermophilus (Amelunxen 1966; Singleton et al. 1969; Biesecker et al. 1977;
Harris et al. 1980; Walker et al. 1980; Skarzynski et al. 1987), the extreme thermophilic bacteria Thermus aquaticus (Hocking and
Harris, 1973, 1976; Jecht et al. 1994; Rehaber and Jaenicke, 1992; Singleton et al. 1969; Tanner et al. 1996) and Thermatoga
maritime (Wrba et al. 1990; Tomschy et al. 1994; Korndorfer et al. 1995), and several genera of Archaebacteria (Fabry et al. 1989;
Zwickl et al. 1990; Arcari et al. 1993; Jones et al. 1995). Second, glycolysis is a universal metabolic process. Consequently, GAPDH is present
in nearly every living organism (Amelunxen 1976). Thus, the model system can be used to illustrate evolutionary diversity. The third advantage is
that purified GAPDH is commercially available and relatively inexpensive. GAPDH from mesophilic sources such as rabbit, pig, yeast, and human
erythrocytes, as well as thermostable GAPDH from B. stearothermophilus may be purchased from Sigma Chemical Company. Finally,
GAPDH activity is easily assayed. GAPDH is an essential glycolytic enzyme that catalyzes the reversible oxidative phosphorylation of
D-glyceraldehyde 3-phosphate to 1,3-bisphosphoglycerate. This reaction requires the presence of the coenzyme NAD+, which is concomitantly
reduced to NADH. Since NADH absorbs strongly at 340 nm, but NAD+ does not, enzyme activity can be measured simply and accurately by
UV spectroscopy (Velick 1955).
It has been proposed that enhanced stability of GAPDH from thermophilic sources may be primarily a result of additional ion pairs that are absent in
enzymes from mesophilic sources (Harris and Walker 1977; Skarzynski et al. 1987; Tomschy et al. 1994; Korndorfer et al. 1995). Crystal
structures of GAPDH isolated from at least three thermophilic sources - B. stearothermophilus, T. aquaticus, and
T. maritime - contain additional ion pairs that are not present in the crystal structures of GAPDH from some mesophilic organisms.
While the presence of these salt links has been verified by X-ray crystallography (Figure 1), their direct stabilizing effect in solution has yet to be
confirmed. Clearly, additional research needs to be performed in order to resolve the matter.
This research compares the effects of neutral salts on the rate of irreversible thermal inactivation of GAPDH from two mesophiles (chicken and rabbit)
to that of two thermophiles (T. aquaticus and B. stearothermophilus). The theoretical basis of this study is that
solvent-accessible ionic bonds will be susceptible to destabilization in solutions of high ionic strength. This charge-screening effect occurs as a
result of the presence of a large number of free ions in solution that are able to cluster around surface charges on the protein. This has the effect of
shielding these surface charges from participating in their normal electrostatic interactions, thus removing any enhanced stability they may normally
If the enhanced thermostability of T. aquaticus or B. stearothermophilus GAPDH is primarily dependent upon surface ion pairs, then
preincubation at an elevated temperature in 1.0 M salt buffer would be expected to inactivate these enzymes at a higher rate than identical
preincubation in buffer without added salt. It should be noted that this model does not account for salt-specific effects, but predicts that the
stability of thermophilic GAPDH should be a simple function of ionic strength. Hence, it is essential to include mesophilic GAPDH as a control,
in order to account for any salt-specific behavior of these enzymes.
Materials and Methods
Enzyme Preparation and Storage
GAPDH (E.C. 188.8.131.52) from rabbit muscle, chicken muscle, and the
moderate thermophile B. stearothermophilus was purchased
from Sigma Chemical Company (St. Louis, MO) as a lyophilized powder.
All preliminary work was done with the rabbit enzyme. T. aquaticus
GAPDH was purified from E. coli strain W3CG + M3B5A, kindly
provided by Dr. Ralph Hecht (Tanner et al. 1996). Strain
W3CG does not produce E. coli GAPDH when tetracycline is
present in the medium. All purified enzymes were stored in glycerol/buffer
solution (50% glycerol, 15 mM sodium arsenate, 7.5 mM sodium pyrophosphate,
pH 8.5) at -10° C.
Purification of T. aquaticus GAPDH
Successful purification of T. aquaticus GAPDH from E. coli
strain W3CG + M3B5A (Ganter and Pluckthun 1990) consisted of four
primary steps: Sonication, incubation at 90°C for 30 min, chromatofocusing,
and ultrafiltration (Figure 2). These steps were performed as described
in Tanner et al. (1994).
Cultures were incubated overnight at 37° C in minimal media 63 (0.1
M KH2PO4, 15 mM (NH4)2SO4,
0.8 mM MgSO4, 2 x 10-6 M FeSO4, pH
adjusted to 7.0 with KOH) supplemented with glucose (0.2%), ampicillin
(100 mg/mL), and tetracycline (50 mg/mL). The cells were collected
by centrifugation, resuspended (10 mM Tris-HCl pH 7.6, 1 mM EDTA,
1mM b-mercaptoethanol), and disrupted by sonication with a Branson
S75 Sonifier. Following disruption, the crude extract was centrifuged
at 17,210 x g (12,000 RPM in a Sorvall SS-34 rotor) for 30 minutes
at 4° C. The supernatant was removed and incubated for 30 minutes
at 90° C. Denatured proteins were removed by a second SS-34 centrifugation.
Activity measurements indicated the presence of thermostable GAPDH
in the supernatant, which was subsequently applied to a chromatofocusing
column (Sluyterman and Wijdenes 1981) for further purification.
Chromatofocusing was performed with 25 mL of DEAE-Sephacel (Pharmacia)
packed in a Pharmacia K9/30 column and equilibrated with 25 mM His-HCl
pH 6.2. Immediately following sample application, GAPDH was eluted
(5 mM acetate, 5 mM His-HCl, 5 mM glutamate, pH 4.0). Elutant was
collected in 3.0 mL fractions into tubes containing 1/10 volume 1.0
M Tris-HCl, pH 8.7. The fractions were analyzed for protein concentration
by measuring absorbance at 280 nm and were also assayed for GAPDH
activity (Figure 3). Fractions showing maximum activity were pooled
and concentrated by ultrafiltration (Blatt 1971). Laemmli SDS-PAGE
(Garfin 1990) was used to analyze purification efficiency. Based on
estimates of protein concentration from absorbance at 280 nm and GAPDH
activity, the ultrafiltration yielded a final product that was 115-fold
purified over the crude extract obtained from sonication (Table 1).
Preincubation solutions of lyophilized GAPDH were prepared by diluting
stock enzyme solutions (2 mg/mL) to a concentration of 30-60 mg/mL
in preincubation buffer (30 mM sodium arsenate, 15 mM sodium pyrophosphate,
0.1 mM EDTA, 5 mM b-mercaptoethanol, pH 8.5). For assays with the
T. aquaticus enzyme, GAPDH preincubation solutions were prepared
identically, except that the purified GAPDH was at a concentration
of 250 mg/mL (15 mM His-HCl, 150 mM Tris-HCl, 50% glycerol, pH 7)
prior to dilution. Preincubation solutions containing salt were prepared
by adding salt to the indicated concentrations prior to addition of
enzyme. For all experiments, pH was adjusted to 8.5 before the addition
of enzyme. A standard pH of 8.5 was chosen, based on the method of
Velick (1955) for assaying GAPDH activity (below), in order to control
for variation in pH caused by the addition of different salts. The
salts used for preincubation were (NH4)2SO4,
K2SO4, NH4Cl, KCl, KF, and NaCl.
Enzyme preincubations were carried out in Eppendorf tubes. In each
case, the preincubation tubes were submerged above the level of enzyme
solution in a water bath at constant temperature. For rabbit and chicken
GAPDH, the temperatures were 40°, 45°, 50°, and 60° C, whereas 70°
C was the preincubation temperature for B. stearothermophilus
GAPDH and 95° C for T. aquaticus GAPDH. The rate of irreversible
inactivation for each enzyme under these conditions can be found in
Tables 2 and 3. Following preincubation, enzyme solutions were immediately
cooled on ice and assayed for activity.
GAPDH Activity Assays
To determine enzymatic activity, the method of Velick (1955) was employed.
Solutions composed of 3-6 mg GAPDH in 2.9 mL of reaction buffer (27
mM sodium arsenate, 13.5 mM sodium pyrophosphate, 3.5 mM DTT, 0.3
mM NAD+, pH 8.5) were equilibrated to a temperature of
25° C in a thermostatted cuvette attached to a circulating water bath.
At thermal equilibrium, 100 mL of substrate (15 mM DL-glyceraldehyde
3-phosphate) was added, and the change in absorbance at 340 nm was
observed. Specific activity was calculated based on the linear regression
slope of the DA340 versus time plot.
Calculation of First-Order Rate Constants of Irreversible Inactivation
Zale and Klibanov (1986) presented a model that predicts a first order
process for irreversible inactivation of enzymes. In this model, it
is assumed that for an enzyme to make a transition from the native
(N), catalytically active state to the irreversibly inactivated state
(I), it must pass through a reversibly inactivated transition state
(R) (Klibanov 1983; Zale and Klibanov 1984). The key features of this
model are that the N to R transition is a reversible equilibrium,
whereas the R to I transition is irreversible.
Reversible denaturation of enzymes is caused by temperature-induced
conformational changes (Ahern and Klibanov 1985). For the purposes
of this research, it was important to consider only the overall process
from the N state to the I state. Hence, prior to assaying for enzymatic
activity, every enzyme solution was "snap cooled" on ice to allow
any reversibly inactivated molecules to revert to the native state.
The rate constant measured is thus a measure of irreversible
inactivation. Rate constants were determined directly from the linear
regression slope of the logarithm of percent activity as a function
of time of preincubation. All values reported have a linear regression
correlation of at least 0.98.
Inactivation of GAPDH from Rabbit Muscle
Preincubation of rabbit GAPDH (rGAPDH) in 1.0 M solutions of KCl
or NH4Cl at 40° C resulted in an approximately four-fold
increase in the rate of irreversible thermal inactivation relative
to preincubation without added salts. These salts destabilize the
enzyme against thermal inactivation since the percent remaining
line in Figure 4A is below the line for no salt added. Conversely,
preincubation in 1.0 M (NH4)2SO4,
1.0 M KF, or 0.5 M K2SO4 at 40° C stabilized
rGAPDH greater than five-fold against irreversible inactivation
since the percent remaining line with these salts in Figure 4A is
above the line for no salt added. At 45° C, a similar trend was
observed (Figure 4B). Preincubation in the presence of 1.0 M (NH4)2SO4
again inhibited thermal inactivation greater than 10-fold, whereas
preincubation in 1.0 M solutions of NaCl, KCl, or NH4Cl
increased the rate of irreversible thermal inactivation three- to
four-fold. The absolute rate constants for thermal inactivation
of rGAPDH are shown in Table 2.
In order to determine whether (NH4)2SO4
is able to protect against rGAPDH inactivation under more severe conditions,
we measured the rate of irreversible thermal inactivation at higher
temperatures. A solution of 1.0 M (NH4)2SO4
effectively stabilized the enzyme at both 50° C (Figure 5A) and 60°
C (Figure 5B), resulting in a 10-15 fold decrease in the rate of inactivation
in each case (Table 2). Once again, both KCl and NaCl actually increased
the rate of inactivation, although it is a more modest two-fold effect.
This suggests that the protective effects of (NH4)2SO4
are specific to the chemical structure and not solely due to increasing
the ionic strength of the enzyme environment. (NH4)2SO4
was also able to protect another mesophilic GAPDH isolated from chicken
muscle (cGAPDH) against irreversible thermal inactivation (data not
Irreversible Inactivation of B. stearothermophilus GAPDH
GAPDH from the moderate thermophile B. stearothermophilus (BsGAPDH)
was found to maintain 100% activity at 60° C over a time period of
1 hour (data not shown), in agreement with published results (Hocking
and Harris 1976). Preincubation of BsGAPDH at 70° C resulted in greater
than 90% irreversible enzyme inactivation within 30 minutes (Figure
6A). Similar to the mesophilic rGAPDH or cGAPDH, BsGAPDH was stabilized
against inactivation by 1.0 M (NH4)2SO4
but the effect was more pronounced with the BsGAPDH. Interestingly,
1.0 M KCl also stabilized BsGAPDH against irreversible thermal inactivation,
whereas it was found to destabilize the mesophilic GAPDH.
Irreversible Inactivation of T. aquaticus GAPDH
GAPDH from the extreme thermophile T. aquaticus (TaGAPDH) was
found to maintain 100% activity at 60° C and at 80° C over a time
period of 2 hours (data not shown). When incubated at 95° C, TaGAPDH
was approximately 90% inactivated within 30 minutes (Figure 6B). TaGAPDH
was completely protected from irreversible thermal inactivation after
30 minutes at 95° C by 1.0 M concentrations of (NH4)2SO4
or NH4Cl. The rate of irreversible inactivation of TaGAPDH
at 95° C was also decreased by KCl (three-fold) or KF (five-fold).
In fact, the only salt tested that was unable to stabilize TaGAPDH
was 1.0 M NaCl, which had no detectable effect. The absolute rate
constants for thermal inactivation of TaGAPDH are shown in Table 3.
In comparing these results, it is seen that the mesophilic enzymes
can be either stabilized or destabilized by preincubation in the presence
of salts. In contrast, none of the salts tested resulted in destabilization
of the thermophilic enzymes. Specifically, it was observed that (NH4)2SO4
stabilizes both the two mesophilic and the two thermophilic enzymes
against thermal inactivation. In contrast, KCl has a destabilizing
effect with the mesophilic enzymes but a stabilizing effect with both
The initial goal of this research was to test the hypothesis that
the thermostabilization of GAPDH from the moderate thermophile B.
stearothermophilus and the extreme thermophile T. aquaticus
is a result of additional ion pairs not found in mesophilic homologs
of this enzyme. The technique used to achieve this goal was a charge-screening
assay. By this model, if ion pairs are the primary determinant of
increased thermal stability, then incubation of the thermophilic
proteins in solutions of high ionic strength should result in decreased
thermal stability due to competition by ions in the salt solution
with ion-pairs present in the enzyme.
We recognized that the charge-screening model is limited by the
necessity to neglect salt-specific effects on protein structure
and function. Thus, several experiments were performed on the mesophilic
rGAPDH to examine this possibility. The results show that rGAPDH
is stabilized against irreversible thermal inactivation by (NH4)2SO4,
K2SO4, and KF, and is destabilized by the
presence of KCl, NaCl, or NH4Cl. It can be seen that
the neutral salt effects on rGAPDH (Figure 7) are roughly consistent
with the lyotropic series (von Hippel and Schleich 1969). In this
series, various ions are ranked in order of increasing effectiveness
in disordering the native conformation of a globular protein.
The rough agreement between the behavior of the rabbit enzyme and
the effects of neutral salts on globular proteins reported by others
was taken as an indication that GAPDH would be an appropriate model
system on which to use the charge-screening technique. The thermal
inactivation experiments were repeated on TaGAPDH, and it was determined
that this enzyme was stabilized against inactivation by (NH4)2SO4,
KF, KCl, and NH4Cl. The presence of NaCl in the solvent
was found to have no significant effect on enzyme stability. TaGAPDH
was not tested in the presence of K2SO4.
Taken together, these results clearly show that the thermostability
of TaGAPDH is not reduced as the ionic strength of the solvent is
increased, but rather its resistance to inactivation is enhanced.
This is precisely the opposite of the effect predicted by the charge-screening
model if solvent exposed ion pairs were an additional source of enzyme
thermostabilization. In addition, TaGAPDH shows strong deviations
from the lyotropic series, demonstrating that salt-specific effects
are different between rGAPDH and TaGAPDH.
Subsequent analysis of cGAPDH indicates that this mesophilic GAPDH
also follows the lyotropic series, at least with respect to preincubation
in the salts KCl and (NH4)2SO4. BsGAPDH
was found to behave more like the T. aquaticus enzyme in that
it was stabilized by both (NH4)2SO4
These results suggest there is perhaps some conservation of salt-specific
effects within the categories of thermophilic and mesophilic GAPDH.
Two possible interpretations are presented here. First, it may be
the case that the mechanisms of evolution to or from the thermophilic
state tend to include specific chemical changes in the macromolecular
structure that result in corresponding changes in enzyme sensitivity
to solvent salt composition. In other words, the types of mutations
that result in thermostability may also often result in halophilic
A second explanation for the differences in the behavior of thermophilic
and mesophilic GAPDH may have to do with the different types of environments
in which these two classes of organisms have evolved. Thermophilic
organisms have been isolated from extreme environments such as hot
springs and deep-sea hydrothermal vents. Since the concentration of
inorganic salts is typically higher in these types of environments
than in mesophilic climes, it is not surprising that, in general,
thermophilic enzymes may be stabilized by salts, whereas the same
salts can have either stabilizing or destabilizing effects on mesophilic
enzymes. Ions that are characteristic of geothermal hot springs include
Na+, K+, Li+, Ca+2, Mg+2,
Cl-, F-, HCO3-, and SO4-2.
The ultimate goal of this research was to examine whether the stability
of some thermophilic GAPDHs in solution is attained primarily via
ion pairs that are absent in mesophilic versions of this enzyme. This
goal was achieved, with the result that no evidence was found in support
of the hypothesis that TaGAPDH and BsGAPDH are stabilized by extra
solvent-exposed ion pairs. It should be noted that our results do
not rule out the possibility that additional ion pairs are important
for providing increased thermal stability in thermophilic GAPDH. It
is possible that salt-specific effects observed in our charge-screening
assay are sufficient to mask the predicted decrease in stability (and
increase in the rate of inactivation) following preincubation in high
salt solution. An alternative approach would be to use site-directed
mutagenesis to target specific amino acid residues present in thermophilic,
but not mesophilic, versions of the enzyme. This type of approach,
when combined with charge-screening data, could provide powerful evidence
for or against the theory that increased thermal stability is the
result of ion pairs.
The question of how some proteins are able to achieve extreme thermal
stability is important both because of its potential industrial and
practical applications and because of the fundamental physical and
chemical processes involved. The enzyme GAPDH offers many advantages
as a model system to study this problem. This research has provided
insight into the different behaviors of mesophilic and thermophilic
GAPDHs when incubated in solvents of various compositions and has
laid the groundwork for several possible avenues of future work in
this area of biochemistry.
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Journal of Young
Investigators. 2002. Volume Six.
Copyright © 2002 by Matt Kaeberlein and JYI. All rights reserved.