The catalytic mechanism of the hotdog-fold enzyme superfamily 4-hydroxybenzoyl-coa thioesterase from arthrobacter sp. Strain SU

Article abstract of DOI:10.1021/bi301059m

The hotdog-fold enzyme 4-hydroxybenzoyl-coenzyme A (4-HB-CoA) thioesterase from Arthrobacter sp. strain AU catalyzes the hydrolysis of 4-HB-CoA to form 4-hydroxybenzoate (4-HB) and coenzyme A (CoA) in the final step of the 4-chlorobenzoate dehalogenation pathway. Guided by the published X-ray structures of the liganded enzyme (Thoden, J. B., Zhuang, Z., Dunaway-Mariano, D., and Holden H. M. (2003) J.Biol. Chem. 278, 43709-43716), a series of site-directed mutants were prepared for testing the roles of active site residues in substrate binding and catalysis. The mutant thioesterases were subjected to X-ray structure determination to confirm retention of the native fold, and in some cases, to reveal changes in the active site configuration. In parallel, the wild-type and mutant thioesterases were subjected to transient and steady-state kinetic analysis, and to ¹⁸O-solvent labeling experiments. Evidence is provided that suggests that Glu73 functions in nucleophilic catalysis, that Gly65 and Gln58 contribute to transition-state stabilization via hydrogen bond formation with the thioester moiety and that Thr77 orients the water nucleophile for attack at the 4-hydroxybenzoyl carbon of the enzyme-anhydride intermediate. The replacement of Glu73 with Asp was shown to switch the function of the carboxylate residue from nucleophilic catalysis to base catalysis and thus, the reaction from a two-step process involving a covalent enzyme intermediate to a single-step hydrolysis reaction. The E73D/T77A double mutant regained most of the catalytic efficiency lost in the E73D single mutant. The results from ³¹P NMR experiments indicate that the substrate nucleotide unit is bound to the enzyme surface. Kinetic analysis of site-directed mutants was carried out to determine the contributions made by Arg102, Arg150, Ser120, and Thr121 in binding the nucleotide unit. Lastly, we show by kinetic and X-ray analyses of Asp31, His64, and Glu78 site-directed mutants that these three active site residues are important for productive binding of the substrate 4-hydroxybenzoyl ring.

Full text of DOI:10.1021/bi301059m

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pubs.acs.org/biochemistry
The Catalytic Mechanism of the Hotdog-fold Enzyme Superfamily
4‑Hydroxybenzoyl-CoA Thioesterase from Arthrobacter sp. Strain SU
Feng Song,†,# James B. Thoden,‡ Zhihao Zhuang,†,⊥ John Latham,† Michael Trujillo,§
Hazel M. Holden,*,‡ and Debra Dunaway-Mariano*,†
†Department of Chemistry and Chemical Biology, University of New Mexico, Albuquerque, New Mexico 87131, United States
‡Department of Biochemistry, 433 Babcock Drive, University of Wisconsin, Madison, Wisconsin 53706-1544, United States
§Scientific Laboratory Division, New Mexico Department of Health, Albuquerque, New Mexico 87131, United States
S
* Supporting Information
ABSTRACT: The hotdog-fold enzyme 4-hydroxybenzoyl-
coenzyme A (4-HB-CoA) thioesterase from Arthrobacter sp.
strain AU catalyzes the hydrolysis of 4-HB-CoA to form 4-
hydroxybenzoate (4-HB) and coenzyme A (CoA) in the final
step of the 4-chlorobenzoate dehalogenation pathway. Guided
by the published X-ray structures of the liganded enzyme
(Thoden, J. B., Zhuang, Z., Dunaway-Mariano, D., and Holden
H. M. (2003) J. Biol. Chem. 278, 43709−43716), a series of
site-directed mutants were prepared for testing the roles of
active site residues in substrate binding and catalysis. The
mutant thioesterases were subjected to X-ray structure
determination to confirm retention of the native fold, and in
some cases, to reveal changes in the active site configuration. In parallel, the wild-type and mutant thioesterases were subjected to
transient and steady-state kinetic analysis, and to 18O-solvent labeling experiments. Evidence is provided that suggests that Glu73
functions in nucleophilic catalysis, that Gly65 and Gln58 contribute to transition-state stabilization via hydrogen bond formation
with the thioester moiety and that Thr77 orients the water nucleophile for attack at the 4-hydroxybenzoyl carbon of the enzyme-
anhydride intermediate. The replacement of Glu73 with Asp was shown to switch the function of the carboxylate residue from
nucleophilic catalysis to base catalysis and thus, the reaction from a two-step process involving a covalent enzyme intermediate to
a single-step hydrolysis reaction. The E73D/T77A double mutant regained most of the catalytic efficiency lost in the E73D single
mutant. The results from 31P NMR experiments indicate that the substrate nucleotide unit is bound to the enzyme surface.
Kinetic analysis of site-directed mutants was carried out to determine the contributions made by Arg102, Arg150, Ser120, and
Thr121 in binding the nucleotide unit. Lastly, we show by kinetic and X-ray analyses of Asp31, His64, and Glu78 site-directed
mutants that these three active site residues are important for productive binding of the substrate 4-hydroxybenzoyl ring.
rganic acids comprise ∼20% of the Escherichia coli
reviews see refs 13 and 14). The well-studied α/β-hydrolase-
fold family employs an Asp/Glu-His-Ser catalytic triad to
catalyze the hydrolysis of amides and oxygen esters in addition
to the comparatively less challenging thioester substrates.15 The
hotdog-fold family contributes more than half of the cellular
thioesterases, but hotdog-fold amidases or O-esterases have yet
to be discovered.
The hotdog-fold thioesterase is a small (∼19 kDa) protein
comprised of a five- or six-stranded β-sheet that wraps a long α-
helix16 (hence, the name hotdog-fold17). Two subunits
associate to form the minimal catalytic unit. The active site is
located at the subunit interface and is a composite of residues
from each subunit. The thioesterase catalytic scaffold appears to
have been invented at least twice during the evolution of the
hotdog-fold family as evidenced by the 4-hydroxybenzoyl-CoA
O
metabolome.1 The carboxylate group increases the
solubility of the metabolite in the aqueous environment of
the cell, and it provides a source of binding energy needed for
metabolite−protein association. Organic acids are activated for
acyl transfer, Claisen condensation, Michael addition, and β-
elimination reactions by conversion to the corresponding
thioester,2−4 a reaction that is catalyzed by ATP-dependent
synthetases or ligases.5 The most common thiol unit used for
thioester formation is the pantetheine phosphate moiety of
coenzyme A (CoA) or holo-acyl carrier proteins (holo-ACP).
Thioesterases catalyze the hydrolysis of the thioester
metabolite as needed to release the chemically transformed
organic acid. Thioesterases also function in a housekeeping
mode to release the CoA or holo-ACP from pathway
intermediates that accumulate in stalled pathways (for
examples, see refs 6−10). Thioesterases have primarily evolved
within two large enzyme superfamilies families: the α/β-
hydrolase-fold11 family and the hotdog-fold family12 (for recent
Received: August 5, 2012
Published: August 8, 2012
© 2012 American Chemical Society
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Scheme 1. Three Chemical Steps of the 4-Chlorobenzoate Dehalogenation Pathway Catalyzed by 4-Chlorobenzoate: CoA
Ligase, 4-Chlorobenzoyl-CoA Dehalogenase and 4-Hydrobenzoyl-CoA Thioesterase, Respectively
(4-HB-CoA) thioesterases from Pseudomonas sp. strain CBS3
and Arthrobacter sp. strain AU (UniProt accession # Q04416),
which are the archetypes of two distinct clades of the
family.16,18,19 Both thioesterases function in the final step of
the 4-chlorobenzoate dehalogenation pathway (see Scheme 1),
which is operative in specialized strains of soil-dwelling
bacteria.20 Remarkably, these two 4-HB-CoA thioesterases are
not homologous in sequence and they do not use the same
catalytic machinery, yet their tertiary structures are highly
similar.
The Arthrobacter sp. strain AU 4-hydroxybenzoyl-CoA
thioesterase clade, which is comprised of a wide variety of
aryl- and acyl-CoA and aryl- and acyl-ACP thioesterases, is by-
far the largest and most diversified of the two clades. The goal
of the work reported herein was to determine the mechanisms
by which the Arthrobacter sp. strain AU 4-HB-CoA thioesterase
binds 4-HB-CoA and catalyzes its hydrolysis. This particular
thioesterase was selected as the mechanistic prototype because
it proved to be an ideal system for structure determination,
transient kinetic studies, 18O-solvent labeling experiments, and
site-directed mutagenesis.
The starting point for our studies was the examination of the
reported X-ray structures of the thioesterase bound with its
products 4-hydroxybenzoate (4-HB) and CoA, with the inert
substrate analogue 4-hydroxybenzyl-CoA (CH2−S replaces
OC−S) or with the inert substrate analog 4-hydroxyphe-
nacyl-CoA (4-HP-CoA; OC−CH2−S replaces OC−S)18
(PDB accession codes 1Q4S, 1Q4U, 1Q4T, respectively). The
thioesterase catalytic site is comprised of invariant residues
contributed from the central α-helix N-terminus (His64,
Gly65) and connecting loop (Gln58) from one subunit
(subunit A) and the midsection of the central α-helix from
the opposing subunit (subunit B) (Glu73*, Thr77*, Glu78*)
(Figure 1A). The back wall of the 4-hydroxybenzoyl binding
pocket is formed by the N-terminal region of subunit A
(Asp31). The 4-HB-CoA pantetheine arm snakes through a
narrow hydrophobic tunnel that links the catalytic site to the
protein surface. The nucleotide unit is located on the protein
surface where subunits A (Arg102) and B (Arg150*) join and it
is also positioned to interact with subunit C (Ser120** and
Thr121**) (Figure 1B). In this paper, we report on an in depth
experimental investigation of the roles played by the key active
site residues (depicted in Figure 1C) in substrate recognition
and catalysis. We provide evidence that Arg102, Arg150,
Ser120, and Thr121 contribute to the substrate binding energy
through favorable electrostatic interaction with the phosphoryl
groups, that Asp31, His64, and Glu78 assist in orienting the 4-
hydroxybenzoyl ring, that Glu73 functions in nucleophilic
catalysis, that Gly65 and Gln58 contribute to transition-state
stabilization via hydrogen bond formation with the thioester
moiety and that Thr77 orients the water nucleophile for attack
at the 4-hydroxybenzoyl carbon of the anhydride intermediate.
We also report that the replacement of Glu73 with Asp
switches the function of the carboxylate residue from
nucleophilic catalysis to base catalysis and thus, the reaction
from a two-step process involving a covalent enzyme
a
intermediate to a single-step hydrolysis reaction.
MATERIALS AND METHODS
■
Materials. Wild-type recombinant Arthrobacter sp. strain SU
4-HB-CoA thioesterase was prepared as previously reported.23
The thioesterase site-directed mutants were prepared by using a
PCR based strategy as is detailed in the Supporting
Information. The substrates 4-HB-CoA24 and the 4-hydroxy-
dithiobenzoyl-CoA25 were prepared according to published
procedures, as were the inhibitors 4-hydroxybenzyl-CoA and 4-
HP-CoA18 and the radiolabeled substrate [14C]4-HB-CoA
(benzoyl ring 14CO).23
Steady-State kcat, Km, and Ki Determinations. The initial
velocity of the 4-HB-CoA thioesterase-catalyzed hydrolysis of
4-HB-CoA was measured by monitoring the decrease in
solution absorption at 300 nm (Δε = 11.8 mM−1 cm−1).
Reactions were carried out in 50 mM K+HEPES (pH 7.5, 25
°C) containing wild-type or mutant 4-HB-CoA thioesterase
and varying concentrations of 4-HB-CoA (Km − 10Km). The
inhibition data were measured using 200 and 400 μM 4-HB, 18
and 36 μM CoA or 2Ki concentration of 4-hydoxybenzyl-CoA
and 4-hydoxyphenacyl-CoA (for which linear inhibition was
previously demonstrated18). For all measurements, the initial
velocity data were analyzed using eqs 1, 2, or 3 and the
computer program KinetAsyst (IntelliKinetics, PA):
V = Vmax[S]/([S] + Km)
(1)
V = Vmax[S]/(Km(1 + [I]/Kis) + [S])
(2)
V = Vmax[S]/(Km(1 + [I]/Kis) + [S](1 + [I]/Kii)
(3)
where V = initial velocity, Vmax = maximum velocity, [S] =
substrate concentration, Km = Michaelis constant, [I] =
inhibitor concentration, Kis = the slope inhibition constant,
and Kii = the intercept inhibition constant. The kcat was
calculated from Vmax/[E] where [E] is the enzyme concen-
tration (determined using the Bradford method26).
Stopped-Flow UV−vis Transient Kinetic Determina-
tions of Multiple and Single Turnover Reactions. A
DX.17MV sequential stopped-flow spectrometer (Applied
Photophysics, Leatherhead, U. K.) with a light path of 10
mm and a mixing time of 3 ms was used to mix buffered wild-
type or mutant thioesterase with 4-HB-CoA and to monitor the
hydrolysis reaction at 300 nm (Δε = 11.8 mM−1 cm−1). For the
wild-type thioesterase-catalyzed multiple turnover reactions, the
reaction solutions initially contained 115 μM 4-HB-CoA and
16, 21, or 31 μM enzyme (in 0.15 M KCl/50 mM K+HEPES
(pH 7.5, 25 °C). For the mutant thioesterase-catalyzed multiple
turnover reactions, the reaction solutions initially contained 80
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or 100 μM 4-HB-CoA and 20 or 30 μM enzyme (in 0.15 M
KCl/50 mM K+HEPES (pH 7.5, 25 °C).
For single turnover reactions, the thioesterase and 4-HB-CoA
were present at 40 μM and 20 μM, respectively. The data were
fitted to a single (eq 4) or double exponential equation (eq 5)
Y = Y0 + A exp(−kt)
(4)
(5)
t
Y = Y0 + A1 exp(−k1t) + A2 exp(−k2t)
t
Yt and Y0 are the absorbance at time t and zero, A, A1, and A2
are amplitudes, and k, k1, and k2 are rate constants.
Rapid Quench Transient Kinetic Determinations. A
Rapid-quench instrument from KinTek Instruments was used
to mix buffered wild-type or mutant thioesterase with [14C]4-
HB-CoA and then with 0.2 M HCl to terminate the reaction.
For the multiple turnover reactions, mixtures initially contained
10 μM enzyme and 50 μM [14C]4-HB-CoA in 0.15 M KCl/50
mM K+HEPES (pH 7.5, 25 °C), whereas for the single
turnover reactions, enzyme and [14C]4-HB-CoA concentrations
were 50 μM and 5 μM, respectively. The enzyme was
precipitated from the quenched reaction solution by vigorous
mixing with 200 μL CCl4 using a Vortex mixer, and then
collected by (micro)centrifugation of the mixture (14000 rpm
for 1 min). The (upper) water phase was injected into an
HPLC for separation of the [14C]4-HB-CoA and [14C]4-HB as
described previously.27 The eluent was collected using a
fraction collector and analyzed by liquid scintillation counting.
Control reactions (lacking enzyme) were carried out in parallel
to establish the background radioactivity associated with the
[14C]4-HB fraction. The background radioactivity (∼2%) was
taken into account when calculating the concentrations of
substrate and product remaining in each quenched reaction
mixture.
The single turnover time course data were fitted to a single
(eq 4) or double exponential equation (eq 5). Yt and Y0
represent the concentrations of [14C]4-HB-CoA or [14C]4-HB
at time t and zero, respectively.
Kd Determination. A FluoroMax-2 fluorometer (290 nm
excitation; 334 nm emission) was used to monitor intrinsic
protein fluorescence quenching for 50 mM K+HEPES/0.2 M
KCl/1 mM DTT (pH 7.5, 25 °C) solutions containing wild-
type or mutant 4-HB-CoA thioesterase and varying concen-
trations of 4-HB-CoA or 4-HP-CoA ligand. For a typical
titration experiment, 1 μL aliquots of ligand were added to a 1
mL solution of 1 μM thioesterase, and the fluorescence
intensity at 334 nm was measured following each addition. The
fluorescence data, collected at ligand concentrations ranging
from 0 to 12 μM, were fitted to eq 6 using the Kaleida Graph
computer program for nonlinear regression analysis.
Figure 1. (A) The catalytic site of the Arthrobacter sp. strain SU 4-HB-
CoA thioesterase bound with 4-HB and CoA (PDB accession 1Q4S).
Only the AB dimer is shown. The nitrogen atoms are colored blue, the
oxygen atoms red and the sulfur atom yellow. The subunit A carbon
atoms are colored gray, whereas the subunit B carbon atoms are
colored cyan. (B) The nucleotide binding site of the Arthrobacter sp.
strain SU 4-HB-CoA thioesterase bound with 4-hydroxybenzyl-CoA
(PDB accession 1Q4U). The ABCD tetramer is shown. The coloring
scheme is the same as that stated in (A) but also includes the green-
colored carbon atoms of subunit C and wheat colored-carbon atoms of
subunit D. (C) The ChemDraw depiction of the proposed binding
interactions between the thioesterase and the 4-HB-CoA substrate.
The residues from subunits B and C are marked with * and **,
respectively, and the hydrogen bonds are represented with dashed
lines.
ΔF = (ΔFmax[E]){(Kd + [E] + [S])
−
(Kd + [E] + [S])2 − 4[E] }/2
(6)
where [S] is the total ligand concentration, [E] is the total
enzyme concentration, Kd is the apparent dissociation constant
of the enzyme−ligand complex, ΔF is the observed change in
fluorescence intensity, and ΔFmax is the maximum change in
fluorescence intensity.
Determination of kcat pH Rate Profile in H2O and D2O.
The kcat pH rate profiles for Arthrobacter sp. strain 4-HB-CoA
thioesterase-catalyzed hydrolysis of 4-HB-CoA in H2O and
D2O were measured by using a dual buffer system consisting of
50 mM acetate and 50 mM MES (pH 4.0−5.6); 50 mM MES
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and 50 mM HEPES (pH 5.6−8.0); 50 mM HEPES and 50 mM
TAPS (pH 8.0−9.1); 50 mM TAPS and 50 mM CAPSO (pH
9.1−9.6). The enzyme activity at overlapping pH values was
tested to ensure equal enzyme activity in the different buffer
systems. The stability of the thioesterase at the acidic and basic
pH values was verified by carrying out preincubation test
experiments.
were able to hydrolyze the ligand, generating the various
product complexes that were observed. Specific unit cell
parameters, data collection, and refinement statistics are
presented in Table SI1.
RESULTS AND DISCUSSION
■
Transient Kinetic Experiments Provide Evidence For a
Two-Step Chemical Pathway. Pre-Steady-State Multiple
Turnover Reaction. The time course for pre-steady-state wild-
type thioesterase-catalyzed 4-HB-CoA hydrolysis was examined
under multiple-turnover conditions so that the rate of the first
turnover and that of subsequent turnovers might be compared.
The reaction time courses were first determined by monitoring
the decrease in solution absorbance at 300 nm (Δε = 11.8
mM−1 cm−1 for the thioester cleavage) with a stopped-flow UV-
vis spectrophotometer (Figure 2A). The stopped-flow
absorbance traces for the reaction of 115 μM 4-HB-CoA with
16, 21, and 31 μM wild-type thioesterase are biphasic and are
suggestive of a “burst” of substrate consumption approximating
a single turnover of substrate on the enzyme (i.e., ∼16, 21, 31
μM calculated from the amplitude derived from the fit to a
double exponential equation) followed by a slow, linear phase
that derives from the subsequent turnover events (rate
constants derived from data fitting to a double exponential
equation are reported in Table 1).
Steady-State Multiple Turnover Time Courses. The
time courses for 0.003 μM thioesterase-catalyzed 4-HB-CoA
hydrolysis of 4-HB-CoA (1.5, 2.7, and 5.7 μM) in 50 mM
K+HEPES (pH 7.5, 25 °C) were monitored at 300 nm (Δε =
11.8 mM−1 s−1) to completion. The data were fitted by curve
simulation using the KinTek Corporation Global Kinetic
Explorer (www.kintek-corp.com/KGExplorer/index.php).28
Solvent 18O-Labeling Experiments. Stock solutions of
wild-type or mutant thioesterase in 97% H218O (Spectra)
containing 0.15 M KCl/10 mM K+HEPES (pH 7.5) were
prepared by lyophilizing 2 mM or 0.02 mM thioesterase in 0.15
M KCl/10 mM K+HEPES (pH 7.5) and then dissolving the
resulting powder with the desired volume of H218O. The
thioesterase activity was measured to ensure that full activity
was retained. The enzyme stock solution was combined with 4-
HB-CoA in 97% H218O to generate a 200−400 μL reaction
solution that for a typical experiment contained 2.0 mM
thioesterase and 0.2 mM 4-HB-CoA (test reaction) or 0.02 mM
thioesterase and 0.2 mM 4-HB-CoA thioesterase (control
reaction), in 0.15 M KCl/10 mM K+HEPES (pH 7.5) at 25 °C.
Following a 15 min incubation period the reaction solution was
filtered using a 10 kDa micro separation filter (Pall Filtron).
The filtrate was acidified by adding 5 μL of 6 N HCl and
extracted four times with 1 mL portions of ethyl acetate. The
ethyl acetate extracts were combined, dried over anhydrous
sodium sulfate and the solvent was evaporated in vacuo. The
solid was dissolved in 100 μL of absolute methanol and
subjected to GC-MS analysis using a Saturn GC-MS
spectrometer equipped with a Restek XTI-5 column.
Next, the time course for the multiple turnover of [14C]4-
HB-CoA (50 μM) catalyzed by wild-type thioesterase (10 μM)
was measured using rapid (acid) quench techniques coupled
with HPLC separation of the 14C-labeled reactant and 4-HB
product released from the denatured enzyme (Figure 2B). The
time course shown in Figure 2B is biphasic, with the fast phase
approximating a single turnover of substrate on the enzyme
(i.e., ∼10 μM) (rate constants derived from data fitting to a
double exponential equation are reported in Table 1)
The burst-phase kinetics could be rationalized in terms of fast
hydrolysis of the thioester in the enzyme−substrate complex
31P NMR Spectral Determinations. The 31P NMR spectra
were recorded on a Bruker AC-F 500 NMR spectrometer at
202.5 MHz. Samples were contained in 5 mm NMR tubes, and
spectra were recorded at 5 °C under conditions of proton
decoupling. The sample solution contained 10% D2O for use in
signal locking. The instrument was calibrated using an 85%
phosphoric acid standard.
b
followed by slow product release. Alternatively, the burst-
phase kinetics might reflect fast conversion of the enzyme−
substrate complex to a chemical intermediate in which the
thioester chromophore is absent (stopped-flow) and which
hydrolyzes to 4-HB in the acid quench solution (rapid quench),
followed by slow regeneration of the catalytically active form of
the enzyme.
X-ray Structure Determinations. Arthrobacter sp. strain 4-
HB-CoA thioesterase crystals were grown as reported
previously.18 Briefly, the mutant proteins were concentrated
to approximately 17−20 mg/mL at pH 7 (10 mM HEPES) in
the presence of 150 mM KCl and 1 mM DTT. Either 4-HP-
CoA or 4-HB-CoA were added to a concentration of 1 mM.
Crystals were grown at room temperature via hanging drop
vapor diffusion using 15−20% PEG-3400 and 200 mM LiCl
buffered at pH 7 with 100 mM MOPS. Crystals were
transferred to cryoprotectant solutions containing 30% PEG-
3400, 200 mM KCl, 250 mM LiCl, 15% ethylene glycol, and
100 mM MOPS at pH 7. 4-HP-CoA or 4-HB-CoA were added
to a concentration of 1 mM.
X-ray data were collected at 100 K with a BRUKER HI-Star
area detector system equipped with Goebel optics. The data
were processed and scaled with SAINT (Bruker AXS Inc.). The
structures were solved using Fourier difference techniques and
refined with REFMAC.29 Iterative model building was
performed with COOT.30 When crystallized in the presence
of 4-HB-CoA, the mutant proteins E73D, E73Q, and Q58A
On the basis of the X-ray structure of the thioesterase bound
with its products 4-HB and CoA (Figure 1A), a likely candidate
for a chemical intermediate is the anhydride formed by Glu73
displacement of CoA from 4-HB-CoA. Specifically, the
structure shows that the Glu73 positioned for nucleophilic
attack at the CO and that there is no room for a water
nucleophile.19 The enzyme-anhydride intermediate lacks the
ring-conjugated thioester group. Its formation would therefore
account for initial drop in absorbance at 300 nm as was
observed in the stopped-flow experiment (Figure 2A). In
addition, an anhydride is chemically labile, and in the acid
quench solution it is likely to undergo hydrolysis to form 4-HB,
thus accounting for the burst of [14C]4-HB formation observed
in the rapid quench experiment (Figure 2B). Consequently, our
next experiments focused on testing the formation of this
putative thioesterase-anhydride intermediate.
Solvent Deuterium Kinetic Isotope Effect. In order to
distinguish between the posited two-step chemical pathway and
a single-step chemical pathway with rate-limiting product
release, we first tested whether the chemical reaction or product
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