Journal of the American Chemical Society
Article
binds the carboxylate of 2-PGA, may alter the ability of the
enzyme to modulate the negative charge at the C1 of substrate
and, hence, the pKa of the reactive carbon at C2. Two
hydrophobic networks extend from the backbone of Leu343
out to the solvent interface, and we propose that in the native
enzyme the precision of this structural motif must be
maintained in order to facilitate a transfer of thermal energy
from the protein−solvent interface toward the catalytic base as
well as Mg(1), both of which will control the ability of the
substrate to undergo deprotonation.
Region B (raspberry and bright red) includes peptides 2−23,
32−49, 64−77, and 113−137 at the N-terminal domain, with
ΔEa(HX) values of −8, −9, −6, and −15 kcal mol−1,
respectively, and peptide 373−383, which is a loop in the
TIM barrel with an ΔEa(HDX) value of −2 kcal mol−1 (Table
2 and Figure 8). This region includes the loop (L1, bright red)
that closes over the active site pocket in the presence of
substrate and Mg(2). The measured changes in enthalpic
barriers, reflecting the impact of mutation on local protein
flexibility, are expected to be mirrored in the capability of the
protein to position key active site components. A meticulous
control of protein flexibility within this region is likely salient
to accessing the reactive conformation once substrate is bound.
The loop dynamics, through Ser39, are proposed to directly
tune the donor−acceptor distances and electrostatic inter-
actions among the catalytic residues and the substrate in
response to thermal transfer arising at the protein/solvent
interface from regions represented by peptides 64−77, and
113−137 and 2−23. As both the substrate and Ser39
coordinate the Mg2+ ions, dynamic motions at Ser39 will
affect the Lewis acidity of the metal ions that influence the
acidity of 2-PGA at C2 (see Figure 1b). This acidity is
expected to be one of the key catalytic features controlling the
(partially) rate-determining transfer of a proton from the
substrate to the active site base.
Peptides 2−23 and 64−77 in the N-terminal domain
surround peptide 32−49, with multiple contact points. Peptide
372−383 in the TIM barrel domain is hydrogen-bonded to the
L1 loop via Gly376 to Ser36 in both the open and closed forms
of the crystal structure (Figure S6). We hypothesize that the
small increased flexibility observed in 372−383 for L343Ala is
a reverberation of the enhanced flexibility of the surrounding
region through the hydrogen-bonding residues and additional
close contacts. On the basis of the large overall increase in
flexibility in region B upon mutation, this region will need to
be well-structured for optimal reactivity to ensue. Significantly,
a site selective connectivity between L1 and the protein/
solvent interface emerges as a hitherto unrecognized element
of catalysis, implicating a thermally activated positioning and
electrostatic optimization of the carboxylate of the substrate
toward rate-limiting proton loss from its α-carbon (see Figure
3 for the structure-based positioning of the substrate).
Region C (raspberry) is located on the front of the TIM
barrel, opposite the N terminal domain, and includes 15 amino
acids in a flexible loop that undergoes a conformational shift
upon substrate binding (Figure 2, L3). The peptide that
comprises this loop, 263−279, has an ΔEa(HDX) value of −12
kcal mol−1, yet is ca. 30 Å away from Leu343Ala. We are
currently uncertain how this region is triggered to increase its
flexibility upon mutation, beyond hypothesizing that it is linked
to the conformational shifts that occur on substrate binding. Its
behavior is possibly related to the adjacent loop (L2), which
contains His159 that binds the substrate; however, L2 itself
shows no detectable difference in flexibility by HDX-MS
analysis.
Region D (dark blue) is located in the dimer interface, at the
connection of two ß sheets by a short loop (peptide 22−31)
and over 10 Å from the other monomer. D is the only region
to show a significant decrease in flexibility, ΔEa(HDX) = 5.0
kcal mol−1, upon the Leu343Ala mutation. This rigidification,
over 30 Å from the Leu343Ala mutation site, may represent a
structural compensation for the increased flexibility within the
adjacent region B.
The remainder of the protein, shown in white in Figure 8,
shows little to no detectable change in Ea(HDX) in our
experimental time regime upon the introduction of the
catalytically impairing mutation Leu343Ala. This region
includes the TIM barrel strands in the center of the protein
and the barrel face opposite the active site. It is made up of 17
type II peptides and four type I peptides. This region
demonstrates clearly that not all HDX-detected motions will
be impacted by activity-altering mutations. Interestingly, the
differences seen in enolase, between the opposing faces of the
TIM barrel, are similar to those observed in murine adenosine
deaminase, subsequent to mutation of a hydrophobic side
chain proximal to substrate.17 One emerging feature of TIM
barrels is the presence of a stable surface on the rear face of the
barrel.17,68 The front of the barrel contains the loops and
catalytic residues that control substrate specificity and
reactivity. From this study and the earlier findings with
adenosine deaminase,17 the front of the barrel also contains the
thermally activated protein networks that arise at the protein/
solvent interface in a reaction-specific manner.
Linking Loop Closures in Proteins to Solvent-
Exposed Thermal Networks. In this work we used
temperature-dependent HDX-MS to analyze an enzyme for
which X-ray crystallography had previously detected a
substrate-initiated loop closure that consolidates key active
site elements to initiate catalysis.26,34 Comparison of the
regions of temperature-dependent HDX perturbation in
enolase with the regions of conformational change, as seen
in the substrate-bound and substrate-free X-ray structures
(PDB 1ONE and 1EBH), shows similarities in the TIM barrel
region. The primary conformational change seen from X-ray
crystallography is the opening and closing of L1 with
additional effects arising in L2 and L3 (Figure 2). Two of
the nine peptides that show temperature-dependent perturba-
tions upon the Leu343Ala mutation contain the L1 and L3
regions: peptide 32−49 (L1) and peptide 263−279 (L3). As
discussed earlier, the changes in L3 that arise in Leu343Ala are
far from the active site and do not appear to be engaged in a
thermal network that connects the solvent-accessible surface to
the active site (as seen for regions A and B, Figure 8). The role
of this region of protein in catalysis is not well-understood at
the moment. The importance of the primary loop closure in
enolase is much better understood. L1 has recently been
examined through molecular dynamics, in a study that
differentiates substrate- and product-liganded complexes. Li
and Hammes-Schiffer found that the substrate-liganded
enolase strongly prefers a closed conformation, whereas in
the presence of product the loop is more like that of the
substrate-free enzyme, with a high degree of flexibility and a
preference for the open conformation. These differences are
attributed primarily to changes in the hydrogen-bonding
network in the active site and to the interactions of Ser39
with Mg(2).69
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J. Am. Chem. Soc. 2021, 143, 785−797