Identification of a long-range protein network that modulates active site dynamics in extremophilic alcohol dehydrogenases

Article abstract of DOI:10.1074/jbc.M113.453951

A tetrameric thermophilic alcohol dehydrogenase from Bacillus stearothermophilus (ht-ADH) has been mutated at an aromatic side chain in the active site (Trp-87). The ht-W87A mutation results in a loss of the Arrhenius break seen at 30 °C for the wild-type enzyme and an increase in cold lability that is attributed to destabilization of the active tetrameric form. Kinetic isotope effects (KIEs) are nearly temperature-independent over the experimental temperature range, and similar in magnitude to those measured above 30 °C for the wild-type enzyme. This suggests that the rigidification in the wild-type enzyme below 30 °C does not occur for ht-W87A. A mutation at the dimer-dimer interface in a thermolabile psychrophilic homologue of ht-ADH, ps-A25Y, leads to a more thermostable enzyme and a change in the rate-determining step at low temperature. The reciprocal mutation in ht-ADH, ht-Y25A, results in kinetic behavior similar to that of W87A. Collectively, the results indicate that flexibility at the active site is intimately connected to a subunit interaction 20 A away. The convex Arrhenius curves previously reported for ht-ADH (Kohen, A., Cannio, R., Bartolucci, S., and Klinman, J. P. (1999) Nature 399, 496-499) are proposed to arise, at least in part, from a change in subunit interactions that rigidifies the substrate-binding domain below 30 °C, and impedes the ability of the enzyme to sample the catalytically relevant conformational landscape. These results implicate an evolutionarily conserved, long-range network of dynamical communication that controls C-H activation in the prokaryotic alcohol dehydrogenases.

Full text of DOI:10.1074/jbc.M113.453951

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 288, NO. 20, pp. 14087–14097, May 17, 2013
© 2013 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A.
Identification of a Long-range Protein Network That
Modulates Active Site Dynamics in Extremophilic
Alcohol Dehydrogenases*
Received for publication, January 16, 2013, and in revised form, March 20, 2013 Published, JBC Papers in Press, March 22, 2013, DOI 10.1074/jbc.M113.453951
Zachary D. Nagel‡1, Shujian Cun‡§2, and Judith P. Klinman‡§¶3
From the Departments of ‡Chemistry and ¶Molecular and Cell Biology, and the §California Institute for Quantitative Biosciences
(QB3), University of California, Berkeley, California 94720
Background: The role of protein flexibility in the C-H activation step, catalyzed by homologous thermophilic and psychro-
philic alcohol dehydrogenases, is addressed.
Results: Mutation at the substrate-binding site, or at a dimer interface, alters kinetic properties and protein oligomeric
structure.
Conclusion: Active site flexibility is correlated with subunit interactions 20 Å away.
Significance: A long-range network of catalytically relevant, dynamical communication is identified.
A tetrameric thermophilic alcohol dehydrogenase from
According to the corresponding state hypothesis, proteins
evolve to strike a balance between stability and conformational
Bacillus stearothermophilus (ht-ADH) has been mutated at an
aromatic side chain in the active site (Trp-87). The ht-W87A flexibility (1). This idea has been used to explain the frequent
observation that proteins are often just stable enough to oper-
ate under physiological conditions (2). It has been suggested
that proteins do not evolve to maximum stability because of the
requirements of the cell to achieve efficient protein synthesis
and turnover, and the need for proteins to undergo rapid con-
formational sampling to execute their biological function
(2–4). The role of conformational sampling in protein function
has become the focus of intense research in the field of enzy-
mology, as a large body of experimental data continues to illu-
minate the link of protein motions to bond cleavage events at
enzyme active sites (5–16).
mutation results in a loss of the Arrhenius break seen at 30 °C for
the wild-type enzyme and an increase in cold lability that is
attributed to destabilization of the active tetrameric form.
Kinetic isotope effects (KIEs) are nearly temperature-indepen-
dent over the experimental temperature range, and similar in
magnitude to those measured above 30 °C for the wild-type
enzyme. This suggests that the rigidification in the wild-type
enzyme below 30 °C does not occur for ht-W87A. A mutation at
the dimer-dimer interface in a thermolabile psychrophilic
homologue of ht-ADH, ps-A25Y, leads to a more thermostable
enzyme and a change in the rate-determining step at low tem-
perature. The reciprocal mutation in ht-ADH, ht-Y25A, results
in kinetic behavior similar to that of W87A. Collectively, the
results indicate that flexibility at the active site is intimately con-
A corollary of the corresponding state hypothesis is that the
introduction of stability in excess of that required under phys-
iological conditions, due to a change in temperature or muta-
tion, may adversely affect protein function by impairing func-
tional conformational flexibility. Extensive investigations of
proteins as a function of temperature support the expectation
that proteins become increasingly rigid with decreasing tem-
perature, and that enzymes evolved to operate at high temper-
atures exhibit sluggish activity relative to their mesophilic and
psychrophilic counterparts at reduced temperature (3, 17–23).
Evidence continues to emerge that enzymes have evolved in
such a way as to optimize the influence of protein dynamics on
the chemical steps of catalysis (see for example, Refs. 24 and 25).
Most enzyme reactions can be fit to the Arrhenius equation
(Equation 1),
˚
nected to a subunit interaction 20 A away. The convex Arrhe-
nius curves previously reported for ht-ADH (Kohen, A., Cannio,
R., Bartolucci, S., and Klinman, J. P. (1999) Nature 399, 496–
499) are proposed to arise, at least in part, from a change in
subunit interactions that rigidifies the substrate-binding
domain below 30 °C, and impedes the ability of the enzyme to
sample the catalytically relevant conformational landscape.
These results implicate an evolutionarily conserved, long-range
network of dynamical communication that controls C-H activa-
tion in the prokaryotic alcohol dehydrogenases.
k ϭ AeϪE /RT
(Eq. 1)
a
* This work was supported, in whole or in part, by National Institutes of
Health Grants GM025765 (to J. P. K.) and GM008295 (to Z. D. N.) and the
National Science Foundation Grant MCB0446395 (to J. P. K.).
1 Present address: The Samson Lab, Bldg. 56–230, Massachusetts Institute of
Technology, Cambridge, MA 02139.
where k is the rate constant, A is the Arrhenius pre-factor, Ea is
the energy of activation, and R is the molar gas constant. Gen-
erally, a plot of ln(k) against 1/T yields a straight line. However,
for some enzymes, a transition to more sluggish catalysis has
been seen to occur abruptly below a threshold temperature in a
manner that is distinct from either a change in the rate-deter-
mining step or a decrease in enzyme stability (5, 24, 26). All of
2 Present address: The Hecht Lab, Biodesign Institute, Arizona State Univer-
sity, Tempe, AZ 85287.
3 To whom correspondence should be addressed. Tel.: 510-642-2668; Fax:
510-642-8369; E-mail: klinman@berkeley.edu.
MAY 17, 2013•VOLUME 288•NUMBER 20
JOURNAL OF BIOLOGICAL CHEMISTRY 14087

Long-range Protein Dynamics and Catalysis in ADH
these enzymes and most other enzymes that have been shown previously demonstrated to rigidify below 30 °C in ht-ADH (21).
to exhibit Arrhenius breakpoints are multimeric (see for exam- We conclude that intersubunit contacts in the family of tetra-
ple, Refs. 27 and 28 and references therein), raising the possi- meric prokaryotic alcohol dehydrogenases can alter the protein
bility of alterations in subunit interactions or protein oligo- conformational landscape (31), thereby controlling the active
meric structure as the origin of the phenomenon.
site configurations that are directly linked to Arrhenius behav-
One of the best-characterized examples of such behavior is ior and the properties of hydride tunneling between substrate
an alcohol dehydrogenase from Bacillus stearothermophilus and cofactor.
(ht-ADH).4 This enzyme shows a break in its Arrhenius plot at
EXPERIMENTAL PROCEDURES
30 °C that is accompanied by a significant increase in the
enthalpy of activation and concomitant rapid decrease in rate
Materials—Benzyl alcohol was purchased from Fisher Scien-
with temperature. In addition, the temperature dependence of tific, and purified by vacuum distillation prior to use. The ␣,␣-
the kinetic isotope effect (KIE) is seen to undergo a transition d2-benzyl alcohol (99.5% D) was purchased from CDN isotopes,
from temperature-independent at elevated temperature to found to be chemically pure within the detection limits of
ϩ
temperature-dependent below 30 °C (5). These changes have GC-MS, and thus, used without further purification. NAD
been attributed to distinct protein conformational substates and NADH were purchased from Sigma and used without fur-
above and below the break, with experimental support for this ther purification.
idea coming from hydrogen-deuterium exchange that demon-
Cloning of ps-ADH Gene—The DNA sequence for alcohol
strates a local increase in flexibility above 30 °C within the sub- dehydrogenase from Moraxella sp. TAE123 (ps-ADH) was
strate-binding domain of the enzyme (21). A psychrophilic obtained from the National Center for Biotechnology Informa-
ortholog from Moraxella TAE sp. 123 (ps-ADH) with very tion website. The gene was synthesized commercially, received
high-sequence identity (61%) has been shown, by contrast, to be in a pDrive cloning vector, and subsequently subcloned into a
highly thermolabile (29). The ps-ADH does not show an Arr- pET-24b expression vector. Correct insertion of the gene was
henius break, and exhibits local flexibility in the substrate-bind- confirmed by DNA sequencing.
ing domain at 10 °C that is greater than that measured for ht-
ADH at the same temperature (30).
Preparation of Mutants—Site-directed mutagenesis was car-
ried out as previously described (22), using the QuikChange Strat-
It has been shown previously that both the Arrhenius behav- agene kit for site-directed mutagenesis, with the following primers
ior and KIEs for ht-ADH are sensitive to amino acid substitu- and their reverse complements: ht-W87F, 5Ј-CCGCGTTG-
ϩ
tions for conserved bulky hydrophobic residues in the NAD
GAATTCCTTTCTTATATTCTGCATGCG-3Ј; ht-W87L, 5Ј-
cofactor binding pocket (31, 32). We have now analyzed a var- CGCGTTGGAATTCCTTTGTTATATTCTGCATGC-3Ј; ht-
iant at a conserved residue in the substrate-binding pocket of W87A, 5Ј-CCGCGTTGGAATTCCTGCGTTATATTCTGC-
ht-ADH (ht-W87A) that is shown to abrogate the Arrhenius ATGC-3Ј; ht-Y25A, 5Ј-GAAGTAGAAAAACCAACCATTT-
break in the experimental temperature range. The KIE for CAGCTGGAGAAGTATTAGTCCGC-3Ј; ps-A25Y, 5Ј-CCC-
ht-W87A at all temperatures resembles that of the wild-type AACGCCAGGTTATGGCGAGATAGTGG-3Ј. The mutated
enzyme above 30 °C, with a kcat that is moderately reduced codon is underlined. Primers were designed using the program
(about 5-fold). Due to the absence of a break in its activity, kcat PrimerX and purchased from Operon. Plasmids were isolated
for W87A approaches that of the wild-type enzyme at reduced and sequenced to confirm the mutations and the intactness of
temperature. However, the apparent enhancement of activity the remainder of the gene.
for ht-W87A relative to wild-type at low temperatures is
Expression and Purification of ht-ADH—Purification and
accompanied by a loss of stability that results from dissociation expression of ht-ADH was carried out as previously described
of the tetramer into inactive dimers and monomers. A recipro- (21) with some modifications. For ht-W87A, an AMP-Sephar-
cal relationship emerges from a mutant ps-ADH, in which sub- ose affinity column (Amersham Biosciences) replaced the Cibi-
unit interactions are strengthened. The ps-ADH (ps-A25Y) cron blue column because it had a higher affinity for ht-ADH.
variant introduces an intersubunit interaction present at the The improved resolution of this column renders subsequent
corresponding position in wild-type ht-ADH; we find that this additional purification by size exclusion chromatography
leads to a marked stabilization of the psychrophilic enzyme that unnecessary. The heat treatment step (15 min at 60 °C) was also
is accompanied by the emergence of an Arrhenius breakpoint at eliminated for ht-W87A because this mutant was found to lose
about 20 °C. Because the ht-ADH active site mutant disrupts significant activity under these conditions (see “Results” and
the subunit interface, and the ps-ADH subunit interface muta- “Discussion”). A number of minor changes were introduced
tion impairs catalysis at the active site, we further interrogated into the subsequent preparation of ht-Y25A. These included
ht-ADH by mutating its tyrosine at the subunit interface, Y25A, inoculation of the stock into an autoinducible medium (Magic
uncovering kinetic behavior very similar to W87A. It is, thus, Medium, Invitrogen) with 50 ␮g/ml of kanamycin and 0.25 mM
possible to identify an approximately 20-Å pathway of dynam- ZnSO4. The cells were aerobically grown for about 16 h at 37 °C,
ical communication between the two targeted residues (Tyr-25 and then collected by microcentrifuge. The cell pellet was lysed
and Trp-87), comprised of the very same peptides that were by a mixture of gentle non-ionic detergents (Bugbuster௡, Nova-
gen), and the cell extract was heated at 60 °C for 15 min. Then
the insoluble components were separated from the liquid phase
by centrifugation at 4 °C, and the supernatant was mounted
onto a fast-flow DEAE anion exchange column (GE Health-
4 The abbreviations used are: ht-ADH, high temperature-alcohol dehydro-
genase; FPLC, fast protein liquid chromatography; FRET, fluorescence
energy transfer; ps, psychrophilic; KIEs, kinetic isotope effects.
14088 JOURNAL OF BIOLOGICAL CHEMISTRY
VOLUME 288•NUMBER 20•MAY 17, 2013

Long-range Protein Dynamics and Catalysis in ADH
TABLE 1
Kinetic parameters at 30 °C for ADH variants
؉
kcat
Km, benzyl alcohol
Km, NAD
KIE for kcat
Ϫ1
s
mM
ht-ADHa
ht-W87F
ht-W87L
ht-W87A
ht-Y25A
ps-ADHb
ps-A25Y
24.9 (Ϯ2.5)
6.1 (Ϯ0.5)
2.2 (Ϯ0.1)
5.5 (Ϯ0.4)
14.0 (Ϯ1.0)
6.9
6.9 (Ϯ0.5)
5.2 (Ϯ0.8)
18 (Ϯ2.2)
1.5 (Ϯ0.3)
7.2 (Ϯ1.7)
29.4
1.1 (Ϯ0.1)
2.1 (Ϯ0.3)
4.0 (Ϯ0.7)
0.2 (Ϯ0.05)
1.0 (Ϯ0.5)
3.2 (Ϯ0.2)
3.4 (Ϯ0.6)
3.0 (Ϯ0.3)
3.2 (Ϯ0.6)
2.9 (Ϯ0.2)
3.2
3.8 (Ϯ0.2)
16 (Ϯ1.5)
0.5 (Ϯ0.06)
3.7 (0.6)
a From Ref. 5.
b From Ref. 22.
care). The column was washed by 60 mM NaCl, and the protein
Fluorescence Kinetics—Fluorescence spectra were moni-
was eluted by 200 mM NaCl. After buffer exchange by centrifu- tored at 10 and 30 °C as a function of time following the addi-
gal filters (Amicon௡, Millipore), the harvested protein was tion of 10 ␮M enzyme to a solution containing 100 mM N-bu-
ϩ
passed through an NAD -affinity column (Blue Sepharose௡, tylformamide, and 5 eq of NADH (50 ␮M). Samples were
ϩ
GE Healthcare) and eluted by 5 mM NAD from the column. excited at 295 nm, and both the excitation and detection slits
The purified sample underwent buffer exchange and concen- were set to 1 nm. Fluorescence emission spectra were collected
tration by centrifugal filters.
at 1–2-min intervals, and the intensity recorded at 338 nm, the
Purification and Expression of ps-ADH—Because ps-ADH maximum for protein fluorescence, and at 410 nm for enzyme-
inactivates rapidly above 30 °C, Escherichia coli were grown to bound NADH, reflecting fluorescence energy transfer (FRET)
an A600 of ϳ0.6 at 37 °C, and then cooled to 17 °C prior to from tryptophan to cofactor. Little or no fluorescence signal at
induction with 0.5 mM isopropyl thiogalactoside. Expression of 410 nm is expected from free NADH in solution for two rea-
ps-ADH was monitored by gel electrophoresis, and found to sons. The excitation wavelength (295 nm) gives negligible
reach a maximum within 48 h. Purification of ps-ADH was direct excitation of NADH (␭max(excitation) ϭ 340 nm) and
carried out using the protocol established for ht-ADH with the resonance energy transfer from protein tryptophan side chains
following modifications. 100 mM NaCl was maintained should be very weak for free NADH due to the 1/r6 distance
throughout the purification to prevent aggregation, and the dependence of FRET. Additionally, the fluorescence signal
60 °C treatment was eliminated. An empty vector expression from free NADH is ϳ6-fold weaker than that for protein-bound
control yielded negligible endogenous ADH activity, so that the NADH, due to quenching in solution.
activity detected under conditions used can be assumed to arise
solely from ps-ADH.
FPLC Size Exclusion Chromatography—Protein oligomeriza-
tion was estimated using a Superdex 200 FPLC column, cali-
Kinetic Assays—Kinetic data were collected for the oxidation brated with commercially available molecular mass standards
of benzyl alcohol (or its ␣,␣-d2 isotopologue) to benzaldehyde (from 6,000 to 690,000 Da). The elution profile was monitored
ϩ
with concomitant reduction of NAD to NADH, as previously by UV absorption at 280 nm and activity assays were carried out
described (5). With the exception of ht-W87A, enzyme stocks for individual fractions. Chromatography and fraction collec-
were maintained at 4 °C with no detectable loss of activity over tion were conducted at 4 °C. These conditions (50 mM phos-
the course of several hours. W87A stock solutions were prein- phate, pH 7, with 150 mM NaCl, and 500 ␮g of protein) differ
cubated at 30 °C and an enzyme concentration of 100 ␮M, in the from previously reported preparative size exclusion chroma-
presence of 1 mM NADH, in 50 mM potassium phosphate tography, which was carried out at higher protein, lower salt,
buffer, pH 7.0, for 30 min prior to use in kinetic assays (see and 1 bar pressure. In all instances, activity was detected solely
“Results” and “Discussion” below regarding temperature-de- in the fractions containing tetrameric protein.
pendent inactivation and reconstitution). These solutions were
Circular Dichroism (CD)—Spectra were collected using an
then further diluted as needed for kinetic assays, and main- Aviv 410 spectropolarimeter in a quartz cuvette with a 1-mm
tained at room temperature in 1 mM NADH to prevent activity path length and a protein concentration of 0.5 mg/ml in 50 mM
loss. The final dilution of enzyme into the activity assays phosphate, pH 7.0. The temperature was controlled by a Peltier
reduced the concentration of NADH to Յ10 ␮M.
temperature controller. Data points were taken every 1 nm. The
Inactivation Kinetics of ht-W87A—The kinetics of activity signal is reported in units of mean residue elipticity.
loss upon incubation of ht-W87A was measured at multiple
RESULTS
concentrations of enzyme and in the presence and absence of
cofactor. Aliquots were removed as a function of time and their
Kinetic and Structural Properties of W87A—Steady-state
activity determined under standard assay conditions, which kinetics, carried out at 30 °C for variants of ht-ADH with alter-
ϩ
were 2.5 mM NAD and 5 mM benzyl alcohol, 50 mM potassium nate hydrophobic side chains (at position 87), indicate rela-
phosphate buffer, pH 7.0, at 20 °C. For incubation at low tively small changes in kcat (less than about 10-fold) with no
enzyme concentration and 0 °C (Fig. 2, panel A, lowest curve), obvious trends across the series (Table 1). The measured KIEs
ht-W87A was incubated under the conditions of the kinetic are all similar to the value obtained for the wild-type enzyme. A
assay. In this case, the reaction was initiated by the addition of detailed analysis of both rates and KIEs as a function of temper-
cofactor instead of the addition of enzyme, and the activity ature was carried out for ht-W87A because it represented the
measured at 4 °C. Conditions were otherwise identical.
most drastic structural perturbation in the series, and was the
MAY 17, 2013•VOLUME 288•NUMBER 20
JOURNAL OF BIOLOGICAL CHEMISTRY 14089

Long-range Protein Dynamics and Catalysis in ADH
only variant to exhibit cold lability (see below). An Arrhenius ternary complex with the reduced form of the cofactor (NADH)
plot of kcat shows a complete loss of the breakpoint observed at and the aldehyde analog inhibitor N-butylformamide (Fig. 2,
30 °C for the wild-type enzyme (Fig. 1). The activation param- panel C). Finally, ht-W87A activity is reconstituted more slowly
eters for ht-W87A are similar to those measured above 30 °C in the presence of the inhibitor complex at 10 °C than observed
for the wild-type enzyme (Table 2). In addition, the KIE for at 30 °C (Fig. 2, panel D). Despite the slightly slower reconsti-
ht-W87A is seen to be nearly temperature-independent through- tution than in the presence of N-butylformamide, NADH alone
out the experimental temperature range, in contrast to the wild- was found to give the largest absolute increase in specific activ-
type enzyme, which exhibits a change in the temperature depend- ity (not shown), possibly due to carryover of inhibitor into the
ence of the KIE below 30 °C (Fig. 1, inset). (Full tabulation of the assay. Incubation of ht-W87A at 200 ␮M enzyme and 1 mM
kinetic data for ht-W87A and the other mutants can be found in NADH at 30 °C for 30 min was, therefore, used to generate
Table 3.)
stable and reproducible enzyme activity for use in kinetic
In the course of purification, ht-W87A was seen to exhibit assays. The presence of NADH was sufficient to prevent activity
cold lability that was not observed in any other variants. To loss over several hours in 2–20 ␮M stock solutions of ht-W87A
establish conditions for steady-state kinetics, the temperature maintained at room temperature. Kinetic assays were linear for
and concentration dependence of this phenomenon was inves- at least 1 min and linearly dependent on enzyme concentrations
tigated (Fig. 2). Wild-type ht-ADH incubated at 0 °C and an between 10 and 55 °C, ruling out the possibility of rapid inacti-
enzyme concentration of 20 ␮M showed no activity loss even vation upon dilution, or during the course of data collection.
after 24 h. By contrast, a freshly thawed sample of ht-W87A
Additional characterization of ht-W87A was aimed at deter-
diluted to 20 ␮M lost 50% of its activity within about 5 h, with mining the mechanism of inactivation and reconstitution.
more rapid loss at lower enzyme concentration (Fig. 2, panel A). Fluorescence monitored as a function of time under reconsti-
When the same sample was incubated at moderately elevated tuting conditions at 10 °C revealed a time-dependent decrease
temperatures, an increase in activity was observed, with the in fluorescence intensity (Fig. 2, panel D). The kinetics of fluo-
most rapid reconstitution seen at 30 °C (Fig. 2, panel B). Slower rescence quenching were correlated with the kinetics of recon-
reconstitution was seen at 40 °C, and the enzyme was inacti-
TABLE 3
vated upon prolonged incubation at 50 °C (data not shown). It
Kinetic parameters for ht-W87A, ps-A25Y, and ht-Y25A
was also found that the presence of cofactor increases the rate
a
T
°C
kcat
s
Error
Dkcat
Error
of reconstitution, with the most rapid reconstitution seen for a
Ϫ1
ht-W87A
12.5
15
1.5
0.1
0.1
0.2
0.7
1.8
1.2
2.5
3.2
3.1
2.9
3.6
3.2
2.9
3.0
0.2
0.4
0.4
0.5
0.6
0.2
0.3
1.6
20
2.5
28.5
38
5.2
14.0
23.0
34.7
45
52
ps-A25Y
5
0.5
1.0
1.5
2.7
3.2
3.8
7.0
7.9
13.0
0.01
0.04
0.04
0.1
2.4b
2.8
0.1
0.1
11.5
16
20
25
29
39
46
52
0.2
0.3
3.7
0.6
1.0
0.9
3.5
3.6
0.6
0.6
1.9
ht-Y25A
10
20
30
40
50
2.9
0.4
0.1
1.0
2.5
5.7
2.8
3.4
2.7
2.4
2.9
0.2
0.6
0.1
0.2
0.3
7.4
13.0
25.2
51.1
a D
FIGURE 1. Arrhenius plot for ht-W87A between 10 and 52 °C. The temper-
ature dependence of kcat (filled circles, left axis), KIE (open squares, right axis),
and the corresponding plot for wild-type ht-ADH (inset) are presented.
k
refers to the primary KIE for oxidation of benzyl alcohol relative to its
cat
␣,␣-d2-isotopolog.
b KIEs were only measured at the temperatures indicated.
TABLE 2
Activation parameters in kcal/mol for wild-type ht-ADH and mutants for the oxidation of benzyl alcohol
ht-ADH,
ht-ADH
ht-W87A
ps-ADH
ps-A25Y
ht-Y25A
5–30 °Ca
30–65 °Ca
12–52 °Cb
5–35 °Cc
20–52 °Cb
7.2 (Ϯ0.3)
Ϫ9.6 (Ϯ0.3)
NDe
10–50 °Cb,d
12.1 (Ϯ0.5)
Ϫ4.0 (Ϯ0.5)
0.5 (Ϯ0.1)
⌬H‡
21.2 (Ϯ1.0)
5.2 (Ϯ2.4)
0.9 (Ϯ0.05)
14.5 (Ϯ0.4)
Ϫ1.4 (Ϯ0.8)
0.1 (Ϯ0.01)
14.8 (Ϯ0.4)
Ϫ1.8 (Ϯ0.4)
0.2 (Ϯ0.01)
9.2 (Ϯ0.6)
Ϫ7.3 (Ϯ1.5)
4.5 (Ϯ1.5)
T⌬S‡
Ea(D) Ϫ Ea(H)
a From Ref. 5.
b Data were fit to a single line.
c From Ref. 22.
d The data were analyzed as a single line or as two separate slopes with a very subtle break. The resulting difference between the separate enthalpies was less than the errors
generated for a single slope.
e ND, not determined.
14090 JOURNAL OF BIOLOGICAL CHEMISTRY
VOLUME 288•NUMBER 20•MAY 17, 2013

Long-range Protein Dynamics and Catalysis in ADH
FIGURE 2. Inactivation and reconstitution of ht-W87A. A, percentage of
activity remaining after incubation at 0 °C for 20 ␮M wild-type ht-ADH (trian-
gles, dashed line), 20 ␮M ht-W87A (squares, solid line), or 0.5 ␮M ht-W87A (cir-
cles, dotted line). B, percentage of activity remaining for ht-W87A after incu-
bation at 0 (circles, dotted line), 10 (squares, solid line), 20 (up triangles, dashed
line), or 30 °C (down triangles, solid gray line). C, time-dependent reconstitu-
tion of activity at 30 °C as a percentage of the activity extrapolated at t ϭ ϱ,
withnoaddition(circles, dottedline)orinthepresenceof1mM NADϩ (squares,
solid gray line), 1 mM NADH (up triangles, dashed line), or 1 mM NADH ϩ 1 mM
N-butylformamide (down triangles, solid line). D, time-dependent reconstitu-
tion of activity at 10 °C in the presence of 1 mM NADH ϩ 1 mM N-butylforma-
mide (circles, dotted line), superimposed over time-dependent changes in flu-
orescence emission intensity at 338 nm under the same conditions (squares,
solid line).
stitution at 10 (Fig. 2, panel D) and 30 °C, where both phenom-
ena occur more rapidly. The fluorescence emission spectra are
presented in Fig. 3A. A CD spectrum for ht-W87A collected for
a sample cold-adapted overnight at 4 °C, and which showed no
detectable activity, was nearly superimposable with a highly
active sample that had been incubated under reconstituting
conditions (Fig. 3B). Size exclusion chromatography indicated
that the dominant oligomeric state for wild-type ht-ADH is a
tetramer, whereas ht-W87A eluted primarily as a monomer
(Fig. 4A). A similar experiment conducted in the presence of 1
mM NADH and 1 mM N-butylformamide, which were found to
protect the enzyme from cold inactivation (data not shown)
resulted in ht-W87A eluting almost exclusively as the tetramer
(Fig. 4B). In all cases, activity assays performed on fractions
eluting from the size exclusion column indicated that only the
tetramer was active.
FIGURE 3. Spectroscopic characterization of ht-W87A. A, time-dependent
fluorescence emission spectrum for ht-W87A. Arrows represent the direction
of change observed as a function of time (min). The fluorescence data in Fig.
2D were generated from the spectra shown herein. Small, but significant
changes were detected in two distinct regions of the fluorescence spectrum.
Protein fluorescence (␭max ϳ 333 nm) was seen to decrease as a function of
time. FluorescenceduetoNADHsimultaneouslybecameblueshifted, asseen
by an increase in the intensity at 410 nm. B, CD spectra at two temperatures
for ht-W87A. Samples were incubated overnight at 20 ␮M enzyme at 0 °C
(heavy black line) or for 1 h at 30 °C and 200 ␮M enzyme, with a spectrum
collectedimmediatelyfollowingdilutionto20␮M at30 °C(graylinewithblack
dots). Difference spectrum, fine black line.
Characterization of a ps-A25Y Variant—Turning to the ps-
ADH variants, it was found that these enzymes are also only
active as a tetramer. The wild-type enzyme dissociated into an
inactive monomeric form when subjected to FPLC size exclu-
sion chromatography at 4 °C, and the extreme thermolability
reported previously was confirmed by incubation at 50 °C (Fig.
5). The availability of x-ray structures for both ht-ADH and
FIGURE4. Analysisofht-W87AbyFPLCsizeexclusionchromatography. A,
elutionprofileofwild-typeht-ADH(black)andht-W87A(gray)at4 °Cshowing
peaks associated with the tetramer (about 11 ml), dimer (about 13 ml), and
monomer (about 15 ml). B, elution profile of ht-W87A at 4 °C in the presence
(black) or absence of 1 mM NADH and 100 mM N-butylformamide. High back-
ground absorption (about 0.85 absorbance units) due to NADH was sub-
tracted from the data collected in the presence of the cofactor to superim-
ps-ADH, together with the high sequence identity between pose the two traces for comparison.
these proteins (61%), afforded the opportunity to search for
structural differences at their subunit interfaces. One striking trast its behavior to wild-type ps-ADH. The ps-A25Y mutation
feature is the substitution of a tyrosine at position 25 in ht-ADH significantly stabilized the tetrameric form of the enzyme at
by alanine in ps-ADH. We therefore prepared ps-A25Y to con- 4 °C, and led to a dramatic increase in its thermostability at
MAY 17, 2013•VOLUME 288•NUMBER 20
JOURNAL OF BIOLOGICAL CHEMISTRY 14091

Long-range Protein Dynamics and Catalysis in ADH
FIGURE 5. Stability and FPLC size exclusion chromatography of ps-ADH
variants. A, inactivationof20 ␮M ps-ADH(opensquares, blackline)orps-A25Y
(closed squares, gray line) at 50 °C. B, elution profile at 4 °C for wild-type ps-
ADH (black) and ps-A25Y (gray) showing peaks associated with the tetramer
(about 11 ml), dimer (about 12.5 ml), and monomer (about 14 ml). Peaks
between the column dead volume (about 7 ml) and the tetramer represent
higher order aggregates.
50 °C (Fig. 5). As a result of the increased thermostability, it was
possible to measure steady-state kinetics up to 52 °C. An Arr-
henius plot for ps-A25Y indicates a breakpoint near about
20 °C. Below this temperature, activity falls more rapidly with
temperature, and the KIE on kcat becomes progressively smaller
(Fig. 6A), in contrast to the wild-type ps-ADH behavior. The
activation parameters for ps-A25Y above the breakpoint
(between 20 and 52 °C) are similar to those measured for wild-
type ps-ADH (Table 2). No attempt was made to estimate the
same parameters below the breakpoint for ps-A25Y because of
the apparent kinetic complexity suggested by the decrease in
KIE with temperature.
Contrasting the Behavior of ht-Y25A to ht-W87A—The recip-
rocal responses of ht-ADH to mutation at its active site and
ps-ADH to mutation at its subunit interface suggested that
ht-Y25A would produce an enzyme form very similar to
ht-W87A. This prediction is confirmed by the kinetic behavior
of ht-Y25A, where the breaks at 30 °C in rate and temperature
dependence of the KIE for wild-type (Fig. 1, inset) are lost in
ht-Y25A (Fig. 6B). The extreme susceptibility of ht-W87A to
dissociation at low temperature is, however, not observed for
ht-Y25A, with the fraction of tetramer being highly dependent
on the concentration of enzyme analyzed by FPLC. The propor-
tion of the enzyme found in the tetrameric form varied from
about 81% at 0.5 ␮g to 44% at 0.3 ␮g, to a level below the limit of
detection at 0.02 ␮g of protein analyzed in a 0.5 ml loading
volume. In the case of ht-W87A, FPLC data (Fig. 4A, gray trace)
were carried out at extremely high protein concentrations (500
␮g) in an effort to suppress protein dissociation; nonetheless,
the dominant enzyme form remained a monomer. This indi-
cates the much larger effect of an active site mutant on oligo-
meric protein stability than the subunit interface mutant Y25A.
FIGURE 6. Arrhenius plots for ADH variants with amino acid substitutions
atthe subunit interface. A, Arrhenius plots for ps-A25Y between 5 and52 °C.
Temperaturedependenceof kcat (filledcircles, solidline, leftaxis)andKIE(open
squares, right axis) are shown. B, Arrhenius plot for ht-Y25A between 10 and
50 °C. Thetemperaturedependenceofkcat (filledcircles, solidline, leftaxis)and
the KIE (open squares, right axis) are shown.
s
s
Ϫ1; for the majority of chemical reactions, AOBS falls near 1013
Ϫ1 (34). Recent characterization of ht-ADH variants mutated
in the cofactor-binding pocket revealed an enhanced Arrhenius
break, with values for AOBS as large as 1025 Ϫ1 (31). A detailed
s
characterization of these enzymes has led to the conclusion that
the unusual Arrhenius curves observed for ht-ADH variants
DISCUSSION
Activation Parameters and KIEs Indicate That a Transition reflect a reversible distribution of the protein conformational
Observed for ht-ADH at 30 °C Is Absent in ht-W87A and landscape into inactive, low energy states at low temperature.
ht-Y25A—Two kinetic signatures define the Arrhenius break The present data indicate that the Arrhenius break is com-
for the wild-type ht-ADH. First, an increase in the enthalpy of pletely absent for ht-W87A, as well as ht-Y25A, with values for
activation ⌬H‡ by about 7 kcal/mol is accompanied by an equiv- ⌬H‡ and T⌬S‡ throughout the temperature range approximat-
alent increase in T⌬S‡ below 30 °C. Fitting the kinetic data ing the values measured above 30 °C for wild-type. We con-
below this temperature to the Arrhenius equation (Eq. 1) clude that ht-Y25A and ht-W87A do not sample the low energy
results in an unusually large Arrhenius prefactor AOBS ϳ 1017 inactive states in the experimental temperature range.
14092 JOURNAL OF BIOLOGICAL CHEMISTRY
VOLUME 288•NUMBER 20•MAY 17, 2013

Long-range Protein Dynamics and Catalysis in ADH
A second signature of the Arrhenius break in the wild-type
enzyme is a transition in the temperature dependence of the
KIE. Above the 30 °C breakpoint, the wild-type enzyme exhibits
a nearly temperature-independent KIE: the difference between
the energy of activation for protium and deuterium, Ea(D) Ϫ
Ea(H), approaches zero (Table 2). By contrast, below the break-
point temperature, the KIE becomes temperature-dependent,
with increasing values for Ea(D) Ϫ Ea(H). The ht-ADH enzyme
is thus one of a growing number of enzymes catalyzing hydro-
gen transfer with KIEs that are nearly temperature-indepen-
dent under native conditions, but become temperature-depen-
dent upon the introduction of a generic perturbation, such as
mutation, protein surface modification, or as in this case, a
change in temperature (23). This phenomenon has increasingly
been attributed to a failure of perturbed enzymes to sample
conformational substates that can support ideal quantum tun-
neling conditions for hydrogen transfer at the active site. In the
cases of both ht-W87A and ht-Y25A, the absence of a detecta-
ble break toward more temperature-dependent KIEs at low
temperature indicates that conformational sampling remains
nearly optimal for hydrogen tunneling throughout the experi-
mental temperature range.
Both ht-ADH and ps-ADH Are Active Exclusively in the
Tetrameric Form, and Both the Cold Lability of ht-W87A and
the Thermolability of ps-ADH Result from Dissociation of the
Active Form of the Enzyme—The cold lability observed in
ht-W87A is concentration-dependent, with dilute enzyme
becoming inactivated more rapidly (Fig. 2A). These inactiva-
tion kinetics are consistent with dissociation of the tetrameric
form of the enzyme into inactive, lower order oligomers. Size
exclusion chromatography confirmed that the ht-W87A
tetramer dissociates to a much larger extent than the wild-type
enzyme (Fig. 4A), and activity assays indicated that the dimer
and monomer are both inactive (not shown). Further evidence
that the inactivation of ht-W87A at low temperature arises
from reversible dissociation of the tetramer comes from the
observation that enzymatic activity can be reconstituted from
fractions eluting as inactive monomeric enzyme upon incuba-
tion at 30 °C in the presence of cofactor under the same condi-
tions used to reconstitute cold-inactivated enzyme in Fig. 2 (not
shown). Moreover, when size exclusion chromatography was
performed at 4 °C in the presence of NADH and N-butylforma-
mide, which prevent cold inactivation, ht-W87A eluted almost
exclusively as a tetramer, and no activity was associated with
minor fractions eluting as monomer or dimer (Fig. 4B). Simi-
larly, when ps-ADH was subjected to size exclusion chromatog-
raphy (also at 4 °C), activity was only associated with the tetra-
meric form of the enzyme. Compared with ht-ADH, a greater
proportion of the psychrophilic enzyme elutes as dimer and
monomer.
The Catalytic Properties of the ht-W87A Variant More
Closely Resemble Those of ps-ADH Than ht-ADH, But the
Enzyme Is Not Suited for Low Temperature Catalysis Because of
Cold Lability—Extensive comparative studies of the kinetic
properties of homologous enzymes from organisms adapted to
different temperature niches have revealed several trends that
extend to ps-ADH and ht-ADH. Thermophilic enzymes are typ-
ically more stable, and exhibit larger values for ⌬H‡ and more neg-
ative values for ⌬S‡ relative to their mesophilic and psychrophilic
counterparts (22, 35–39). In keeping with these trends, ps-ADH
shows a smaller ⌬H‡ and more negative ⌬S‡ than ht-ADH (22).
Also, whereas the ps-ADH is extremely thermolabile, the ht-ADH
is stable to the extended incubation at 65 °C.
Fluorescence and CD Spectra Indicate No Major Changes
in Secondary Structure Upon Dissociation of the ht-ADH
Tetramer into Monomers—CD spectra collected for warm-
adapted (active) ht-W87A as well as cold-inactivated enzyme
were almost superimposable (Fig. 3B), indicating that the loss of
activity is not a result of significant unfolding of the enzyme
upon dissociation of the tetramer. Tryptophan fluorescence
spectra collected under conditions that reconstitute activity
indicated a time-dependent increase in fluorescence quenching
(Fig. 2D) that correlates with the restoration of enzymatic activ-
ity. Examination of the x-ray structure for ht-ADH indicates
that Trp-49 forms a ␲-stacking interaction with Phe-272 of an
adjacent subunit (Fig. 7B). The formation of this interaction
upon oligomerization could lead to a portion of the observed
protein fluorescence quenching (43). Fluorescence energy
transfer to the bound NADH cofactor was detected simultane-
These trends in kinetic properties are likely a consequence of
co-evolutionary constraints on protein flexibility and stability.
Although some studies indicate exceptions are possible (40),
enzymes that have evolved to function at elevated temperature
tend to be more rigid and less active at low temperature than their
psychrophilic counterparts (3, 25, 30, 41, 42). As discussed in the
Introduction, ht-ADH and ps-ADH can be included in this trend
(30), and importantly, a decrease in local flexibility below 30 °C
correlates with the Arrhenius break in ht-ADH (21).
ously at ␭
ϭ 410 nm, and a gradual blue-shift of this
emission
The ht-W87A mutation converts ht-ADH to an enzyme with
properties intermediate between ht-ADH (Ͼ30 °C) and
ps-ADH. The mutant retains at all temperatures the lower ⌬H‡
and more negative ⌬S‡ associated with the wild-type enzyme at
high temperature. Thus, ht-W87A activity falls off less steeply
with temperature, and ht-W87A and ps-ADH yield similar rate
constants at low temperature. However, whereas it remains
more resistant to heat inactivation than ps-ADH, ht-W87A
sacrifices thermostability relative to ht-ADH. Collectively, the
data suggest that ht-W87A is overall a more flexible catalyst
than the wild-type enzyme, failing to undergo rigidification in
the experimental temperature range, but paying for increased
flexibility via a decrease in stability.
signal with a similar time dependence as the quenching of tryp-
tophan fluorescence (Fig. 3A) suggests that the formation of
subunit interactions directly alters the active site microenvi-
ronment. Taken together, the spectroscopic data support the
conclusion that ht-W87A retains the overall subunit struc-
ture of the wild-type enzyme, and that minor structural changes
are responsible for converting the inactive dimer to the active
tetramer form.
Increased ps-A25Y Thermostability Is Due to Strengthened
Intersubunit Interactions, However the Enzyme Becomes Less
Suited for Catalysis at Low Temperatures Because of a Change
in the Rate-limiting Step—The observed relationship between
oligomeric integrity and activity in ht-W87A suggested that
MAY 17, 2013•VOLUME 288•NUMBER 20
JOURNAL OF BIOLOGICAL CHEMISTRY 14093

Long-range Protein Dynamics and Catalysis in ADH
dissociation of the wild-type ps-ADH lead to retention of the
tetramer in the ps-A25Y mutant (Fig. 5B).
Kinetic characterization of ps-A25Y at 30 °C shows this
enzyme is only modestly less active than ps-ADH, and similar
values for the KIE indicate that the chemical step remains rate-
determining (Table 1). In the elevated temperature range for
ps-A25Y (between 20 and 50 °C), activation parameters are also
similar to those measured for ps-ADH between 5 and 35 °C
(Table 2).
Below 20 °C, ps-A25Y and ps-ADH exhibit divergent behav-
ior. A breakpoint emerges in the ps-A25Y Arrhenius point, with
an apparent increase in the enthalpy of activation, reminiscent
of the ht-ADH behavior (Fig. 6A). However, examination of the
KIE on kcat (Dkcat) reveals a trend toward smaller values at lower
temperature, opposite what is seen for ps-ADH (22). As a result,
the “break” in the ps-A25Y Arrhenius plot likely reflects
increasing contributions to kcat from steps other than hydride
transfer; by contrast, the hydride transfer step appears to be
fully rate-limiting at all temperatures for ps-ADH. Although
ps-A25Y is much more stable than ps-ADH, the mutant is sig-
nificantly less active than ps-ADH at temperatures below 20 °C,
consistent with the hypothesis that the need to maintain flexi-
bility at reduced temperature places a limit on the stability of
psychrophilic enzymes.
The Mutant ht-Y25A Exhibits Similar Kinetic Characteristics
to ht-W87A, However, Its Quaternary Structure Is More Resist-
ant against Cold Dissociation—The ␲-stacking interaction
between Tyr-25 residues located on opposing subunits appears
to stabilize their association in the ps-A25Y variant and the
wild-type ht-ADH. Given these observations, the ht-Y25A var-
iant was expected to exhibit stability and kinetic properties
resembling those of ht-W87A. Similar to ht-W87A and the
wild-type ps-ADH, the ht-Y25A variant does not yield a signif-
icant Arrhenius break at 30 °C (Fig. 6B). The activation param-
eters across the experimental temperature range (10–50 °C)
resemble those observed for the wild-type ht-ADH above 30 °C
(Table 2). This observation provides further support for the
notion that Arrhenius behavior can be controlled at least in part
by intersubunit interactions. However, unlike ht-W87A, which
undergoes a significant loss of quaternary stability at low tem-
FIGURE 7. A, x-ray structure of ht-ADH and a model for the origin of the Arr-
henius break. Peptides undergoing a transition in flexibility at 30 °C are illus-
trated in color. Trp-87 (green, near the end of peptide #4), the active site zinc
(crimson sphere), and the alcohol analog trifluoroethanol (cyan) abut a con-
tiguous ␤ raft, which forms a contact with the same structural feature in the
adjacent subunit via tyrosine 25 (red) near the C-terminal end of peptide #1.
At right, three conformational landscapes of the wild-type ht-ADH tetramer
arerepresentedinschematicform. Atelevatedtemperature(circles), aflexible
tetramer exhibits weakly temperature-dependent KIEs. Below a transition
temperature of 30 °C (dotted line), strong intersubunit interactions lead to a
more rigid conformational landscape (squares), exhibiting temperature-de-
pendentKIEs. Thesesubstatesareinequilibriumwithanadditionallandscape
involving low energy, inactive forms of enzyme. B, x-ray structure of ht-ADH.
One dimer is colored black and the monomers comprising the other, blue and
yellow to emphasize interactions at the dimer-dimer interface where Tyr-25 is
shown in green. The interaction between Trp-49 and Phe-272 is highlighted
by a black circle. Residues from the subunit on the left are colored blue, those
from the right subunit are yellow. On the right side of panel B, the image is
rotated 90°. A ␤ sheet interaction between the subunits is seen at the center
of the image, flanked by the Trp-49/Phe-272 interaction.
thermolability in ps-ADH may arise from weakened subunit perature (Fig. 4A), the ht-Y25A tetramer shows a more modest
interactions. Homology modeling to ht-ADH indicates a ps- propensity to dissociate in a concentration-dependent manner
ADH structure nearly superimposable with that of the thermo- (not shown). The contrasting stabilities of ht-W87A, ps-ADH,
philic enzyme, which we regard as a dimer of dimers (44). Sub- and ht-Y25A at the reduced temperatures indicate that an
units interact strongly at a “dimer interface” in the dimeric increase in local flexibility at the active site (expressed as a loss
eukaryotic alcohol dehydrogenases (45); however, the interac- of the Arrhenius break) does not require, a priori, a large desta-
tions that join two such dimers at what we term the “dimer- bilization of intersubunit interactions.
dimer interface” are relatively sparse. Notably, a ␲-stacking
Reciprocal Effects of Mutation to ht-ADH and ps-ADH Impli-
interaction at the tetramer interface between Tyr-25 and its cate a Network of Communication between the Active Site of the
counterpart in an adjacent subunit is lost in ps-ADH, as the Enzyme and the Subunit Interface—Because the active site of
psychrophilic enzyme contains alanine at position 25 (Fig. 7B). the tetrameric bacterial alcohol dehydrogenases does not lie at
We hypothesized that restoration of this interaction in the psy- a subunit interface, the exclusive activity of the tetrameric state
chrophilic enzyme would confer significant stabilization. Upon must reflect subtle structural changes that propagate from the
incubation at 50 °C, ps-A25Y is seen to be dramatically more dimer-dimer interface to the active site of the enzyme upon
stable than the wild-type enzyme, retaining nearly 75% activity formation of intersubunit interactions. The ps-A25Y mutant
after 20 min, at which time ps-ADH is completely inactivated indicates that the effects of strengthened subunit interactions
(Fig. 5A). Analysis by size exclusion chromatography further can propagate to the active site in a deleterious manner, by the
shows that the experimental conditions promoting extensive introduction of a new rate-limiting step. Reciprocally, the
14094 JOURNAL OF BIOLOGICAL CHEMISTRY
VOLUME 288•NUMBER 20•MAY 17, 2013

Long-range Protein Dynamics and Catalysis in ADH
ht-Y25A mutant with a weakened subunit interaction appears the enormously inflated Arrhenius pre-factors measured for a
to prevent the enzyme from falling into a subset of low energy, series of ht-ADH mutants at reduced temperatures (31). The
catalytically inactive conformers that has been invoked previ- molecular basis for these Arrhenius breakpoints has been elu-
ously to account for inflated Arrhenius prefactors in other vari- sive, although a clue may lie in the observation that the transi-
ants of ht-ADH (31). In the case of ht-W87A, the introduction tion from low to high temperature conformational equilibria in
of a packing defect at the active site results in a significant weak- ht-ADH requires both a large positive ⌬H°, and a large positive
ening of subunit interactions at low temperature, indicating ⌬S° (31), as have been measured for the dissociation of protein
that changes in active site geometry likewise propagate to inter- subunits (33).
actions at the subunit interface. Thus, the aggregate data indi-
The results presented herein, taken together with extensive
cate bi-directional communication between the active site and previous characterization of the ht-ADH support a model for
the dimer-dimer interface in the bacterial alcohol dehydroge- the observed kinetic properties, presented in Fig. 7A. At ele-
nases that modulates the conformational flexibility required for vated temperatures, ht-ADH adopts a subset of relatively flex-
efficient hydrogen tunneling. The five regions of protein (iden- ible conformational substates that support efficient tunneling.
tified within peptides 1, 2–4, and 7) that have previously been Below 30 °C, the enzyme adopts more rigid conformational
shown by way of hydrogen-deuterium exchange experiments to substates that still support catalysis, however, with altered tun-
rigidify below 30 °C in ht-ADH directly connect Tyr-25 to neling parameters. The ability of the enzyme to adopt these
Trp-87 via a contiguous ␤ sheet (Fig. 7A), presenting a pathway conformers depends upon intersubunit interactions at position
by which small perturbations at either position may exert struc- 25. As the temperature decreases, the rigidified subunits can
tural and dynamical changes at the other position (21).
enter into a second, lower energy interaction with one another
The functional link between structural features at the active that forces the enzyme into a catalytically inactive conforma-
site and the subunit interface is reminiscent of allostery, tional space via K2. As this occurs, the rate of catalysis decreases
wherein the role of ligand binding as the allosteric signal has rapidly with temperature, resulting in inflated Arrhenius
been replaced by a change in temperature. Furthermore, the parameters (31). We propose that the packing defect caused by
lack of major rearrangements in the secondary structure of the the ht-W87A mutation introduces flexibility into the protein
protein as a function of temperature (31), or upon disruption of that overwhelms the stabilizing effect of interactions at Tyr-25.
intersubunit interactions (W87A) indicates that conforma- This prevents the enzyme from rigidifying at reduced temper-
tional sampling may carry the “allosteric” signal in place of a ature, so that it is able to sample a conformational subspace
large change in the average conformation of the protein. Trans- compatible with efficient tunneling at all temperatures (K1 in
mission of an allosteric signal via dynamic fluctuations, and in the model approaches zero). In complementary fashion, the
the absence of detectable conformational change, has been pro- absence of a ␲-stacking interaction at the subunit interface in
posed for other proteins recently and has been discussed in ht-Y25A likewise prevents the enzyme from rigidifying at
detail (46). The present data show that prokaryotic alcohol reduced temperature (K1 in the model again approaches zero).
dehydrogenases have evolved to tune the long-range interac- Because ht-Y25A remains more cold-stable than the ht-W87A
tions between the subunit interface and the active site in sup- mutation, the latter appears to weaken additional intersubunit
port of optimal catalysis within their respective temperature interactions, possibly through a process of mild deformation of
niches.
the protein about the resulting cavity in the active site. This
Convex Arrhenius Curves Observed for ht-ADH and ps-A25Y process can be prevented or reversed by the binding of NAD
Appear to Arise from a Temperature-dependent Change in Sub- cofactor, NADH, or a combination of NADH and the aldehyde
unit Interactions—Although the Arrhenius break for ps-A25Y analog inhibitor, N-butylformamide. Cofactor binding to alco-
arises from kinetic complexity, it contributes to a pattern in hol dehydrogenase is known to induce a conformational
which stronger subunit interactions tend to be associated with change. This could offset the extreme destabilizing effect of the
convex Arrhenius curves. Weakened subunit interactions in ht-W87A by introducing intersubunit interactions that are
ps-ADH and ht-W87A, or the direct removal of an intersubunit unavailable in the conformational substates adopted in the
interaction in the case of ht-Y25A, are all associated with the absence of cofactor. In the case of ps-A25Y, an increase in sub-
absence of an Arrhenius break. A similar relationship has been unit interactions analogous to the process represented by K2 is
observed recently for a thermophilic, dimeric dihydrofolate suggested to take place below 20 °C. Because the enzyme
reductase (27). In those experiments, the wild-type enzyme remains catalytically active, the consequence of these interac-
showed a modest breakpoint at 25 °C below which both ⌬H‡ tions must be to slow down a step other than hydride transfer
and the temperature dependence of the KIE increased, as seen (possibly product release). The observation of Arrhenius breaks
with ht-ADH. The introduction of a mutation at the subunit in multimeric enzymes may, thus, be a consequence of a tem-
interface resulted in an active monomeric enzyme, and abro- perature-dependent change in the strength of subunit interac-
gated the break. However, unlike the ht-ADH and more similar tions, which imposes restrictions on the conformational land-
to ps-A25Y, the break in the Arrhenius plot was found to arise scape, and thereby distorts the active site away from its optimal
from a change in the rate-determining step. Convex Arrhenius geometry. The ability of subunit interactions to regulate
curves in which the hydrogen transfer step remains rate-deter- enzyme activity in a reversible and temperature-dependent
mining at all temperatures have been proposed to arise from manner may also serve a previously unrecognized biological
conformational pre-equilibria (28, 47, 48), and we have shown role, presenting a strategy by which organisms can respond
that impaired conformational sampling can be used to model rapidly to temperature changes at the level of enzyme activ-
MAY 17, 2013•VOLUME 288•NUMBER 20
JOURNAL OF BIOLOGICAL CHEMISTRY 14095

Long-range Protein Dynamics and Catalysis in ADH
Soc. 134, 1738–1745
ity without the need for cycles of protein degradation and
replenishment.
16. Glowacki, D. R., Harvey, J. N., and Mulholland, A. J. (2012) Exciton dy-
namics. Electrons take an unexpected turn. Nat. Chem. 4, 169–176
17. Tang, K. E., and Dill, K. A. (1998) Native protein fluctuations. The confor-
mational-motion temperature and the inverse correlation of protein flex-
ibility with protein stability. J. Biomol. Struct. Dyn. 16, 397–411
Conclusion—An evolutionary trade-off among stability, local
flexibility, and catalytic activity is evident in ps-ADH, ht-ADH,
and their respective mutant variants. The data support a model
that involves communication between the dimer-dimer inter- 18. Fields, P. A., and Somero, G. N. (1998) Hot spots in cold adaptation.
Localized increases in conformational flexibility in lactate dehydrogenase
face and the enzyme active site in prokaryotic alcohol dehydro-
A4 orthologs of Antarctic notothenioid fishes. Proc. Natl. Acad. Sci. U.S.A.
genases. The two enzyme variants for which a catalytically del-
95, 11476–11481
eterious rigidification appears to occur, ht-ADH and ps-A25Y,
19. Vihinen, M. (1987) Relationship of protein flexibility to thermostability.
share the feature of relatively strong intersubunit interactions.
In both cases, subunit interactions propagate to the active site,
with ht-ADH exhibiting impairment of hydride transfer, and
ps-ADH a change in rate-limiting step. Reciprocally, attenuated
subunit interactions in ps-ADH, ht-Y25A, and ht-W87A per-
mit hydrogen tunneling to proceed unimpaired at reduced tem-
perature. These observations provide new insight into the
molecular origins of unusual Arrhenius curves seen in ht-ADH
and other oligomeric proteins. A more detailed understanding
of the molecular basis for subtle, correlated changes between
catalysis and protein flexibility may yield novel targets for the
disruption of protein-protein interactions and the inhibition of
enzymatic activity.
Protein Eng. 1, 477–480
20. Varley, P. G., and Pain, R. H. (1991) Relation between stability, dynamics
and enzyme activity in 3-phosphoglycerate kinases from yeast and Ther-
mus thermophilus. J. Mol. Biol. 220, 531–538
21. Liang, Z. X., Lee, T., Resing, K. A., Ahn, N. G., and Klinman, J. P. (2004)
Thermal-activated protein mobility and its correlation with catalysis in
thermophilic alcohol dehydrogenase. Proc. Natl. Acad. Sci. U.S.A. 101,
9556–9561
22. Liang, Z. X., Tsigos, I., Bouriotis, V., and Klinman, J. P. (2004) Impact of
protein flexibility on hydride-transfer parameters in thermophilic and
psychrophilic alcohol dehydrogenases. J. Am. Chem. Soc. 126, 9500–9501
23. Nagel, Z. D., and Klinman, J. P. (2009) A 21st century revisionist’s view at
a turning point in enzymology. Nat. Chem. Biol. 5, 543–550
24. Heyes, D. J., Levy, C., Sakuma, M., Robertson, D. L., and Scrutton, N. S.
(2011) A twin-track approach has optimized proton and hydride transfer
by dynamically coupled tunneling during the evolution of protochloro-
phyllide oxidoreductase. J. Biol. Chem. 286, 11849–11854
REFERENCES
25. Oyeyemi, O. A., Sours, K. M., Lee, T., Kohen, A., Resing, K. A., Ahn, N. G.,
and Klinman, J. P. (2011) Comparative hydrogen-deuterium exchange for
a mesophilic vs thermophilic dihydrofolate reductase at 25 °C. Identifica-
tion of a single active site region with enhanced flexibility in the mesoph-
ilic protein. Biochemistry 50, 8251–8260
1. Somero, G. N. (1978) Temperature adaptation of enzymes. Biological op-
timization through structure-function comrpomises. Annu. Rev. Ecol.
Syst. 9, 1–29
2. Somero, G. N. (1995) Proteins and temperature. Annu. Rev. Physiol. 57,
43–68
26. Anandarajah, K., Schowen, K. B., and Schowen, R. L. (2008) Hydrogen
tunneling in glucose oxidation by the Archaeon Thermoplasma acidophi-
lum. Int. J. Res. Phys. Chem. Chem. Phys. 222, 1333–1347
3. Za´vodszky, P., Kardos, J., Svingor, A., and Petsko, G. A. (1998) Adjustment
of conformational flexibility is a key event in the thermal adaptation of
proteins. Proc. Natl. Acad. Sci. U.S.A. 95, 7406–7411
27. Loveridge, E. J., Rodriguez, R. J., Swanwick, R. S., and Allemann, R. K.
(2009) Effect of dimerization on the stability and catalytic activity of dihy-
drofolate reductase from the hyperthermophile Thermotoga maritima.
Biochemistry 48, 5922–5933
4. Feller, G. (2010) Protein stability and enzyme activity at extreme biological
temperatures. J. Phys. Condes. Matter 22, 323101–323117
5. Kohen, A., Cannio, R., Bartolucci, S., and Klinman, J. P. (1999) Enzyme
dynamics and hydrogen tunnelling in a thermophilic alcohol dehydrogen-
ase. Nature 399, 496–499
28. Massey, V., Curti, B., and Ganther, H. (1966) A temperature-dependent
conformational change in D-amino acid oxidase and its effect on catalysis.
J. Biol. Chem. 241, 2347–2357
6. Benkovic, S. J., and Hammes-Schiffer, S. (2003) A perspective on enzyme
catalysis. Science 301, 1196–1202
29. Tsigos, I., Velonia, K., Smonou, I., and Bouriotis, V. (1998) Purification and
characterization of an alcohol dehydrogenase from the Antarctic psych-
rophile Moraxella sp. TAE123. Eur. J. Biochem. 254, 356–362
30. Liang, Z. X., Tsigos, I., Lee, T., Bouriotis, V., Resing, K. A., Ahn, N. G., and
Klinman, J. P. (2004) Evidence for increased local flexibility in psychro-
philic alcohol dehydrogenase relative to its thermophilic homologue. Bio-
chemistry 43, 14676–14683
7. Hammes-Schiffer, S., and Benkovic, S. J. (2006) Relating protein motion to
catalysis. Annu. Rev. Biochem. 75, 519–541
8. Fan, F., and Gadda, G. (2007) An internal equilibrium preorganizes the
enzyme-substrate complex for hydride tunneling in choline oxidase. Bio-
chemistry 46, 6402–6408
9. Henzler-Wildman, K., and Kern, D. (2007) Dynamic personalities of pro-
teins. Nature 450, 964–972
31. Nagel, Z. D., Dong, M., Bahnson, B. J., and Klinman, J. P. (2011) Impaired
protein conformational landscapes as revealed in anomalous Arrhenius
prefactors. Proc. Natl. Acad. Sci. U.S.A. 108, 10520–10525
10. Benkovic, S. J., Hammes, G. G., and Hammes-Schiffer, S. (2008) Free-
energy landscape of enzyme catalysis. Biochemistry 47, 3317–3321
11. Klinman, J. P. (2009) An integrated model for enzyme catalysis emerges
from studies of hydrogen tunneling. Chem. Phys. Lett. 471, 179–193
12. Pudney, C. R., Hay, S., Levy, C., Pang, J., Sutcliffe, M. J., Leys, D., and
Scrutton, N. S. (2009) Evidence to support the hypothesis that promoting
vibrations enhance the rate of an enzyme catalyzed H-tunneling reaction.
J. Am. Chem. Soc. 131, 17072–17073
32. Nagel, Z. D., Meadows, C. W., Dong, M., Bahnson, B. J., and Klinman, J. P.
(2012) Active site hydrophobic residues impact hydrogen tunneling dif-
ferently in a thermophilic alcohol dehydrogenase at optimal versus non-
optimal temperatures. Biochemistry 51, 4147–4156
33. Weber, G. (1995) Revisited, enthalpy of association of protein subunits. J.
Phys. Chem. 99, 1052–1059
13. Bhabha, G., Lee, J., Ekiert, D. C., Gam, J., Wilson, I. A., Dyson, H. J.,
Benkovic, S. J., and Wright, P. E. (2011) A dynamic knockout reveals that 34. Forst, W. (1973) Theory of unimolecular reactions, Academic Press, New
conformational fluctuations influence the chemical step of enzyme catal-
ysis. Science 332, 234–238
York
35. Lonhienne, T., Gerday, C., and Feller, G. (2000) Psychrophilic enzymes.
Revisiting the thermodynamic parameters of activation may explain local
flexibility. Biochim. Biophys. Acta 1543, 1–10
14. Schramm, V. L. (2011) Enzymatic transition states, transition-state ana-
logs, dynamics, thermodynamics, and lifetimes. Annu. Rev. Biochem. 80,
703–732
36. D’Amico, S., Collins, T., Marx, J. C., Feller, G., and Gerday, C. (2006)
Psychrophilic microorganisms. Challenges for life. EMBO Rep. 7, 385–389
37. Feller, G. (2007) Life at low temperatures. Is disorder the driving force?
Extremophiles 11, 211–216
15. Stojkovic´, V., Perissinotti, L. L., Willmer, D., Benkovic, S. J., and Kohen, A.
(2012) Effects of the donor-acceptor distance and dynamics on hydride
tunneling in the dihydrofolate reductase catalyzed reaction. J. Am. Chem.
14096 JOURNAL OF BIOLOGICAL CHEMISTRY
VOLUME 288•NUMBER 20•MAY 17, 2013

Long-range Protein Dynamics and Catalysis in ADH
38. Nagel, Z. D., and Klinman, J. P. (2006) Tunneling and dynamics in enzy-
contribute to the low quantum yield of a conserved, buried tryptophan.
matic hydride transfer. Chem. Rev. 106, 3095–3118
Proteins 40, 112–125
39. Lam, S. Y., Yeung, R. C. Y., Yu, T. H., Sze, K. H., and Wong, K. B. (2011) 44. Ceccarelli, C., Liang, Z. X., Strickler, M., Prehna, G., Goldstein, B. M.,
A rigidifying salt-bridge favors the activity of thermophilic enzyme at
high temperatures at the expense of low-temperature activity. PLoS
Biol. 9, 1–8
Klinman, J. P., and Bahnson, B. J. (2004) Crystal structure and amide H/D
exchange of binary complexes of alcohol dehydrogenase from Bacillus
stearothermophilus. Insight into thermostability and cofactor binding.
Biochemistry 43, 5266–5277
40. Merkley, E. D., Parson, W. W., and Daggett, V. (2010) Temperature de-
pendence of the flexibility of thermophilic and mesophilic flavoenzymes
of the nitroreductase fold. Protein Eng. Des. Sel. 23, 327–336
41. Kohen, A., and Klinman, J. P. (2000) Protein flexibility correlates with
45. Eklund, H., Samma, J. P., Walle´n, L., Bra¨nde´n, C. I., Akeson, A., and Jones,
T. A. (1981) Structure of a triclinic ternary complex of horse liver alcohol
dehydrogenase at 2.9-Å resolution. J. Mol. Biol. 146, 561–587
degree of hydrogen tunneling in thermophilic and mesophilic alcohol de- 46. Tzeng, S. R., and Kalodimos, C. G. (2011) Protein dynamics and allostery.
hydrogenases. J. Am. Chem. Soc. 122, 10738–10739
42. Wolf-Watz, M., Thai, V., Henzler-Wildman, K., Hadjipavlou, G., Eisen- 47. Truhlar, D., and Kohen, A. (2001) Convex Arrhenius plots and their in-
messer, E. Z., and Kern, D. (2004) Linkage between dynamics and catalysis terpretation. Proc. Natl. Acad. Sci. U.S.A. 98, 848–851
An NMR view. Curr. Opin. Struct. Biol. 21, 62–67
in a thermophilic-mesophilic enzyme pair. Nat. Struct. Mol. Biol. 11, 48. Limbach, H. H., Lopez, J. M., and Kohen, A. (2006) Arrhenius curves of
945–949
hydrogen transfers. Tunnel effects, isotope effects and effects of pre-equi-
libria. Philos. Trans. R. Soc. Lond. B Biol. Sci. 361, 1399–1415
43. Nanda, V., and Brand, L. (2000) Aromatic interactions in homeodomains
MAY 17, 2013•VOLUME 288•NUMBER 20
JOURNAL OF BIOLOGICAL CHEMISTRY 14097