Transition State Analysis of the Reaction Catalyzed by the Phosphotriesterase from Sphingobium sp. TCM1

Article abstract of DOI:10.1021/acs.biochem.9b00041

Organophosphorus flame retardants are stable toxic compounds used in nearly all durable plastic products and are considered major emerging pollutants. The phosphotriesterase from Sphingobium sp. TCM1 (Sb-PTE) is one of the few enzymes known to be able to hydrolyze organophosphorus flame retardants such as triphenyl phosphate and tris(2-chloroethyl) phosphate. The effectiveness of Sb-PTE for the hydrolysis of these organophosphates appears to arise from its ability to hydrolyze unactivated alkyl and phenolic esters from the central phosphorus core. How Sb-PTE is able to catalyze the hydrolysis of the unactivated substituents is not known. To interrogate the catalytic hydrolysis mechanism of Sb-PTE, the pH dependence of the reaction and the effects of changing the solvent viscosity were determined. These experiments were complemented by measurement of the primary and secondary 18-oxygen isotope effects on substrate hydrolysis and a determination of the effects of changing the pK_a of the leaving group on the magnitude of the rate constants for hydrolysis. Collectively, the results indicated that a single group must be ionized for nucleophilic attack and that a separate general acid is not involved in protonation of the leaving group. The Br?nsted analysis and the heavy atom kinetic isotope effects are consistent with an early associative transition state with subsequent proton transfers not being rate limiting. A novel binding mode of the substrate to the binuclear metal center and a catalytic mechanism are proposed to explain the unusual ability of Sb-PTE to hydrolyze unactivated esters from a wide range of organophosphate substrates.

Full text of DOI:10.1021/acs.biochem.9b00041

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Article
Transition State Analysis of the Reaction Catalyzed by
the Phosphotriesterase from Sphingobium sp. TCM1
Andrew N. Bigley, Dao Feng Xiang, Tamari Narindoshvili,
Charlie W Burgert, Alvan C. Hengge, and Frank M. Raushel
Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.9b00041 • Publication Date (Web): 07 Feb 2019
Downloaded from http://pubs.acs.org on February 11, 2019
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Transition State Analysis of the Reaction Catalyzed by the
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Phosphotriesterase from Sphingobium sp. TCM1
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Andrew N. Bigleyδ, Dao Feng Xiangδ, Tamari Narindoshviliδ,
Charlie W. BurgertΨ, Alvan C. HenggeΨ*, and Frank M. Raushelδ*
δDepartment of Chemistry, Texas A&M University, College Station TX 77843, United States
ΨDepartment of Chemistry and Biochemistry, Utah State University, Logan UT 84322,
United States
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ABSTRACT
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Organophosphorus flame-retardants are stable toxic compounds used in nearly all
durable plastic products and are considered major emerging pollutants. The
phosphotriesterase from Sphingobium sp. TCM1 (Sb-PTE) is one of the few enzymes known
to be able to hydrolyze organophosphorus flame-retardants such as triphenyl phosphate
and tris(2-chloroethyl) phosphate. The effectiveness of Sb-PTE for the hydrolysis of these
organophosphates appears to arise from its ability to hydrolyze unactivated alkyl and
phenolic esters from the central phosphorus core. How Sb-PTE is able to catalyze the
hydrolysis of the unactivated substituents is not known. To interrogate the catalytic
hydrolysis mechanism of Sb-PTE, the pH-dependence of the reaction and the effects of
changing the solvent viscosity were determined. These experiments were complemented
by measurement of the primary and secondary 18-oxygen isotope effects on substrate
hydrolysis and a determination of the effects of changing the pKa of the leaving group on
the magnitude of the rate constants for hydrolysis. Collectively, the results indicated that a
single group must be ionized for nucleophilic attack and that a separate general acid is not
involved in protonation of the leaving group. The Brønsted analysis and the heavy atom
kinetic isotope effects are consistent with an early associative transition state with
subsequent proton transfers not being rate limiting. A novel binding mode of the substrate
to the binuclear metal center and a catalytic mechanism are proposed to explain the
unusual ability of Sb-PTE to hydrolyze unactivated esters from a wide range of
organophosphate substrates.
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INTRODUCTION
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The phosphotriesterase from Sphingobium sp. TCM1 (Sb-PTE) is unique among
phosphotriesterase enzymes in that it will hydrolyze organophosphate flame-retardants
and plasticizers.1, 2 Unlike the better-known organophosphate nerve agents, the
organophosphate flame-retardants lack the typical labile leaving group, which gives rise to
the neurotoxicity of the nerve agents.3 These compounds are commonly used in plastics
and foams and are produced at rates of hundreds of tons per year.4 While not neurotoxins,
organophosphate flame-retardants have been noted as potential carcinogens and are
developmental toxins in animal and human models.5-7 The widespread use of these
compounds has led them to be considered as major emerging pollutants with widespread
contamination of soil, water, and air.4, 8
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The potential to utilize Sb-PTE for bioremediation of organophosphorus flame
retardants has encouraged efforts to characterize the enzymatic activity of this enzyme.1, 2
The structure of Sb-PTE was recently determined to have a 7-bladed β-propeller protein
fold with a binuclear Mn+2 metal center embedded at the top of the central core of the
enzyme (Figure 1).9 The substrate binding site is found directly above and essentially
perpendicular to the plane of the metal center.
Sb-PTE has been shown to catalyze the hydrolysis of phosphotriesters via a direct
nucleophilic attack of hydroxide (or water) on the phosphorus of the bound substrate.10 It
is highly probable that the nucleophilic water is initially coordinated by the two divalent
cations bound in the active site of enzyme.9 However, other phosphotriesterase enzymes
generally exhibit a very strong dependence on the pKa of the leaving group.11-13 For
example, the phosphotriesterase from Pseudomonas diminuta (Pd-PTE) has a 100,000-fold
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reduction in catalytic activity when p-nitrophenol is substituted with phenol as the leaving
group.14 With Sb-PTE, there is only ~2-fold difference in the rate of hydrolysis between
dibutyl p-nitrophenyl phosphate and dibutyl phenyl phosphate.2 One simple explanation is
that Sb-PTE effectively delivers a proton to the leaving group during the course of the
reaction, but how that proton might be delivered is unclear. Further complicating the
study of Sb-PTE is the observation that this enzyme can hydrolyze any one of the three
ester bonds within a given substrate resulting in multiple reaction products.15 However,
the ability to cleave a simple alkyl ester leaving group appears to require at least one other
substituent attached to the central phosphorus core with a pKa <11. With some substrates
the hydrolysis of a methyl ester can proceed at the same rate as hydrolysis of a p-
nitrophenyl ester within the same compound.15 How Sb-PTE is able to catalyze the
hydrolysis of simple alkyl esters at the same rate as an activated phenolate ester has yet to
be determined.
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As an initial probe of the activation mechanism for the nucleophilic water molecule
and to test the potential of additional groups within the active site that may function as a
general acid for the direct protonation of the leaving group, the pH-rate profile of Sb-PTE
was obtained. The degree of charge development in the transition state during substrate
hydrolysis was addressed by a systematic variation of the pKa of the leaving group ester,
and by kinetic isotope effects in the leaving group and in the nonbridging oxygen positions
of the substrate paraoxon. The rate limiting steps during product formation were probed
by measuring the effects of solvent viscosity and the substitution of D2O for H2O during
enzyme-catalyzed hydrolysis of the substrate paraoxon.
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Glu 407
His 389
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His 475
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Wat 3
His 317
His 258
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Wat 1
α
β
Wat 4
Wat 2
Glu 201
Figure 1. Binuclear metal center of Sb-PTE. The plane of the metal center is defined by the
coordinating nitrogen atoms from four histidine residues (green oval). The α-metal is
coordinated by Glu201, His389, Glu407, His475, the bridging hydroxide (Wat 1) and an
additional water ligand (Wat 2). The β-metal is coordinated by Glu201, His258, His317, the
bridging hydroxide (Wat 1) and two additional waters (Wat 3 and Wat 4) from solvent.
MATERIALS and METHODS
Materials. Unless otherwise stated, the bacterial growth medium was purchased
from Research Products International and general chemicals were obtained from
Sigma/Aldrich. Paraoxon (5) and tributyl phosphate (27) were purchased from
Sigma/Aldrich (Scheme 1). DNA sequencing was conducted by Eton Biosciences Inc.
Compounds 19-27 were synthesized by previously reported methods.2
Chemical Synthesis of Substrates. Phosphotriesters 1-5 and 7-18 were
synthesized with modifications of previously published methods.11 For compounds 1-5, 7-
11, and 13-18 a stirred reaction mixture containing 5 mmol phenol (or substituted phenol)
and 6 mmol of triethylamine in 20 mL of anhydrous dichloromethane was cooled to 0 °C
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and diethyl chlorophosphate (97%, 6 mmol) added dropwise. The reaction was allowed to
warm to ambient temperature (23 °C) and then stirred for an additional 18 h. The reaction
was quenched by the addition of 2.0 mL of water. The organic phase was washed with 0.1
N HCl and saturated NaHCO3, dried over NaSO4, and then the solvent was removed by
rotary evaporation. The product was purified by silica gel chromatography (hexanes:
EtOAc mixtures) yielding pure compounds in yields of 42 - 65%. The physical constants
are provided in the Supplementary Information.
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For compound 12, diethyl chlorophosphate (0.29 mL, 97%, 2 mmol) was added
dropwise to a stirred reaction mixture of 4-hydroxybenzamide (0. 27 g, 2 mmol) and K2CO3
(0.28 g, 2 mmol) in 7.0 mL of anhydrous tetrahydrofuran. The reaction was stirred for 24 h,
filtered, and then 20 mL of EtOAc were added. The organic phase was washed with 0.1 N
HCl and saturated NaHCO3 solution, dried over NaSO4, and the solvent removed by rotary
evaporation. The residue was purified by silica gel chromatography (EtOAc:MeOH, 20:1),
yielding 100 mg (19%) of a white solid product. Organophosphate triesters are potentially
toxic and thus the appropriate safety precautions must be used when handling these
compounds. The structures of the substrates used for this investigation are presented in
Scheme 1.
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O
O
O
EtO P OEt
O
O
EtO P OEt
O
O
EtO P OEt
O
O
EtO P OEt
O
O
EtO P OEt
O
O
EtO P OEt
O
O
EtO P OEt
O
EtO P OEt EtO P OEt
O
O
F
F
F
F
F
F
F
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7
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F
F
F
NO2
NO2
F
NO2
NO2
F
CN
O
H
O
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O
O
O
O
O
EtO P OEt
O
O
O
O
EtO P OEt
O
O
EtO P OEt
O
EtO P OEt EtO P OEt
EtO P OEt
O
EtO P OEt
EtO P OEt
O
EtO P OEt
O
O
O
O
F
F
F
F
Cl
F
OCH3
O
OCH3
O
NH2
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17
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O
O
O
BuO P OBu
O
O
O
O
O
O
O
BuO P OBu
S
BuO P OBu
O
BuO P OBu
O
BuO P OBu BuO P OBu BuO P OBu BuO P OBu
BuO P OBu
O
O
O
O
O
F
F
F
F
O
F
F
F3C
NO2
O
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Scheme 1: Structures of organophosphate triesters used as substrates for Sb-PTE.
Expression and Purification of Sb-PTE. The gene for wild-type Sb-PTE
(gi|664819019; UniProt id: A0A077JBW9) was cloned into a pET30a(+) plasmid using a
codon optimized sequence to enable expression of the C-terminal histidine-tagged Sb-PTE
as previously described.2 The plasmid was transformed into E. coli BL21(DE3) cells (EMD
Millipore), and single colonies were used to inoculate 6-mL cultures of LB medium, which
were grown for 8 h at 37 °C. The 6-mL cultures were subsequently used to inoculate 1-L
cultures of Terrific Broth, supplemented with 1.0 mM MnAc2 and then grown at 27 °C for
20 h. The cells were harvested by centrifugation and stored at -80 °C. Frozen cell pellets
(∼10 g) were resuspended in 50 mL of binding buffer (20 mM HEPES, pH 7.9, 0.5 M NaCl,
and 10 mM imidazole). Following the disruption of cells by sonication using a Branson
Sonifier 450, the solution was clarified by centrifugation (15,300 x g for 20 min). The
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supernatant solution was passed through a 0.45 µm syringe filter and subjected to Ni-
affinity chromatography using a 5-mL HisTrap FF column (GE Healthcare) attached to an
NGC liquid chromatography system (Bio-Rad) previously equilibrated with binding buffer.
The column was washed with 30 column volumes of binding buffer, and then Sb-PTE was
eluted with a linear gradient of 10 - 250 mM imidazole. The fractions with paraoxonase
activity were combined and concentrated. The elution buffer was exchanged with 20 mM
HEPES, pH 8.0, containing 100 mM NaCl, using a PD-10 desalting column (GE Healthcare).
The protein purity was judged to be >95% by SDS-PAGE using a Mini-PROTEIN TGX Gels
(Bio-Rad). The yield of Sb-PTE was ~10 mg/L of cell culture.
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Determination of Extinction Coefficients. Extinction coefficients for hydrolysis of
the modified phenolic groups from compounds 1-21 were determined at either pH 9.0 or
10.0. Generally, solutions of the substituted phenol were prepared in methanol and then
diluted in the appropriate buffer with a final concentration of 10% MeOH. The UV spectra
were recorded using a 1-cm quartz cuvette and the spectra of an identical concentration of
the corresponding phosphotriesters were recorded in the same cuvette. The substrate
spectrum was subtracted from the spectrum of the substituted phenol to generate a
difference spectrum, which was used to identify the optimal wavelength for the assay and
to calculate the appropriate extinction coefficient. The extinction coefficients and the
wavelengths use to measure the enzymatic rates of hydrolysis of compounds 1- 21 are
listed in Table S1.
Optimization of Catalytic Activity. The catalytic activity of Sb-PTE was tested with
various buffers using 1.0 mM paraoxon (5) as the substrate. The buffers HEPES, TRIS,
CHES, BICINE, TRICINE, Gly-Gly, TAPS, TABS, and AMPSO were each tested at a
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concentration of 50 mM at pH 8.5. Each buffer was subsequently tested in the presence of
20 - 500 mM of each of the following salts: NaCl, KCl, NH4Cl, or (NH4)2SO4 using 1.0 mM
paraoxon (5) as the substrate.
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pH-Rate Profile. The kinetic constants, kcat and kcat/Km, were measured for Sb-PTE
using paraoxon (5) or diethyl phenyl phosphate (17) as the substrate at pH values ranging
from 5.5 to 10.5 at 30 °C. A buffer system consisting of 40 mM MOPS, 40 mM MES, 40 mM
HEPES, 40 mM CHES, and 100 mM NH4Cl (combination buffer) was used for measurement
at all pH values. Stock buffer solutions (10X) were adjusted to the appropriate pH prior to
dilution. In each case, the reported pH was the measured value at the end of the enzymatic
reaction.
Brønsted Analysis. All kinetic measurements were conducted using a Molecular
Devices SpectraMax 384 Plus 96-well plate reader at 30 °C in 250 μL assay mixtures.
Combination buffer was used for the experiments at pH 10 for compounds 1-11 and 13-21.
Hydrolysis of compound 12 was followed in the same buffer at pH 9.0. Compounds with
diethyl phosphorus centers (1-18) included a final concentration of 5% methanol as a
cosolvent while the dibutyl series (19-27) used 5% DMSO. For the hydrolysis of 1.0 mM
paraoxon by Sb-PTE, the inclusion of 5% methanol in the assay reduces the rate of
hydrolysis by approximately 30%, while the inclusion of 5% DMSO reduces the rate of
hydrolysis by approximately 10%. Hydrolysis of compound 22 was measured by
monitoring the release of 1-butanethiol by the inclusion of 0.3 mM DTNB at pH 8.0 (ΔE412
14 150 M-1 cm-1). Hydrolyses of compounds 23-27 were monitored using a pH-sensitive
colorimetric assay.16 The reactions were conducted in 2.5 mM BICINE buffer, pH 8.3,
=
containing 0.2 M NaCl, 10% DMSO, and 0.1 mM of the pH indicator cresol purple to monitor
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proton release from the hydrolysis reaction. The effective extinction coefficient (ΔOD/M of
H+) was found to be ΔE577 = 1.68 × 103 M−1 cm−1 by titration with acetic acid at pH 8.3, 30 °C.
Product Ratios. 31P-NMR spectroscopy was used to assess the composition of the
products formed from the hydrolysis of compounds 1-27 by Sb-PTE. For each assay the
substrate was dissolved in DMSO and then diluted to 2.0 mM in a final volume of 1.0 mL
containing the combination buffer (pH 10.0) and 5% DMSO. The reactions were initiated
by addition of Sb-PTE (1-5 µM) and allowed to proceed until the reaction was complete.
Enzyme was removed via utrafiltration using a Vivaspin 500 10 kDa MWCO centrifugal
device (GE Healthcare). 100 μL D2O and 50 μL of 100 mM EDTA were added to 650 μL of
the reaction mixture prior to the 31P-NMR spectrum being recorded.
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To determine if there is a pH-dependence to the product ratios, assays with diethyl
phenyl phosphate (17) were conducted at pH 7.0, 8.0, 9.0, and 10.0. Reactions containing
0.1 μM Sb-PTE, 2.0 mM compound 17, combination buffer, and 20% methanol were
incubated at room temperature until the reaction was complete. Enzyme was removed by
centrifugation using a Vivaspin 500 10 kDa MWCO centrifugal device. 100 μL of D2O and 50
μL of 100 mM EDTA were added to 650 μL of the original reaction mixture before
recording the 31P-NMR spectrum.
To determine if there is a concentration dependence to the product ratios, total
hydrolysis reactions were conducted using 2.0 mM, 0.5 mM and 10 µM concentrations of
diethyl m-fluorophenyl phosphate (14). The 2.0 mM reaction was conducted in a volume of
1.0 mL in combination buffer at pH 10. The 0.5 mM reaction was conducted in a volume of
4.0 mL, and the 10 µM reaction was in a total volume of 50 mL. Reactions were initiated by
addition of 0.1 μM Sb-PTE and incubated at room temperature until the reaction was
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complete. The 0.5 mM and 10 µM reaction mixtures were concentrated to approximately
1.0 mL under reduced pressure, and the enzyme removed by ultrafiltration using a
Vivaspin 500 10 kDa MWCO centrifugal device. 100 μL of D2O and 50 μL of 100 mM EDTA
at pH 10 were added to 650 μL of the original reaction mixture before recording the 31P-
NMR spectrum.
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Viscosity Effects. Analysis of the effects of solvent viscosity was conducted using
sucrose as the viscogen and either 110 µM dibutyl phenyl phosphate (21) or 30 µM
paraoxon (5) as the substrate in total hydrolysis assays. Assays were conducted in a
volume of 1.0 mL with the combination buffer to maintain the pH at 10.0. Sb-PTE was
preincubated with 100 µM MnCl2 and reactions initiated by the addition of 60 or 48 nM Sb-
PTE for the hydrolysis of dibutyl phenyl phosphate (21) and paraoxon (5), respectively.
Sucrose was added to final concentrations of 2.0, 4.0, 4.64, 5.5, 10.0, 13.3, 15.6, 21.3, 26.7,
or 30 (w/w%) giving relative viscosities of 1.01, 1.03, 1.04, 1.07, 1.15, 1.2, 1.4, 1.8, 2.1 and
2.4 as previously reported.11 The assays with dibutyl phenyl phosphate (21) contained a
final concentration of 2.5% MeOH while the assays with paraoxon contained 0.25% MeOH.
Solvent Isotope Effects. Solvent isotope effects were measured using paraoxon (5)
and diethyl m-fluorophenyl phosphate (14) as the substrates at pH 8.5 and 9.0. Reactions
were conducted using 35 µM paraoxon (5) or 200 µM diethyl m-fluorophosphate (14) in
total hydrolysis reactions. Reactions were 1.0 mL total volume with 50 mM CHES and 100
mM NH4Cl. The pD of D2O solutions was determined as pD = pH + 0.4 using a standard pH
meter calibrated in H20 buffers.17 The reactions were initiated by the addition of 48 nM Sb-
PTE for paraoxon (5) or 60 nM for diethyl m-fluorophenyl phosphate (14) and followed at
an appropriate wavelength in a cuvette until the reaction was complete. Reactions were
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replicated 2 to 5 times and the reported isotope effects are the average obtained from all
replicates. The solvent deuterium isotope effect on kcat was measured by determining the
steady state reaction kinetics with paraoxon (5) at pH 9.0. Reactions were 250 µL total
volume with 50 mM CHES, 1.0 mM MnCl2, 100 mM NH4Cl and 0.001-1.5 mM paraoxon in
either H2O or D2O. Reactions were initiated by addition of 0.5 µM Sb-PTE.
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Similar reactions were conducted to determine the solvent isotope effects on the
chemical hydrolysis of 35 µM paraoxon (5) and 200 µM diethyl m-fluorophenyl phosphate
(14). Substrate stock solutions were made in D2O with 5% deuterated DMSO. 5 µL of
paraoxon or 50 µL of diethyl m-fluorophenyl phosphate was diluted into either H2O or D2O
and hydrolysis initiated by addition of 50 µL of 40% (w/w) KOD (Sigma Aldrich).
Reactions were 1.0 mL total volume and followed at the appropriate wavelength until
complete.
To evaluate the solvent isotope effect on the product ratio, approximately 2 mg of
paraoxon (5) or diethyl m-fluorophenyl phosphate (14) were hydrolyzed to completion in
a 1.0 mL total volume reaction containing 50 mM CHES (pH 8.5), 100 mM NH4Cl and 5%
deuterated DMSO with either H2O or D2O as the solvent. Substrates were predissolved in
50 µL of deuterated DMSO. Reactions were initiated by the addition of 1.2 µM Sb-PTE.
Once the reaction was complete the enzyme was removed by ultrafiltration using a
Nanosep 10K Omega (Pall Corporation) centrifugal filtration device. 650 µL of the reaction
mixture was added to 100 µL of D2O and the 31P NMR spectra were recorded.
Data Analysis. Steady state kinetic parameters for the release of phenols, alcohols
and thiols were determined by fitting the initial rates to eqn. 1 using the nonlinear least-
squares fitting program in SigmaPlot 10.0, where v in the initial velocity, Et is the enzyme
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concentration, kcat is the turnover number, [A] is the substrate concentration and Km is the
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Michaelis constant.
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9
v/Et = (kcat[A] / (Km + [A])
(1)
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Kinetic constants for the total hydrolysis of substrates by Sb-PTE and the rate
constants for the release of individual substituents from the various phosphotriester
substrates were obtained using equations 2-5, where Tkcat and T(kcat/Km) are the sum of the
kinetic constants for the hydrolysis of all leaving groups catalyzed by Sb-PTE with a given
1
substrate. kcat and 1(kcat/Km) are the observed kinetic constants obtained directly from the
spectroscopically determined hydrolysis of the phenolate (compounds 1-21) or thiolate
ester (compound 22). F is the fraction of the total product formed from the hydrolysis of
the phenolate or thiolate ester as determined from the 31P-NMR analysis of the product
2
ratios. kcat and 2(kcat/Km) are the rate constants for cleavage of the alcohol substituents by
Sb-PTE. An analogous approach was utilized for measurement of the kinetic constants for
the substituted butyl groups in compounds 23-27, except the observed rates correspond to
the total hydrolysis and each component is the obtained by multiplying its fractional
hydrolysis times the total value.
Tkcat = 1kcat/F
T(kcat/Km) = 1(kcat/Km)/F
Tkcat = 1kcat + 2kcat
(2)
(3)
(4)
(5)
T(kcat/Km) = 1(kcat/Km) + 2(kcat/Km)
Plots of log kcat or log (kcat/Km), as a function of the leaving group pKa, were fit to
equations 6 and 7, respectively, where β is the Brønsted coefficient and C is the y-intercept.
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(6)
(7)
log 푘cat = 훽 p푘a + 퐶
log (푘cat/퐾m) = 훽 p푘a + 퐶
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9
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Absorbance values for total hydrolysis reactions were converted to fraction
hydrolyzed according to eqn. 8, where A is absorbance at any time, Ao is the initial
absorbance, and ΔA is the total change in absorbance due to hydrolysis. Data were plotted
as fraction hydrolyzed as a function of time and fit to eqn. 9, where a is the magnitude of
the exponential phase, t is time in seconds and k is the observed exponential rate constant.
Dividing the observed rate constant k by enzyme concentration yields the kcat/Km for the
reaction.
퐹 = 퐴 ― 퐴
표
(8)
∆퐴
퐹 = 푎(1 ― 푒 ―푘푡
)
(9)
For the minimal kinetic mechanism of the Sb-PTE-catalyzed reaction presented in
Scheme 2, the values of kcat and kcat/Km are dependent on the relative values of the
microscopic rate constants as depicted in eqns. 10 and 11.12 The effects of changes in
solvent viscosity on the values of kcat/Km were obtained in the presence of variable
amounts of sucrose. The ratio of kcat/Km in the absence, (kcat/Km)o, and in the presence of
the viscogen, (kcat/Km)η, was plotted as a function of the relative viscosity according to eqn.
12.11 The profiles for the variation of kcat or kcat/Km with pH were fit to equation 13, where
y is the value of kcat or kcat/Km and c is the pH-independent value of y.
(10)
푘cat = (푘3푘5)/(푘3 + 푘5)
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(11)
(12)
푘cat/퐾m = (푘1푘3)/(푘2 + 푘3)
(푘cat/퐾m)o/(푘cat/퐾m)휂 = (푘3휂rel + 푘2)/(푘2 + 푘3)
7
8
9
푐
(13)
log 푦 = log
1 + 10(푝퐾
[
― 푝퐻)
]
푎
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k5
k3
A k1
EP
E+P
EA
E
k2
Scheme 2
Heavy Atom Isotope Effect Measurements. Kinetic isotope effects (KIEs) on the
Sb-PTE-catalyzed hydrolysis of paraoxon were measured using the competitive method,
and thus are isotope effects on kcat/Km.18 Scheme 3 illustrates the three positions where
KIEs were measured in the substrate paraoxon (5) and the designations used.
Scheme 3: Sites on the paraoxon substrate where kinetic isotope effects were measured and
their designations.
Natural abundance paraoxon was used for measurements of 15(kcat/Km). The 18
KIEs, 18(kcat/Km)bridge and 18(kcat/Km)nonbridge, were measured by the remote label method in
which the nitrogen atom in the p-nitrophenyl leaving group is used as a reporter for 18
O
O
fractionation.19 The mixtures of isotopic isomers used for each of the 18O KIEs are shown in
Scheme 4. The synthesis of each of the isotopic isomers was as previously described.19
Reactions for the determinations of isotope effects were performed in triplicate. For each
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reaction ~30 mg of paraoxon was dissolved in 50 mL of CHES, pH 9.0, at 25 °C and 0.5 mL
of a 100 mM stock solution of MnCl2 was added. Sufficient enzyme was then added to give
7
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12
13
14
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from 40% to 60% conversion of the substrate in 2 h.
Scheme 4: (A) The mixture of isotopic isomers of paraoxon used for measurement of
18(kcat/Km)bridge. (B), the mixture of isotopic isomers of paraoxon used for measurement of
18(kcat/Km)nonbridge
.
Reaction progress was monitored periodically by adding an aliquot of the reaction
mixture to phosphate buffer at pH 9.0 and measuring the absorbance at 400 nm. When
reactions had reached at least 40% completion, unreacted paraoxon was extracted with
methylene chloride (3 x 50 mL). The methylene chloride fractions were combined, and
after removal of solvent by rotary evaporation, the residual paraoxon was completely
hydrolyzed by addition of 50 mL of 0.1 N NaOH and stirring for 12 h. The aqueous layer
from the methylene chloride extractions contained the p-nitrophenol liberated by the Sb-
PTE catalyzed reaction. The fraction of reaction of each trial was determined by assaying
both aqueous solutions for p-nitrophenol at 400 nm in basic solution. The aqueous layer
after methylene chloride extraction, containing the enzymatic product p-nitrophenol, was
titrated to pH <5 and extracted with ether (3 x 50 mL). A similar treatment of the
hydrolyzed residual substrate samples was used to isolate the p-nitrophenol produced
from the residual substrate after partial enzymatic reaction. The p-nitrophenol samples
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were purified by sublimation and analyzed by isotope ratio mass spectrometry to
determine the 15N/14N ratios of the nitrophenol samples for the product (Rp), and for the
residual substrate (Rs). Separately, samples of the substrate were subjected to total
hydrolysis and similarly treated to determine the 15N/14N ratio in the original mixed
substrate (Ro). Each KIE experiment permits two independent determinations of the
isotope effect, one from Rs and the other from Rp, using equations 14 and 15.20
10
11
12
13
14
15
16
17
18
19
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isotope effect = log(1 - f ) / log[(1 - f ) (Rs/Ro)]
isotope effect = log(1 - f ) / log[1 - f(Rp/Ro)]
(14)
(15)
For each isotope effect measured, the values calculated from Ro and Rp and from Ro
and Rs were averaged to give the results reported in Table 2. The 15(kcat/Km) is given
directly from these equations. For the 18O isotope effects, the observed KIEs were
corrected for the 15N isotope effect and for incomplete levels of isotopic incorporation (see
Supplemental Information). The equations used for these corrections and their
derivations have been described.21
RESULTS
Optimization of Sb-PTE Assay Conditions. Previous studies of the kinetic
characteristics of Sb-PTE utilized HEPES or CHES buffer.2, 9, 10, 15 While attempting to
determine the pH-dependence of the reactions catalyzed by Sb-PTE it was noted that the
enzyme displayed a ~2-fold greater activity when TRIS was used as the buffer, rather than
HEPES, at pH 8.5. Subsequent tests demonstrated that this increase in activity appeared to
be due to the presence of the primary amine from this buffer, and that the increase in
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activity could be replicated by the inclusion of NH4+ in the assays. It was found that
inclusion of 100 mM NH4Cl resulted in a uniform catalytic activity regardless of the buffer
used. Subsequent assays for the pH-rate profile and Brønsted analysis utilized a
combination buffer, which could be used over a wide range of pH values and was
supplemented with 100 mM NH4Cl.
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14
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pH-Rate Profiles for Sb-PTE. The profiles for the effects of pH on the values of the
catalytic constants for the hydrolysis of paraoxon (5) and diethyl phenyl phosphate (17) by
Sb-PTE were determined (Figure 2). The dependence of the kinetic parameters, kcat and
kcat/Km, with pH are characterized by a single ionization indicated by a slope of +1 below
~7.0 and a plateau above pH 8.0. A fit of the data to eqn. 13 gives the pKa values from the
log kcat profile of 7.9 ± 0.1 and for the kcat/Km profile of 7.4 ± 0.1. Fitting the data for diethyl
phenyl phosphate gives pKa values of 7.3 ± 0.1 and 7.1 ± 0.1 for kcat and kcat/Km, respectively
(data not shown).
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Figure 2: pH-rate profile for the hydrolysis of paraoxon (5) by Sb-PTE at 30 °C. The data
5
6
were fit to eqn. 13. Additional information is provided in the text.
7
8
9
Multiple Products from Single Substrate. A series of diethyl (1-18) and dibutyl
(19-27) phosphotriesters with leaving groups of variable pKa were tested for the
production of alternative products using 31P-NMR spectroscopy. Multiple products were
formed from the hydrolysis of all but two of these compounds by Sb-PTE (Table 1). To
determine if the product ratios varied with pH, experiments were conducted at pH values
ranging from 7-10 with 2.0 mM diethyl phenyl phosphate (17), and the product
composition analyzed by 31P-NMR spectroscopy. It was found that the product ratios were
constant at all of the tested pH values (Figure S1). To determine if the product ratios were
valid for the subsequent analysis of kcat/Km, the product ratios derived from the hydrolysis
of diethyl m-fluorophenyl phosphate (14) were determined using an initial concentration
2.0 mM, 500 µM and 10 µM of this substrate. Reactions with the lowest concentration of
substrate were conducted in a higher reaction volume and subsequently concentrated after
total hydrolysis by Sb-PTE. The products were analyzed by 31P-NMR spectroscopy and it
was found that the product ratios did not vary with the initial substrate concentration
(Figure S2).
10
11
12
13
14
15
16
17
18
19
20
21
22
23
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Steric Effects of Phenyl Substitution. The usefulness of any chemical series for
linear free energy analysis requires the ability to alter the pKa without introducing
substantial steric effects. The catalytic activity of Sb-PTE was measured for the diethyl
phophotriester series (1-18), which utilized substituted phenols as the leaving group
(Table 1). Paraoxon (5) with a p-nitrophenol leaving group is a prototypical substrate for
phosphotriesterase activity. Adding an additional fluoride substitution in the meta-
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position (compound 4) increases activity as expected, but the mono- (compound 2) or di-
(compound 1) substitution of fluoride in the ortho-position results in a loss of activity
despite nearly three units lower pKa. Similarly, the presence of a p-carbonyl substituent
(compounds 7, 9, 11, and 12) results in dramatically more ethyl cleavage than other
compounds with similar pKa values. The unsubstituted phenyl (compound 17) gives a
kcat/Km of 1.5 x 104 M-1s-1 and 23% cleavage of the phenyl group. A para-substitution of
fluoride (16) or chloride (15) diminishes the enzymatic efficiency by more than 4-fold and
further enhances ethyl cleavage. Substitution of the fluoride at the meta-position rather
than the para- position (compound 14) results in an order of magnitude increase over the
unsubstituted phenol and results in the primary product arising from phenol cleavage.
These effects demonstrate that both catalytic activity and product distribution in the
reaction catalyzed by Sb-PTE is sensitive to steric effects from substitution on the phenyl
ring.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
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The dibutyl series of compounds (19-27) showed much smaller steric effects (Table
1). The two compounds that did show apparent steric effects were compounds 20 and 25.
Compound 20, which contains a carbonyl substitution on the phenyl ring, shows ~5-fold
less activity than the unsubstituted phenol. Compound 25 contains a carbonyl substitution
on C-2 of one butyl group, which results in nearly no cleavage of the unsubstituted butyl
groups for this compound.
Brønsted Analysis. The kinetic constants for the enzyme-catalyzed hydrolysis of
the diethyl (1-18) and dibutyl (19-27) series of compounds were measured (Table 1).
Since multiple products were formed from a single substrate in most cases, the kinetic
constants for the total hydrolysis (Tkcat and T(kcat/Km)), and the two possible pairs of
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reaction products; (1kcat and 1(kcat/Km) for the phenol or substituted butanol, and 2kcat and
2(kcat/Km) for ethanol or butanol) were calculated from the spectroscopically determined
rates of product formation and the product ratios determined by 31P-NMR spectroscopy.
The kinetic constants were plotted as a function of the pKa of the most activated leaving
group. For compounds 1-18 the plot of log 1kcat versus the pKa of the leaving phenol shows
a decrease in activity with an increase in pKa (Figure 3a). While there is significant scatter
in the data there is an overall trend with no upper plateau at the lower pKa values. A linear
fit to the data gives a β-value of -0.3 ± 0.1. The plot of log 1(kcat/Km) versus the pKa of the
leaving group also exhibits a decrease in value as the pKa increases with an apparent β-
value of -0.3 ± 0.1 (Figure 3b). For hydrolysis of the ethyl substituent, there is no obvious
dependence of the most activated leaving group pKa for either 2kcat or 2(kcat/Km) (Figures
3c and 3d). The kinetic constants for total substrate hydrolysis exhibit β-values of -0.2 ±
0.1 and -0.2 ± 0.1 for Tkcat and T(kcat/Km), respectively (Figures 3e and 3f).
10
11
12
13
14
15
16
17
18
19
20
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The trends in activity with the pKa of the leaving group were much clearer with the
series of dibutyl phosphate compounds (19-27). In this case the leaving group is either a
phenol (19-21) butylthiol (22) or a substituted butyl alcohol (23-27). For these
compounds the hydrolysis of the leaving group with the lowest pKa exhibits a β-value of -
0.33 ± 0.04 for 1kcat and -0.32 ± 0.05 for 1(kcat/Km) (Figure 4a and 4b). Hydrolysis of the
unactivated butanol group shows a similar trend with the pKa of the most activated group
giving a slope of -0.23 ± 0.07 and -0.23 ± 0.08 for 2kcat and 2(kcat/Km), respectively (Figures
4c and 4d). The rate constants for total hydrolysis (Tkcat and Tkcat/Km)) have β-values of -
0.31 ± 0.04 and -0.30 ± 0.05, respectively (Figures 4e and 4f).
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7
8
9
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12
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18
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21
22
23
24
25
26
27
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29
30
31
32
33
34
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36
37
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39
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Table 1. Kinetics constant for the hydrolysis of phosphotriesters by Sb-PTE.
a
compound pKa
Fb
Km
1kcat
1(kcat/Km)
2kcat
(s-1)
-
2(kcat/Km)
(M-1 s-1)
-
Tkcat
(s-1)
93
T(kcat/Km)
(M-1 s-1)
(mM) (s-1)
(M-1 s-1)
1
2
3
4
5
6
7
8
4.10
5.45
5.53
5.94
7.14
7.38
7.66
7.95
8.05
8.43
8.47
8.56
8.81
9.28
9.38
9.91
10.04
10.29
7.14
8.05
10.04
10.78
11.1
12.5
13.0
13.9
16.1
1.00
0.95
1.00
0.98
0.93
0.95
0.57
0.84
0.49
0.75
0.53
0.20
0.87
0.53
0.03
0.08
0.23
0.16
0.78
0.87
0.85
1.10
0.63
0.46
0.25
0.32
0.95
1.20
0.67
0.24
0.39
1.00
0.55
1.30
0.27
0.41
1.60
1.10
0.44
0.15
0.43
0.14
93
38
167
105
30
68
18
78
0.26
9.5
1.7
0.2
18
8.5 x 10⁴
5.9 x 10⁴
3.6 x 10⁵
4.2 x 10⁵
9.5 x 10⁴
7.2 x 10⁴
1.5 x 10⁴
1.2 x 10⁵
1.1 x 10³
2.4 x 10⁴
1.7 x 10³
2.7 x 10²
1.4 x 10⁴
1.6 x 10⁵
2.9 x 10³
3.7 x 10³
1.5 x 10⁴
8.6 x 10²
3.9 x 10⁴
5.1 x 10³
2.6 x 10⁴
4.0 x 10³
5.5 x 10³
1.0 x 10³
8.5 x 10⁴
6.2 x 10⁴
3.6 x 10⁵
4.3 x 10⁵
1.0 x 10⁵
7.6 x 10⁴
2.6 x 10⁴
1.4 x 10⁵
2.2 x 10³
3.3 x 10⁴
3.2 x 10³
1.4 x 10³
1.6 x 10⁴
3.0 x 10⁵
9.8 x 10⁴
4.9 x 10⁴
6.5 x 10⁴
5.5 x 10³
5.0 x 10⁴
5.8 x 10³
3.1 x 10⁴
4.9 x 10³
9.4 x 10³
3.3 x 10³
1.8
-
2.1
2.4
2.8
13
2.9 x 10³
-
40
167
107
32
71
31
93
0.6
13
3.2
0.8
21
81
40
78
71
2.5
7.5
2.5
4.4
0.42
0.83
0.36
8.0 x 10³
7.2 x 103
3.8 x 10³
1.1 x 10⁴
2.2 x 10⁴
1.1 x 10³
8.2 x 10³
1.5 x 10³
1.1 x 10³
2.1 x 10³
1.4 x 10⁵
9.5 x 10⁴
4.5 x 10⁴
5.0 x 10⁴
4.6 x 10³
1.1 x 10⁴
7.1 x 10²
4.7 x 10³
9.1 x 10²
4.0 x 10³
2.3 x 10³
15
9
0.26
3.2
1.5
0.60
2.7
38
39
72
55
2.1
1.6
0.3
0.7
0.08
0.35
0.25
10
11
12
13
14
15
16
17
18
19
20
21
22
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24
25
26
27
43
1.2
5.9
16
0.38
5.9
2.2
3.7
0.80 0.085 0.34
0.58 0.088 0.48
0.31
0.95
0.26
0.33c
0.11
0.05 0.043
0.17 0.033
0.11
8.6 x 10² 0.002 4.9 x 10¹ 0.045 9.1 x 10²
1.9 x 10² 0.010 5.7 x 10² 0.043 7.6 x 10²
1.0 x 10¹
nd
nd
nd
2.6 x 10¹
nd
3.9 x 10¹
a pKa for compounds 1-21 were obtained from references 12, 14, 19 and 22. pKa values for compounds
22-26 were predicted values calculated with Advanced Chemistry Development (ACD/Labs)
Software V11.02 and published by SciFinder. pKa valued for compound 27 was taken from
reference 23
.
bF is fraction of most activated leaving group hydrolyzed as measured by 31P NMR
c
spectroscopy. Fractional values for 27 were calculated by dividing total observed hydrolysis by a
factor of 3. The standard errors for the reported rate constants are less than ±15%.
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10 11
4
5
6
7
8
9
10 11
pKa leaving group
pKa leaving group
Figure 3. Brønsted plots for diethyl phosphotriester series compounds 1-18. Lines are
best fit to eqns. 6 or 7 giving β-values of -0.3 for both kcat (R2 = 0.35) and kcat/Km (R2 = 0.37)
for phenol leaving group hydrolysis (panels a and b) there is no trend apparent for ethyl
cleavage (panels c and d) and a β-value -0.2 for total hydrolysis for both kcat (R2 = 0.12) and
kcat /Km (R2 = 0.14) (panels e and f).
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0
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-1
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-2
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0
-1
-2
6
8
10
12
14
16 18
6
8
10
12
14
16
18
pKa leaving group
pKa (leaving group)
Figure 4. Brønsted plots for dibutyl phosphotriester series compounds 19-27. Lines are
best fit to equation 6 or 7. β-values are -0.33 (R2=0.92) and -0.32 (R2 = 0.87) for kcat and
kcat/Km, respectively, for activated leaving group (panels a and b), -0.23 (R2 = 0.57) and -0.23
(R2 = 0.57) for butyl leaving group (panels c and d) and -0.31 (R2 = 0.87) and -0.30 (R2 =
0.83) for total hydrolysis rates (panels e and f).
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Solvent Viscosity Effects. To further probe the catalytic mechanism, the solvent
viscosity effects on kcat/Km were measured with both paraoxon (5) and dibutyl phenyl
phosphate (21). In both cases there is a modest viscosity-independent enhancement of the
reaction rate in the presence of sucrose, but the slope of the viscosity plots is negligible for
both substrates (-0.017 ± 0.004 for paraoxon (5) and -0.010 ± 0.006 for dibutyl phenyl
phosphate, 21) (Figure 5).
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1.6
1.4
1.2
1
0.8
1
1.5
2
2.5
Relative Viscosity
Figure 5. Solvent viscosity effects of Sb-PTE for the hydrolysis of paraoxon (5) () and
dibutyl phenyl phosphate (21) ().
Solvent Deuterium Isotope Effects. The solvent kinetic isotope effects on kcat/Km
were measured with paraoxon (5) and diethyl m-fluorophenyl phosphate (14) at pH 8.5
and 9.0. For paraoxon, the leaving group is expected to be deprotonated at both pH values
examined, while for diethyl m-fluorophosphate, the leaving group is expected to be largely
protonated at pH 8.5 and partially deprotonated at pH 9.0. At pH values of 8.5 and 9.0 the
solvent isotope effects for hydrolysis of paraoxon are quite small with values of 0.88 ± 0.03
and 1.05 ± 0.03, respectively. For diethyl m-fluorophenyl phosphate, the solvent isotope
effects are also quite small with values of 1.15 ± 0.01 at pH 8.5 and 0.90 ± 0.1 at pH 9.0.
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Thus, there is essentially no effect on kcat/Km for either substrate when D2O is substituted
for H2O.
7
8
9
To determine if there is a solvent deuterium effects on kcat, steady state kinetics
were measured with paraoxon in H2O and D2O at pH 9.0 and determined to be 1.31 ± 0.02.
This finding was further verified by examining the product ratios for the reactions
catalyzed in either H2O or D2O at pH 8.5 using 31P-NMR. No significant changes to the
product ratios were observed for either substrate when the reaction was conducted in D2O,
suggesting that protonation of the leaving group is not rate limiting for either phenyl or
alkyl cleavage. The enzymatic results are similar to the alkaline hydrolysis of these
compounds where the solvent deuterium isotope effects for paraoxon (5) and diethyl m-
fluorophenyl phosphate (14) are 0.95 ± 0.01 and 0.88 ± 0.05, respectively.
Heavy Atom Isotope Effects. The heavy atom kinetic isotope effects on the Sb-PTE
catalyzed hydrolysis of paraoxon (5) were determined to address the transition state for
cleavage of the P-O bond of the substrate. The primary isotope effect 18(kcat/Km)bridge gives a
measure of the degree of cleavage of the P-O bond, whereas the secondary isotope effect
18(kcat/Km)nonbridge is defined by changes in bond order and to bending and torsional
vibrational modes.18 The secondary 15(kcat/Km) is a measure of charge development on the
nitrophenyl leaving group. The primary and secondary isotope effects for Sb-PTE are
provided in Table 2 for hydrolysis of paraoxon, along with the previously determined
values for the alkaline and Pd-PTE catalyzed hydrolysis of paraoxon (5) and diethyl p-
carbamoylphenyl phosphate (12).
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Table 2. Heavy atom isotope effects for the alkaline and enzymatic cleavage of phosphotriesters.
18
18
15N
O
O
bridge
nonbridge
alkaline hydrolysis
Sb-PTE (paraoxon)
Pd-PTE (paraoxon)
Pd-PTE (compound 12)
1.0007 ± 0.0001a
1.0009 ± 0.0002
1.0060b
1.0102 ± 0.0006
1.0020b
1.0063 ± 0.0001b
1.0036 ± 0.0020
1.0021 ± 0.0004b
1.0181 ± 0.0007b
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1.036 ± 0.002b
aData from reference 24. bData from reference 19
.
DISCUSSION
pH-Rate Profile. The pH-rate profile for Sb-PTE was determined using paraoxon (5)
as the substrate. For both kcat and kcat/Km, the pH-rate profiles exhibit a dependence on a
single ionization with pKa values of 7.9 and 7.4, respectively. Diminution in catalytic
activity at high pH is not observed for the hydrolysis of paraoxon up to pH 10.5. The loss of
activity at the lower pH is consistent with the protonation of the bridging hydroxide that is
proposed to be utilized for nucleophilic attack of the substrate.25 A separate general acid
for protonation of the leaving group is unlikely to be needed for the efficient release of p-
nitrophenolate. However, similar results were obtained for hydrolysis of diethyl phenyl
phosphate (17), a substrate with a poorer phenolic leaving group. The enzyme must be
able to effectively protonate the leaving group for efficient catalysis, but the lack of a loss of
activity at high pH is inconsistant with a separate general acid serving this role, unless it
occurs after the rate-limiting step. In addition, Sb-PTE has been shown to catalyze
hydrolysis of the ethyl and butyl esters in many of the compounds utilized in this
investigation. Therefore, the enzyme must also be able to effectively protonate these
leaving groups since there is no pH dependence on the product ratios determined for these
substrates.
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Brønsted Analysis. Previously, we have shown that Sb-PTE is able to catalyze
the hydrolysis of substrates with relatively unactivated leaving groups with catalytic
efficiencies similar to the cleavage of p-nitrophenol.15 The effect of the pKa of the leaving
group on kcat and kcat/Km was measured with two series of substrates. In the first series a
diethyl phosphate core was utilized with a series of substituted phenols and a second series
utilized a dibutyl phosphate core with substituted phenols and modified butyl substituents.
For the dibutyl phosphate series, the Brønsted β-coefficients for kcat and kcat/Km were
determined to be approximately -0.3 for the net hydrolysis of the substrate. This result is
consistent with the development of a relatively small amount of negative charge in the
leaving group during the transition state.26 For comparison, the β-coefficient for the
alkaline hydrolysis of a similar series of substrates is -0.43.14 The relatively linear Brønsted
plot for this series of substrates is also fully consistent with P-O bond cleavage as the rate-
limiting step in the reaction.11, 19, 27 Interestingly, when the rate of hydrolysis for the un-
activated ester is plotted as a function of the pKa of the more activated group, a linear
relationship is observed with a β-value of -0.23 suggesting that in the transition state there
is a dispersal of charge to the unreactive ester groups as well as to the leaving group.
With the diethyl phosphate series of substrates there is considerable scatter in the
data, likely due to steric constraints in the enzymatic system by the substituted phenols
(Figure 3). Interpretation of these results is more complicated because of the differential
rate constants for substrates having leaving groups of nearly identical pKa values (e.g.
compounds 8 and 9) and significant disparities in the relative magnitude of ethyl versus
phenyl hydrolysis (e.g. compounds 14 and 15). In any event, Sb-PTE can catalyze the
hydrolysis of the ethyl substituent with relatively high efficiency even in substrates with
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significantly better leaving groups. This fact is exemplified with diethyl phenyl phosphate
(17) hydrolysis where the ethyl substituent is hydrolyzed nearly four-fold more often than
the phenyl substituent.
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The Brønsted analysis also suggests that P-O bond cleavage is rate limiting for kcat
and kcat/Km for the dibutyl series of substrates tested for this investigation. The effects of
changes in solvent viscosity on kcat/Km support this conclusion. When sucrose was used to
increase the relative viscosity from 1.1 to 2.5, there was essentially no effect on the relative
magnitude of kcat/Km. Thus, for both paraoxon (5) and dibutyl phenyl phosphate (21) the
rate constant for the dissociation of substrate from the Michaelis complex (k2) is
significantly bigger than the first-order rate constant for the conversion of substrate to
products (k3). Therefore, the substrates for Sb-PTE are not “sticky” and the net rate for
substrate turnover is dictated by the relative magnitude of k3.
Kinetic Isotope Effects with Paraoxon. The kinetic isotope effects (KIEs) report on the
transition state for cleavage of the P-O ester bond in the substrate. Two of the three KIEs
report on the leaving group, while the third reports on the structure of the phosphorus
center in the transition state. The secondary isotope effect 15(kcat/Km) measures negative
charge delocalized into the nitro group due to contributions from a quinonoid resonance
form of the p-nitrophenolate anion involving the nitrogen atom.18 As a result, the 15
K
equilibrium isotope effect (EIE) for deprotonation of p-nitrophenol is normal.28 In enzymes
catalyzing phosphoryl transfer of substrates with this leaving group, this isotope effect has
a maximum of 1.0030 in enzymes lacking general acid catalysis and a late transition state
with extensive P-O bond fission. An earlier transition state, or charge neutralization by
general acid catalysis, reduces the magnitude.18
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