Enzyme architecture: Remarkably similar transition states for triosephosphate isomerase-catalyzed reactions of the whole substrate and the substrate in pieces

Article abstract of DOI:10.1021/ja501103b

Values of (k_cat/K_m)_GAP for triosephosphate isomerase-catalyzed reactions of (R)-glyceraldehyde 3-phosphate and k _catK_HPiK_GA for reactions of the substrate pieces glycolaldehyde and HPO₃^2- have been determined for wild-type and the following TIM mutants: I172V, I172A, L232A, and P168A (TIM from Trypanosoma brucei brucei); a 208-TGAG for 208-YGGS loop 7 replacement mutant (L7RM, TIM from chicken muscle); and, Y208T, Y208S, Y208A, Y208F and S211A (yeast TIM). A superb linear logarithmic correlation, with slope of 1.04 ± 0.03, is observed between the kinetic parameters for wild-type and most mutant enzymes, with positive deviations for L232A and L7RM. The unit slope shows that most mutations result in an identical change in the activation barriers for the catalyzed reactions of whole substrate and substrate pieces, so that the two transition states are stabilized by similar interactions with the protein catalyst. This is consistent with a role for dianions as active spectators, which hold TIM in a catalytically active caged form.

Full text of DOI:10.1021/ja501103b

Communication
pubs.acs.org/JACS
Open Access on 03/03/2015
Enzyme Architecture: Remarkably Similar Transition States for
Triosephosphate Isomerase-Catalyzed Reactions of the Whole
Substrate and the Substrate in Pieces
Xiang Zhai, Tina L. Amyes, and John P. Richard*
Department of Chemistry, University at Buffalo, State University of New York, Buffalo, New York 14260, United States
S
* Supporting Information
binding energy of the nonreacting phosphodianion in enzyme
activation, observed here and for other enzymatic reactions,7,9−13
ABSTRACT: Values of (kcat/Km)GAP for triosephosphate
isomerase-catalyzed reactions of (R)-glyceraldehyde 3-
is a critical difference between reactions catalyzed by
enzymes14,15 and catalysis by small molecules.16
phosphate and kcat/KHP KGA for reactions of the substrate
i
2−
pieces glycolaldehyde and HPO3 have been determined
for wild-type and the following TIM mutants: I172V,
I172A, L232A, and P168A (TIM from Trypanosoma brucei
brucei); a 208-TGAG for 208-YGGS loop 7 replacement
mutant (L7RM, TIM from chicken muscle); and, Y208T,
Y208S, Y208A, Y208F and S211A (yeast TIM). A superb
linear logarithmic correlation, with slope of 1.04 0.03, is
observed between the kinetic parameters for wild-type and
most mutant enzymes, with positive deviations for L232A
and L7RM. The unit slope shows that most mutations
result in an identical change in the activation barriers for
the catalyzed reactions of whole substrate and substrate
pieces, so that the two transition states are stabilized by
similar interactions with the protein catalyst. This is
consistent with a role for dianions as active spectators,
which hold TIM in a catalytically active caged form.
We report here a linear free-energy relationship, with slope of
1.0, between the kinetic parameters for the reactions of GAP and
the pieces GA and HPi catalyzed by wild-type and structural
mutants of TIM. This correlation shows that the transition states
for the two reactions are stabilized by similar interactions with
the protein catalyst and that the reactions proceed through
similar transition states.
The I172 V,17 I172A,17 L232A,17,18 and P168A19,20 mutants of
TIM from Trypanosoma brucei brucei (TbbTIM) and the 208-
TGAG for 208-YGGS loop 7 replacement mutant (L7RM)19,21
of TIM from chicken muscle (cTIM) were examined in earlier
work. The Y208T, Y208S, Y208A, Y208F, and S211A mutants of
yeast TIM (yTIM) were prepared, purified, and characterized as
described in the Supporting Information (SI). The positions of
these amino acid residues are shown in Figure 1, for the complex
between DHAP and TIM from yeast.22 The kinetic parameters
determined for the Y208F and S211A enzyme-catalyzed
riosephosphate isomerase (TIM) catalyzes the stereo-
T
specific and reversible conversion of dihydroxyacetone
phosphate (DHAP) to (R)-glyceraldehyde 3-phosphate (GAP),
by a proton transfer mechanism through enzyme-bound cis-
enediolate intermediates (Scheme 1).1−4 The 12 kcal/mol
Scheme 1
stabilization of the transition state by interactions between TIM
and the remote phosphodianion group of substrate accounts for
∼80% of total transition-state stabilization.5 These binding
interactions not only anchor substrate to the enzyme active site
but also play a role in activating TIM for deprotonation of bound
carbon acid, as shown by the large effect of the deletion of a
phosphodianion gripper loop on kcat for TIM-catalyzed isomer-
ization of GAP6 and by the large activation by exogenous
phosphite dianion (HPi) of TIM-catalyzed deprotonation of
glycolaldehyde (GA)7 and isomerization of [1-13C]-glycolalde-
hyde ([1-13C]-GA) to [2-13C]-GA.8 This utilization of the
Figure 1. A model, from an X-ray crystal structure, of the complex
between TIM from yeast and DHAP (PDB entry 1NEY) showing the
amino acids mutated in this work. The side chains of H95, K12, and N10
play key roles in catalysis of the isomerization reaction.2,3Small
differences in the numbering of the amino acids at TIM from the
different sources used in these studies are noted, where appropriate:
(cTIM or yTIM and TbbTIM): Pro 166 and 168, I170 and 172, L230
and 232.
Received: January 31, 2014
Published: March 3, 2014
© 2014 American Chemical Society
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Communication
isomerization of GAP are in good agreement with the published
values of Sampson and Knowles.23
The second-order rate constants (kcat/Km)obs for TIM-
catalyzed reactions of [1-13C]-GA in D2O and the fractional
yields, (f P)E, of products [2-13C]-GA, [2-13C, 2-2H]-GA, and
[1-13C, 2-2H]-GA (Scheme 2) were determined by monitoring
in a decrease in the kinetic parameters for the TIM-catalyzed
reactions of whole substrates and substrate pieces. This reflects
the destabilization of the respective transition states for the
mutant enzyme-catalyzed reactions, which result from subtle
effects of these mutations on enzyme structure.
Figure 2 shows the dependence on [HPO32−] of kcat/Km for
the reactions of [1-13C]-GA catalyzed by Y208 and S211 mutants
Scheme 2
the disappearance of [1-13C]-GA and the formation of products
by 1H NMR.8 Tables S1−S6 report kinetic and product data for
the reactions catalyzed by wild-type yTIM and by the Y208T,
Y208S, Y208A, Y208F, and S211A mutant enzymes. A significant
yield of [1-13C, 2,2-di-2H]-GA is sometimes observed from the
TIM-catalyzed reactions of [1-13C]-GA,8,17,19 where the
dideuterium-labeled product is formed by a nonspecific
protein-catalyzed reaction.8,24,25 This is a minor product (5−
10% yield) of the Y208T, Y208S Y208A, S211A mutant enzyme-
catalyzed reactions of [1-13C]-GA in the presence of HPi and the
major product (30 − 40% yield) of the reactions catalyzed by the
severely crippled Y208F mutant. When the total yield of [2-13C]-
GA, [2-13C, 2-2H]-GA and [1-13C, 2-2H]-GA [∑(f P)E, eq 1] is
less than quantitative, the second-order rate constants (kcat/Km)
for reactions at the enzyme active site (Scheme 2) were
determined from the observed second-order rate constant (kcat/
Km)obs and the sum of the yields of the three products [∑(f P)E],
using eq 1.17,19
Figure 2. Dependence of kcat/Km for the TIM-catalyzed turnover of the
free carbonyl form of [1-13C]-GA in D2O on [HPO32−] at pD 7.0, 25 °C,
and an ionic strength of 0.10 (NaCl). (A) Reactions catalyzed by the
Y208T, Y208S, Y208A, and S211A mutants of yTIM. (B) Reactions
catalyzed by the Y208F mutant.
Scheme 3
⎡
⎤
kcat
Km
kcat
=
(f )
E
P
⎢
⎣
⎥
⎦
∑
K
m
(1)
obs
Figure 1 shows the active site for a complex between yTIM and
DHAP.22 I172 and L232 from TbbTIM function in a
hydrophobic clamp.26 The basicity of the side chain of E167,
which reacts to deprotonate the carbon acid substrate, is
enhanced by interactions with the side chain of I172.27 Steric
interactions between the side chains of P168 and loop 7, induced
by the ligand-gated conformational change,1,2 force the E167
(Tbb numbering) carboxylate toward the carbon acid sub-
strate.20,28 This conformational change is enabled by formation
of hydrogen bonds between the side-chain hydroxyls of Y208 and
S211 from loop 7, respectively, with the backbone amide
nitrogen of A176 and G173 from loop 6 and a hydrogen bond
between the carbonyl oxygen of A169 and the γ-O of S211. The
I172 V,17 I172A,17 L232A,17,18 and P168A19,20 mutations of
TbbTIM, the 208-TGAG for 208-YGGS loop 7 replacement
mutation (L7RM) of cTIM,19,21 and the Y208 and S211
mutations of yTIM each modify the enzyme structure in the
region of the active site. Most of these structural mutations result
of TIM. The third-order rate constants kcat/KGAKHP (Scheme 3)
i
reported in Table S7 were determined as the slopes of linear plots
of kcat/Km against [HPO32−] for the reactions catalyzed by the
Y208F, Y208A, and S211A mutants or as the slopes of the linear
portions of the plots of data at low [HPO32−] for the reactions
catalyzed by Y208S and Y208T mutants. Table S7 also reports
the kinetic parameters kcat and (kcat/Km)GAP for wild-type and
mutant TIM-catalyzed isomerization of GAP.
The activation barrier for conversion of TIM and GAP to the
transition state for enzyme-catalyzed isomerization of GAP
[ΔG⧧GAP] is defined by the second-order rate constant (kcat/
Km)GAP, while the barrier to formation of the transition state for
the TIM-catalyzed reaction of the substrate pieces GA + HPi
[ΔG⧧GA+HP ] is defined by the third-order rate constant kcat/
i
KHP KGA (Scheme 4). Figure 3 presents the linear logarithmic free
i
energy correlation between the activation barriers for wild-type
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Journal of the American Chemical Society
Communication
dominant inactive open form Eo to an active loop-closed form Ec,
which is reflected by a ∼25-fold increase in concentration of the
active enzyme (Scheme 5).8,17,18 We are uncertain of the
Scheme 4
Scheme 5
explanation for the smaller positive deviation from this linear
correlation of the kinetic parameters for the complex loop 7
replacement mutation of cTIM (Figure 3).
Wolfenden proposed that optimal enzymatic catalysis is
sometimes obtained when the substrate is trapped in a protein
cage, at an active site that provides for maximum enzyme-ligand
contacts.32 This catalytic cage is created when substrate or
transition-state analogs bind to TIM, by the closure of a flexible
gripper loop over the ligand phosphodianion22,26,33−35 and
presumably over the HPi activator. The linear correlation from
Figure 3 provides evidence that dianions play a role as active
spectators in creating the caged catalytic complex. The dianions
are active and serve as a type of glue to hold TIM in a high-energy
closed active form (Ec, Scheme 5), but in a different sense they
are spectators, which provide no direct stabilization of the
transition state for the unactivated reaction19 and which do not
affect the transition-state structure.
The results of previous studies on TIM and the decarbox-
ylation catalyzed by orotidine 5′-monophophate decarboxylase
show that each enzyme is composed of a catalytic domain, which
is competent to carry out the catalyzed reaction, and a dianion
binding domain, where strong binding interactions with dianions
are utilized to activate these enzymes for catalysis.36−38 The
present results provide evidence that interactions between TIM
and a spectator dianion lock the enzyme into an active
conformation that is otherwise present at low concentrations
(Scheme 5). Part of the dianion binding energy is used to drive
desolvation of the carboxylate side-chain of Glu-165 (cTIM and
yTIM) or Glu-167 (TbbTIM), which enhances the side-chain
basicity toward deprotonation of carbon.27 The dianion binding
interactions might also be utilized to organize/position the
catalytic side chains at the enzymatic transition state, consistent
with the notion that there is a high degree of “preorganization” of
these side chains at the active sites of efficient enzyme
catalysts.39−41
Figure 3. Linear free energy relationship, with slope 1.04
between the second-order rate constant [log (kcat/Km)GAP] for wild-type
and mutant TIM-catalyzed isomerization of GAP and the corresponding
0.03,
third-order rate constant [log (kcat/KHPKGA)] for the enzyme-catalyzed
i
reactions of the substrate pieces GA and HPi. Key: Green, TbbTIM;
black, cTIM; red, yTIM.
and mutant-TIM-catalyzed reactions of the whole substrate GAP
and the substrate pieces GA + HPi. This correlation with slope of
1.04 0.03 (95% confidence interval; 0.97−1.11) shows that
most of these mutations, which alter the interactions of ligands
with flexible loops 6 and 7 (Figure 1),29 result in the same
destabilization of the transition states for the catalyzed reactions
of the whole substrate and substrate pieces. We conclude that
these transition states show strikingly similar interactions with
TIM and that by this criteria are remarkably similar. The slope of
1.0 for Figure 3 reflects the constant difference in activation
barriers for the reaction of whole substrate and the substrate in
pieces: ΔΔG⧧ = 6.6 0.3 kcal/mol (Scheme 4). This difference
is the entropic advantage to the binding of the transition state for
the reaction of the whole substrate compared with the transition
state for reaction of the two pieces.30 This result is in good
agreement with other estimates of the catalytic advantage
obtained from covalent attachment of the reactants in a
bimolecular reaction.31
ASSOCIATED CONTENT
* Supporting Information
■
S
Experimental procedures for the preparation and purification of
Y208T, Y208S, Y208A, Y208F and S211A mutants of yTIM;
procedures for determination of the product yields and kinetic
parameters for wild-type and mutant yTIM-catalyzed reactions of
[1-13C]-GA; Tables S1−S6, second-order rate constants and
fractional product yields for the reaction of [1-13C]-GA catalyzed
by wild-type yTIM (Table S1); and, by Y208T, Y208S, Y208A,
Y208F and S211A mutants (Tables S2−S6, respectively); Table
S7, kinetic parameters for wild-type and mutant yTIM-catalyzed
reactions of GAP and of GA and HPi. This material is available
free of charge via the Internet at http://pubs.acs.org.
The large positive deviation of the point for L232A mutant
TbbTIM from the correlation in Figure 3 reflects the 25-fold
larger value of kcat/KHP KGA for the L232A mutant compared to
i
wild-type TIM.17,18 We have proposed that the L232A mutation
results in a 25-fold increase in the equilibrium constant Kc for the
thermodynamically unfavorable conversion of TIM from the
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(34) Davenport, R. C.; Bash, P. A.; Seaton, B. A.; Karplus, M.; Petsko,
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AUTHOR INFORMATION
■
Corresponding Author
jrichard@buffalo.edu
Notes
(37) Goryanova, B.; Goldman, L. M.; Amyes, T. L.; Gerlt, J. A.;
Richard, J. P. Biochemistry 2013, 52, 7500.
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS
■
This work was supported by Grant GM39754 from the National
Institutes of Health.
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