Subsequently, nine directly evolved PON1 variants, selected for increased hydrolytic rates with a fluorogenic diethylphosphate ester, were tested for detoxification of cyclosarin, soman, O
Trang 1organophosphates by directly evolved variants of
mammalian serum paraoxonase
Gabriel Amitai1, Leonid Gaidukov2, Rellie Adani1, Shelly Yishay1, Guy Yacov1, Moshe Kushnir1, Shai Teitlboim1, Michal Lindenbaum1, Peter Bel1, Olga Khersonsky2, Dan S Tawfik2
and Haim Meshulam1
1 Division of Medicinal Chemistry, Israel Institute for Biological Research, Ness Ziona, Israel
2 Department of Biological Chemistry, Weizmann Institute of Science, Rehovot, Israel
Keywords
acetylcholinesterase; detoxification;
organophosphates; paraoxanase;
stereoselective degradation
Correspondence
G Amitai, Department of Pharmacology,
IIBR, PO Box 19, Ness Ziona 74100, Israel
Fax: +972 8 938 1559
Tel: +972 8 938 1591
E-mail: amitai@iibr.gov.il
(Received 4 September 2005, revised 16
February 2006, accepted 23 February 2006)
doi:10.1111/j.1742-4658.2006.05198.x
We addressed the ability of various organophosphorus (OP) hydrolases to catalytically scavenge toxic OP nerve agents Mammalian paraoxonase (PON1) was found to be more active than Pseudomonas diminuta OP hydrolase (OPH) and squid O,O-di-isopropyl fluorophosphatase (DFPase)
in detoxifying cyclosarin (O-cyclohexyl methylphosphonofluoridate) and soman (O-pinacolyl methylphosphonofluoridate) Subsequently, nine directly evolved PON1 variants, selected for increased hydrolytic rates with
a fluorogenic diethylphosphate ester, were tested for detoxification of cyclosarin, soman, O-isopropyl-O-(p-nitrophenyl) methyl phosphonate (IMP-pNP), DFP, and chlorpyrifos-oxon (ChPo) Detoxification rates were determined by temporal acetylcholinesterase inhibition by residual non-hydrolyzed OP As stereoisomers of cyclosarin and soman differ signifi-cantly in their acetylcholinesterase-inhibiting potency, we actually measured the hydrolysis of the more toxic stereoisomers Cyclosarin detoxification was 10-fold faster with PON1 mutants V346A and L69V V346A also exhibited fourfold and sevenfold faster hydrolysis of DFP and ChPo, respectively, compared with wild-type, and ninefold higher activity towards soman L69V exhibited 100-fold faster hydrolysis of DFP than the wild-type The active-site mutant H115W exhibited 270–380-fold enhancement toward hydrolysis of the P–S bond in parathiol, a phosphorothiolate ana-log of parathion This study identifies three key positions in PON1 that affect OP hydrolysis, Leu69, Val346 and His115, and several amino-acid replacements that significantly enhance the hydrolysis of toxic OPs
GC⁄ pulsed flame photometer detector analysis, compared with assay of residual acetylcholinesterase inhibition, displayed stereoselective hydrolysis
of cyclosarin, soman, and IMP-pNP, indicating that PON1 is less active toward the more toxic optical isomers
Abbreviations
ChPo, chlorpyrifos-oxon [O,O-diethyl O-(3,5,6-trichloro-2-pyridyl)phosphate]; cyclosarin, O-cyclohexyl methylphosphonofluoridate; DEPCyC: O,O-diethyl phosphate O-(3-cyano-7-coumarinyl); DFP, O,O-di-isopropyl fluorophosphate; IMP-pNP, O-isopropyl O-(p-nitrophenyl)methyl-phosphonate; OPAA, organophosphorus acid anhydrolase; OP, organophosphate; OPH, organophosphate hydrolase; paraoxon,
O,O-diethyl O-(p-nitrophenyl) phosphate; parathiol, O,O-diethyl S-(p-nitrophenyl) phosphorothiolate; PC, the annotation of PON1 variants screened by the phospho-coumarin DEP-CyC; PFPD, pulsed flame photometer detector; PON1, mammalian paraoxonase (EC 3.1.8.1); soman, O-pinacolyl methylphosphonofluoridate; VX, O-ethyl S-(N,N-di-isopropylaminoethyl) methylphosphonothiolate.
Trang 2Toxic organophosphates (OPs) that serve as nerve
agents, such as O,O-di-isopropyl fluorophosphate
(DFP), soman and cyclosarin (O-cyclohexyl
methyl-phosphonofluoridate), and various insecticides, such as
chlorpyrifos, parathion and their oxo-metabolites,
chlorpyrifos-oxon (ChPo) and paraoxon [O,O-diethyl
O-(p-nitrophenyl) phosphate] (Scheme 1), exert their
toxicity by irreversible inhibition of acetylcholinesterase
[1] Inhibition of acetylcholinesterase results in severe
cholinergic toxic signs caused by increased
concentra-tions of acetylcholine at cholinergic nerve–nerve and
nerve–muscle synapses [1] The treatment of OP
poison-ing is based mainly on therapeutic combination of
anti-cholinergic drugs such as atropine together with
quaternary oxime reactivators of inhibited
acetylcholin-esterase such as 2-pyridinealdoximemethiodide and
tox-ogonin [2–4] The potential use of acetylcholinesterase
and butyrylcholinesterase for stoichiometric scavenging
of toxic OPs and various OP hydrolases (OPHs) as
cata-lytic scavengers has been studied extensively [5–8]
OPHs could also be used for noncorrosive
decontam-ination of sensitive surfaces including human skin [9]
Four groups of hydrolases have been studied with
regard to OP degradation: (a) bacterial (Pseudomonas
diminuta or Flavobacterium sp.) OPH (also known as
phosphotriesterase) was cloned and exhibited hydrolytic
activity toward various nerve agents [10]; (b)
organo-phosphorus acid anhydrolase (OPAA) from
Alteromon-as sp JD6.5 [12], a halophilic prolidase that exhibits
marked hydrolytic activity toward soman, DFP and
cyclosarin [13]; (c) recombinant Loligo vulgaris squid
DFPase cloned by Scharff et al [14] is active toward DFP and other toxic OP compounds; (d) mammalian serum paraoxonases (PON1), isolated from human, and other mammalian sera PON1 is a group of calcium-dependent hydrolases capable of catalyzing the hydro-lysis of various lactones, esters and certain OP compounds [11] The human serum paraoxonase⁄ arylesterase gene (PON1) is a member of a multigene family [15], the primary function of which appears to be lactonase [27–29] The hydrolysis of OPs, including paraoxon which gave PON1 its name, turned out to be
a promiscuous activity of PON1 [20,27,29] The rate of hydrolysis of certain nerve agents such as sarin and soman by human serum PON1 is comparable to that of
Ps diminuta OPH, with bimolecular rate constants (kcat⁄ Km) of 105)106m)1Æmin)1 [16] The catalytic effi-ciency of PON1 in the hydrolysis of sarin and soman and the possibility to re-inject it in humans render PON1 a possible candidate for medical countermeasure against nerve agent poisoning [16] Pertinently, it was estimated that a 10-fold increase in wild-type PON1 cat-alytic activity toward toxic OPs would be sufficient to provide substantial in vivo protection against certain nerve agents [17] It was also noted recently that bacter-ial OPAA and OPH catalyze preferentbacter-ially the hydro-lysis of the less toxic optical isomer of cyclosarin [30] The 3D structure of mammalian PON1 was described at 2.2 A˚ resolution [18] It is a six-bladed b-propeller with
a unique active-site lid which seems also to be involved
in high-density lipoprotein binding [18] Interestingly, the 3D structures of DFPase and PON1 are similar,
Scheme 1 Chemical structure of toxic OP substrates.
Trang 3both showing a secondary structure of a six-bladed
b-propeller [14,18] Using directed evolution, various
variants of PON1 were generated by Aharoni et al [19]
The first series of PON1 variants were evolved for
heterologous expression in Escherichia coli and exhibit
enzymatic properties that are essentially identical with
the serum-purified PON1 [19] The recombinant
vari-ants were subjected to further mutation and selection
with the aim of increasing their activity towards various
substrates [18–20] In particular, a series of PON1
vari-ants were selected after three generations of enhanced
evolution using the fluorogenic OP ester
O,O-diethyl-phosphate O-(3-cyano-7-coumarinyl) (DEPCyC) which
resembles in its structure the oxo-metabolite of the
insecticide coumaphos Certain newly evolved variants
selected with DEPCyC exhibit improved rates of OPH
activity toward DEPCyC and paraoxon compared with
wild-type PON1 by factors of up to 155-fold and
10-fold, respectively [19,20] As noted above, PON1 is a
multifunction enzyme exhibiting lactonase, esterase and
OPH activities [19] It was noted that different
muta-tions affect differently the lactonase, esterase and OPH
activity of PON1 [18,20] The amino-acid residues that
affect the OPH activity are primarily Val346, Leu69,
Lys192 and Ser193, but the effect of mutations on these
positions has thus far only been examined with
para-oxon and DEPCyC [19] It was therefore important to
examine the newly evolved PON1 variants and evaluate
their detoxification activity toward nerve agents and
other toxic OPs In this report, we demonstrate
mark-edly enhanced catalytic activity of certain newly evolved
mammalian PON1 variants mainly toward ChPo, DFP,
cyclosarin and soman We identify the residues that
affect the rate of hydrolysis of nerve agents such as
cyclosarin, DFP and soman, and mutations that
dra-matically enhance their degradation We further
des-cribe the PON1 variant H115W, in which the His115
that catalyzes lactone and ester hydrolysis is mutated to
Trp [21] This variant was found to display unexpectedly
high activity toward parathiol [O,O-diethyl
S-(p-nitro-phenyl) phosphorothiolate], a P–S bond-containing OP
The enantioselectivity of OP hydrolysis by PON1 and
some of its variants is also demonstrated here with
cyclosarin, soman and the sarin analog O-isopropyl
O-(p-nitrophenyl)methylphosphonate (IMP-pNP)
Results
Detoxification of cyclosarin and soman by bacterial
OPH, squid DFPase and mammalian PON1
The rate of enzymatic hydrolysis of cyclosarin, soman,
DFP, ChPo, IMP-pNP, paraoxon and parathiol
(Scheme 1) was determined primarily by measuring the temporal acetylcholinesterase inhibition caused by the residual nonhydrolyzed OP This enzymatic hydrolysis
of OPs measured by the acetylcholinesterase inhibition assay actually reflects detoxification of the more toxic stereoisomers of chiral OPs Our attempts to determine
Kmand kcatvalues for cyclosarin and soman using the acetylcholinesterase inhibition assay were unsuccessful because the rate of hydrolytic detoxification did not increase with increasing substrate concentrations Therefore, the time-course of OP detoxification was analyzed by measuring the initial rates of hydrolysis The first-order initial rate constant (kobs), was calcula-ted from the slope of the linear decrease in ln(% resid-ual OP) with time Eqresid-ual concentrations of OPs as well as OPH, DFPase and PON1 variants were used in all kinetic studies These conditions enable the compar-ison of initial rate constants obtained for OPH, DFPase or PON1 variant relative to wild-type PON1 Thus, changes in OPH activity observed for the newly evolved PON1 variants were evaluated by the ratio
kobs(mutant)⁄ kobs(wild-type)
The hydrolytic activity of recombinant PON1 toward cyclosarin was sevenfold and ninefold higher than that of squid DFPase and Ps diminuta OPH, respectively (at 0.03 mgÆmL)1 enzyme, 10 lm cyclo-sarin, kobs¼ 25.4 · 10)3, 3.8· 10)3 and 2.7· 10)3 min)1, respectively, Fig 1A) Furthermore, PON1 was more active than DFPase and OPH in detoxifying soman, with fourfold higher rates (at 0.03 mgÆmL)1 enzyme, 10 lm soman, kobs¼ 7.5 · 10)3, 1.8· 10)3 and 1.7· 10)3min)1, respectively, Fig 1B) The con-centration of each enzyme was 0.03 mgÆmL)1 or 0.75 lm (when the molecular mass of OPH, DFPase and PON1 is taken as 40 kDa) and OP substrate con-centration was 10 lm All kinetic data obtained for detoxification of cyclosarin and soman using the ace-tylcholinesterase inhibition assay were fitted to a single exponential decay function (Figs 2 and 3)
Modified rates of OP detoxification by newly evolved PON1 variants
The enhanced rate of detoxification of cyclosarin and soman by wild-type PON1 compared with DFPase and OPH (Fig 1) led us to study further PON1 and its vari-ants as catalytic OP scavengers New PON1 varivari-ants were evolved by directed evolution using the
fluorogen-ic OP substrate DEPCyC [19,20] Nine of these variants were evaluated for their hydrolysis of cyclosarin, so-man, DFP, paraoxon, parathiol, IMP-pNP and ChPo (Scheme 1) The most rapid detoxification of cyclosarin was obtained with the single-site mutants V346A
Trang 4(1.2PC) and L69V (1.1PC): kobs¼ 270 · 10)3 and
250· 10-3min)1, respectively, versus 25· 10)3min)1
with wild-type PON1 (Fig 2) The double mutant
L69V⁄ S193P (2.1PC) exhibited a fourfold faster
detoxi-fication rate toward cyclosarin (kobs¼ 92 · 10)3min)1;
Figs 2 and 4) The variants L69V⁄ S138L ⁄ S193P
(3.1PC), L69V⁄ S138L ⁄ S193P ⁄ N287D (3.2PC) and
L69V⁄ S138L ⁄ S193P ⁄ N287D ⁄ V346A (3.2PC⁄ V346A)
displayed 2.5–3.5-fold higher activity than wild-type
PON1 (Figs 2 and 4) The rate of soman hydrolysis by
wild-type PON1 was significantly slower than
hydroly-sis of cyclosarin and DFP (kobs¼ 7.5 · 10)3compared
with 25· 10)3and 17· 10)3min)1, respectively; Figs 2,
3 and 4; time-course for DFP hydrolysis is not shown)
However, the variant V346A (1.2PC) exhibited a
nine-fold enhancement of hydrolysis toward soman
com-pared with wild-type PON1 (kobs¼ 65 · 10)3 and
7.5· 10)3min)1, respectively, Figs 3 and 4) In
addition, the five-site mutant L69V⁄ S138L ⁄ S193P ⁄
N287D⁄ V346A (3.2PC⁄ V346A) catalyzed soman
detoxification twofold faster than wild-type PON1
(Figs 3 and 4) All other variants exhibited equal or slower hydrolytic rates than wild-type PON1 toward soman (Figs 3 and 4) The kinetic data obtained for en-zymatic hydrolysis of soman with all tested PON1 vari-ants indicate the importance of the V346A mutation for the enhancement of cyclosarin and soman hydro-lysis The PON1 variant V346A also exhibited fourfold and sevenfold faster hydrolysis than wild-type PON1 toward DFP and ChPo (kinetic data not shown; see
kobs ratios in Fig 4) The most active variant toward DFP was the single-site mutant L69V, with a 100-fold enhancement over that of wild-type PON1 (kobs¼ 1.7 versus 0.017 min)1, Fig 4) All other multiple mutants (with three to five active-site mutations) yielded faster rates than wild-type PON1 for DFP, cyclosarin and parathiol hydrolysis, but to a lower extent than the sin-gle and double mutants (Fig 4) These multiple muta-tion variants also exhibited lower activity than wild-type PON1 toward soman and ChPo (Figs 3 and 4) Thus, the most universally active PON1 variant toward DFP, cyclosarin, soman and ChPo was the single-site mutant
Fig 1 Time-course of enzymatic detoxification of cyclosarin (A) and soman (B) by Ps diminuta OPH, squid DFPase and mammalian wild-type PON1, measured by the acetylcholinesterase inhibition assay Cyclosarin and soman concentration 10 l M ; 20 m M Tris ⁄ HCl, pH 7.0; enzyme concentration 0.03 mgÆmL)1(0.75 l M ); CaCl21 m M ; 25 C Initial rates of OP detoxification (k obs , min)1mean ± SEM, n ¼ 3) were estimated from the slopes of the linear plot of ln[% OP] versus time All kobsvalues are summarized in the attached table ND, not deter-mined The linear plot is based on points transformed from the initial part (up to 50% of OP hydrolysis) of the experimental nonlinear curve All kinetic experiments were performed in triplicate The curves were fitted by one-phase exponential decay (r 2 ¼ 0.96–0.99) The plots shown are taken from one representative experiment.
Trang 5V346A, which exhibited a 4–11-fold enhanced activity
compared with wild-type PON1 (Fig 4)
It was of particular interest to search for a PON1
variant that could hydrolyze parathiol, a P–S
bond-containing paraoxon congener (Scheme 1) and thereby
to learn about putative residues involved in the
hydro-lysis of the P–S bond in OP insecticides (e.g Demeton,
malathion) and toxic nerve agents such as O-ethyl
S-(N,N-di-isopropylaminoethyl)
methylphosphonothio-late (VX) Therefore, the activity of PON1 variants
with parathiol, paraoxon and cyclosarin was also
compared at higher OP substrate concentration
(100 lm) (Fig 5) The rate of parathiol hydrolysis by
wild-type PON1 was 88-fold slower than with
para-oxon (kobs¼ 6 · 10)4 and 0.053 min)1, respectively)
However, parathiol was hydrolyzed 380-fold faster by
the H115W variant (kobs¼ 0.23 min)1, Fig 5) than by
wild-type PON1
A complete Michaelis–Menten kinetic analysis was
performed with the chromogenic symmetrical OP
sub-strates paraoxon and parathiol using selected PON1
variants Table 1 summarizes the kinetic data obtained for hydrolysis of paraoxon and parathiol by wild-type PON1 and the following variants: H115W, L69V, V346A, L69V⁄ S138L ⁄ S193P ⁄ N287D and L69V⁄ S138L⁄ S193P ⁄ N287D ⁄ V346A Figure 6 shows the kin-etics of hydrolysis of paraoxon and parathiol by H115W and wild-type PON1 It was noted that H115W enhanced the rate of parathiol hydrolysis by 270-fold compared with wild-type PON1 (kcat⁄ Km¼ 1.6· 104 versus 60 m)1Æs)1; Table 1, Fig 7) These results corroborate those obtained for H115W with parathiol as substrate using the acetylcholinesterase inhibition assay (Figs 4 and 5) H115W enhanced paraoxon hydrolysis only 16-fold (kcat⁄ Km¼ 6.4 · 104 versus 4· 103m)1Æs)1; Table 2, Fig 7) All other PON1 variants exhibited 17–28-fold enhancement of parathiol hydrolysis compared with that of the wild-type (Table 2, Fig 7) Similarly, these variants also showed a lower increase in activity toward paraoxon, with a 2–10-fold increase in kcat⁄ Km values (Table 2, Fig 7) These results strongly corroborate the data
Fig 2 Time-course of enzymatic detoxification of cyclosarin by PON1 variants measured by the acetylcholinesterase inhibition assay Cyclo-sarin concentration 10 l M ; PON1 0.03 mgÆmL)1(0.75 l M ); CaCl21 m M ; 20 m M Tris ⁄ HCl, pH 7.0 All experimental kinetics data were fitted
to mono-exponential decay curves drawn on the left (r2¼ 0.98–0.99) Initial rate value for each PON1 variant (first-order rate constant k obs , min)1, mean ± SEM, n ¼ 3) were calculated from the slopes of the linear plots of ln(% OP) versus time shown in the right panel Correlation coefficients (r 2 ) for the linear plots were 0.94–0.99 The kinetic plots shown are taken from a single representative experiment out of three replicates All k obs values are summarized in the attached table ND, not determined.
Trang 6Fig 3 Time-course of enzymatic degradation of Soman by PON1 variants measured by acetylcholinesterase inhibition assay Soman concen-tration 10 l M ; PON1 0.03 mgÆmL)1(0.75 l M ); CaCl 2 1 m M ; 20 m M Tris ⁄ HCl, pH 7.0 All experimental kinetics data were fitted to mono-expo-nential decay curves drawn on the left (r 2 ¼ 0.98–0.99) Initial rate values for each PON1 variant (first-order rate constant k obs , min)1; mean
± SEM, n ¼ 3) were calculated from the slopes of the linear plots of ln(% OP) versus time shown on the right Each k obs value is based on triplicate kinetic measurements The kinetic plots shown are taken from a single representative experiment out of three replicates All k obs values are summarized in the attached table ND, not determined.
Fig 4 Changes in hydrolytic activity (k obs ) toward toxic OP
sub-strates of PON1 variants compared with wild-type PON1 (PON1
0.03 mgÆmL)1, OP 10 l M ) Detoxification was followed by residual
acetylcholinesterase inhibition assay The change in activity of each
PON1 variant versus PON1 wild-type is expressed as the ratio
kobs(mutant) ⁄ k obs (wild-type) drawn on a logarithmic scale The value
of this ratio for wild-type PON1 is 1 The asterisk designates a
value of 1.0 obtained for the L69V variant with ChPo.
Fig 5 Changes in hydrolytic activity (k obs ) of PON1 variants using higher concentrations (100 l M ) of cyclosarin, paraoxon and parathiol
by PON1 variants (PON1, 0.3 mgÆmL)1) Detoxification was fol-lowed by acetylcholinesterase inhibition assay The change in activ-ity is expressed as the ratio k obs (mutant) ⁄ k obs (wild-type) drawn on a logarithmic scale.
Trang 7obtained for paraoxon and parathiol using the
acetyl-cholinesterase inhibition assay (Figs 4 and 5),
confirm-ing that the results obtained by the acetylcholinesterase
inhibition assay at single substrate concentration
clearly reflect the Michaelis–Menten kinetic analysis of
enzymatic activity
Stereoselective degradation of cyclosarin, soman
and IMP-pNP by PON1
As indicated previously, cyclosarin is a racemic
mix-ture of its S and R optical isomers configured around
the phosphorus (P) atom [P(–) and P(+) optical iso-mers] Soman is a mixture of four stereoisomers con-sisting of two pairs of diastereoisomers with two chiral centers: one on the phosphorus atom (P) and a second on the asymmetric carbon (C) atom of the pinacolyl group [P(–)C(+), P(–)C(–), P(+)C(+) and P(+)C(–) stereoisomers] Benschop et al [23] have noted that the pair of soman stereoisomers that are configured with the (–) isomer on the P atom [P(–)C(+⁄ –)] are 20–150-fold more toxic than the P(+)C(+⁄ –) pair of diastereoisomers It was previ-ously noted that Ps diminuta OPH preferentially
Table 1 Michaelis–Menten analysis for the hydrolysis of paraoxon and parathiol by wild-type PON1 and its evolved variants Each value rep-resents the mean of at least two independent experiments Standard deviations were less then 10% of parameter values Values in paren-theses are the x-fold increase in k cat ⁄ K m relative to the wild-type PON1.
kcat (s)1)
KM (m M )
kcat⁄ K M ( M )1Æs)1)
kcat (s)1)
KM (m M )
kcat⁄ K M ( M )1Æs)1)
(18)
S138L S193P N287D
S193P N287D V346A
Fig 6 Kinetics of hydrolysis of paraoxon (A) and parathiol (B) by the PON1 variant H115W and wild-type Hydrolysis of OP substrates was fol-lowed by measuring the increase in p-nitrophenol A405at pH 8 and 25 C Enzymatic parameters with paraoxon and parathiol were determined
by Michaelis–Menten analysis of initial rates {v0¼ k cat [E]0[S]0⁄ ([S] 0 + KM)} Values in parentheses represent molar concentrations of PON1.
Trang 8hydrolyzes the less toxic optical isomers of cyclosarin
[30] and those of p-nitrophenol analogs of sarin and
soman [25] Therefore, it was of interest to examine
the stereoselectivity of cyclosarin and soman
hydro-lysis exerted by PON1 variants We compared the
results of GC⁄ pulsed flame photometer detector
(PFPD) analysis, monitoring the chemical degradation
of all stereoisomers of soman and cyclosarin at
speci-fied time intervals, with those of the residual
acetyl-cholinesterase inhibition assay, measuring its
detoxification rate Table 2 summarizes values of
chemical degradation of soman and cyclosarin
com-pared with its detoxification level at specified time
intervals It was noted that soman is 50% hydrolyzed
by V346A within the first minute (based on GC⁄ PFPD
analysis; Table 2), whereas acetylcholinesterase
inhibi-tion bioassay reveals practically no detoxificainhibi-tion at
this short time interval (1 min) Similarly, cyclosarin was degraded by 50% within the first minute (GC⁄ PFPD analysis; Table 2) compared with less than 5% detoxification measured by acetylcholinest-erase inhibition at this short time interval (Table 2) After 100 min incubation of soman or 15 min incuba-tion of cyclosarin with V346A PON1, each agent was both degraded and detoxified by 91–98% Soman and cyclosarin were 95–98% degraded and detoxified by wild-type PON1 only after 470 and 100 min, respect-ively These data are consistent with faster hydrolysis
of the less toxic optical isomer of cyclosarin [P(+)] and the two less toxic diastereoisomers of soman [P(+)C(+⁄ –)] by V346A Stereoselective hydrolysis of chiral OP esters by PON1 was further demonstrated
by using the sarin analog IMP-pNP as substrate IMP-pNP degradation by wild-type PON1 and V346A was followed using three different analytical methods: quantitative GC⁄ PFPD analysis, direct spec-trophotometric determination of p-nitrophenol released during hydrolysis, and detoxification kinetics measured by acetylcholinesterase inhibition assay Table 3 summarizes the levels of degradation of
Fig 7 Changes in bimolecular rate constants (k 2 ¼ k cat ⁄ K M ) of
paraoxon and parathiol hydrolysis by PON1 variants compared with
wild-type PON1 determined by Michaelis–Menten analysis of the
enzymatic activity The changes in activity of each variant toward
degradation of paraoxon and parathiol are expressed by the ratio
k2(mutant) ⁄ k 2 (wild-type) drawn on a logarithmic scale.
Table 2 Comparison of degradation and detoxification levels of
soman and cyclosarin by wild-type PON1 and V346A PON1 variant
at specified time intervals % Degradation (Deg) was determined
by GC⁄ PFPD analysis and percentage detoxification (Detox) was
determined by residual acetylcholinesterase inhibition assay.
Enzyme ⁄ buffer
Time (min)
% Deg
% Detox Time (min)
% Deg
% Detox Tris, pH 7.0 1–100 < 9 < 5 1 < 9 < 5
PON1 V346A 100 > 91 > 95 15 > 98 98
PON1 wild-type 470 > 95 > 95 100 > 98 98
Table 3 Stereoselective hydrolysis of IMP-pNP by wild-type and V346A PON1 measured in parallel by GC ⁄ PFPD, spectrophotomet-ric and acetylcholinesterase inhibition assays IMP-pNP concentrat-ion 10 l M ; PON1 0.03 mgÆmL)1; 50 m M Tris ⁄ HCl, pH 8, 25 C GC analysis: samples of enzymatic degradation solutions were
extract-ed at specifiextract-ed time intervals with equal volumes of methyl t-butyl ether that were used for quantitative GC analysis Spectrophoto-metric analysis was performed by measuring increases in p-nitro-phenol absorbance acetylcholinesterase inhibition assay was measured by 5 min incubation with a 20-fold dilution aliquot of IMP-pNP sampled from the hydrolysis reaction Deg, Degradation; Detox, detoxification.
Enzyme
Time (min)
% Deg (GC)
% Deg (A400min)1)
% Detox (acetylcholinesterase activity)
Trang 9IMP-pNP by wild-type PON1 and its single mutation
variant V346A (1.2PC) at specified time intervals
using both direct spectrophotometric assay and
GC⁄ PFPD analysis, used for determination of
degra-dation levels of both stereoisomers These degradegra-dation
levels of IMP-pNP were compared with the levels of
detoxification measured by the acetylcholinesterase
inhibition assay (Table 3) Figure 8 shows the
time-course of IMP-pNP detoxification as well as
degrada-tion by wild-type PON1 and its variant V346A using
the acetylcholinesterase inhibition assay and the
spec-trophotometric method, respectively Detoxification of
IMP-pNP by the V346A PON1 variant measured by
acetylcholinesterase inhibition assay fits well to a
sin-gle exponential decay function (Fig 8), whereas the
time-course of p-nitrophenol release induced by
V346A is biphasic (Fig 8) A mono-exponential decay
fit to the experimental detoxification data yields a
sin-gle rate constant k¼ 0.005 min)1 (r2¼ 0.987) An
excellent nonlinear fit (r2¼ 0.999) to the experimental
degradation data measured by p-nitrophenol release
was obtained with the following double exponential
decay function:
%IMP-pNP¼ ½A expðk1tÞ þ ½B expðk2tÞ This fit provides two rate constants k1¼ 0.98 min)1 and k2¼ 0.014 min)1with almost equal spans (A¼ 55 and B¼ 45) consistent with equal amounts of two enantiomers in the racemic mixture The lower rate constant of the biphasic degradation curve (k2) (Fig 8)
is consistent with the first-order rate constant obtained from the acetylcholinesterase inhibition assay reflect-ing IMP-pNP detoxification (k2¼ 0.014min)1 derived from the double exponential decay fit, shown by the left ordinate in Fig 8, and k¼ 0.005 min)1 obtained from detoxification kinetics presented on the right ordinate in Fig 8)
As shown by the spectrophotomertic and GC analy-sis, IMP-pNP was already degraded 40–52% and 52–57% by wild-type and V346A PON1, respectively, within the first minute (second and third row in the third and fourth column of Table 3, Fig 9) In con-trast, no detoxification was observed with the V346A variant within 10 min and up to seven hours with wild-type PON1 as evidenced by the residual acetyl-cholinesterase inhibition assay (fifth column in Table 3, Fig 9) These results are consistent with significantly faster degradation of the less toxic isomer [P(+)] of IMP-pNP compared with its more toxic stereoisomer [P(–)] by wild-type and V346A [23,24] After 3 h in the presence of V346A, IMP-pNP was detoxified by 54% and degraded by 87–98% (third, fourth and fifth col-umn at the 11th row in Table 3, Fig 9) Interestingly, wild-type PON1 degraded IMP-pNP only up to a level
of 50% even after 21 h (Fig 8), whereas the V346A variant caused complete degradation within 4 h (Figs 8 and 9, Table 3) This property of wild-type PON1 was utilized to enzymatically separate the more toxic P(–) stereoisomer of IMP-pNP Racemic IMP-pNP (500 lm) was incubated with wild-type PON1 (0.1 mgÆmL)1) for 2 h The enzymatic reaction was monitored spectrophotometrically by measuring the increase in the absorbance of the released p-nitrophe-nol up to the plateau level obtained at 50% degrada-tion, as demonstrated in Fig 8 After hydrolysis by PON1, the nonhydrolyzed stereoisomer was extracted with methyl t-butyl ether IMP-pNP concentration in methyl t-butyl ether was determined by quantitative
GC analysis The bimolecular rate constant of human acetylcholinesterase by the separated stereoisomer of IMP-pNP was ki¼ 6.3 · 106min)1Æm)1, which is four-fold higher than that of racemic IMP-pNP (ki¼ 1.6· 106min)1Æm)1) These results are consistent with
a 16-fold difference in the rate of human acetylcholin-esterase inhibition by the P(–) compared with P(+) stereoisomer of IMP-pNP
Fig 8 Time-course of IMP-pNP degradation and detoxification by
wild-type and V346A PON1 The spectrophotometric method
meas-uring the increase in A 400 of p-nitrophenol (pNP) was used for
de-gradation kinetics, and the acetylcholinesterase inhibition assay
was used for detoxification kinetics (50 m M Tris ⁄ HCl, pH 8, 25 C).
The left ordinate presnts the scale for residual percentage IMP-pNP
during its degradation determined spectrophotometrically by
p-nitro-phenol release The right ordinate represents the percentage of
putative P(–)IMP-pNP during detoxification as determined by the
acetylcholinesterase inhibition assay.
Trang 10Hydrolysis of all the OPs was measured by the
acetyl-cholinesterase inhibition assay Acetylacetyl-cholinesterase
inhibition was measured by diluting (50–1000-fold) the
intact OP remaining in solution at various time
intervals during the enzymatic hydrolysis The
acetyl-cholinesterase inhibition assay is therefore sensitive to
changes in concentration of the more toxic isomer of
chiral OPs and reflects the rate of detoxification, rather
than degradation, of cyclosarin, IMP-pNP and soman
In the case of symmetric OPs such as DFP, ChPo,
paraoxon and parathiol, the acetylcholinesterase
inhibi-tion assay reflects the rate of both degradainhibi-tion and
detoxification The rates of detoxification of soman and
cyclosarin catalyzed by Ps diminuta OPH, squid
DFPase and PON1 shown in Fig 1 were determined
by calculating the initial rates of hydrolysis The initial
rate (kobs) is equal to the slope of linear dependence of
ln(% acetylcholinesterase inhibition) [parallel with
ln(% residual OP)] with time It is pertinent to note a
recent report on the stereoselective hydrolysis of
cyclos-arin by bacterial OPAA and OPH [30] Hydrolysis was
followed by measuring the fluoride ions released during hydrolysis This study demonstrated a 12–24.3-fold fas-ter rate of hydrolysis by OPH and OPAA for the P(+) isomer than for the P(–) isomer As the acetylcholinest-erase inhibition assay measures exclusively the hydroly-sis of the more toxic stereoisomer P(–)cyclosarin, the time-course profile of cyclosarin detoxification fits bet-ter a single-exponential decay (Fig 1A) rather than a double-exponential profile, as demonstrated previously
by the fluoride-release assay [30] Possible racemization induced by fluoride ions released during hydrolysis is unlikely, as the maximal concentration of fluoride released from 10 lm cyclosarin is not sufficient for the conversion of cyclosarin enantiomers at the time scale used in our study (not shown) The slow phase of P(–)cyclosarin hydrolysis observed by Harvey et al [30]
is consistent with the slow detoxification rate of cyclos-arin by bacterial OPH measured in the present report
by the acetylcholinesterase inhibition assay (kobs¼ 2.7· 10)3min)1; Fig 1)
Comparison of the rate of enzymatic detoxification of cyclosarin and soman using constant substrate and enzyme concentrations clearly demonstrates faster detoxification by wild-type mammalian PON1 than bac-terial OPH and squid DFPase (Fig 1) Therefore, it was
of particular interest to develop and study new PON1 variants with enhanced activity This work describes several PON1 variants with significantly improved detoxification rates toward toxic OP substrates Most notably, the single mutants V346A and H115W exhib-ited higher rates (11–380-fold) of hydrolysis of certain OPs compared with wild-type PON1 The newly evolved PON1 variants could be segregated into four main groups: group 1, H115W showing 270–380-fold enhanced hydrolytic activity toward the P–S bond in pa-rathiol compared with wild-type PON1 (Figs 4, 5 and 7); group 2, the single mutant L69V showing 10–100-fold enhanced activity toward P–F-containing OP com-pounds (i.e DFP, cyclosarin and soman; Figs 2, 3 and 4); group 3, V346A, L69V⁄ S193P ⁄ V346A and the five-site mutant L69V⁄ S138L ⁄ S193P ⁄ N287D ⁄ V346A exhibiting a 4–10-fold higher activity toward both P–O-containing (ChPo) and P–F-P–O-containing OP esters (Fig 4); group 4, includes the variants S193P, L69V⁄ S193P, L69V⁄ S138L ⁄ S193P, L69V⁄ S138L ⁄ S193P⁄ N287D displaying no enhancement or lower activity than wild-type PON1 toward soman and ChPo (Fig 4)
The H115W mutant is an interesting variation His115 and His134 have been proposed as the key cat-alytic residues of PON1 [18] However, Yeung et al [17] have shown that the paraoxonase activity of H115W PON1 is even higher than that of the
wild-Fig 9 Time-course of IMP-pNP degradation and detoxification
elici-ted by wild-type and V346A PON1 at specified time intervals
pre-sented in three dimensions Degradation of IMP-pNP was
measured by GC ⁄ PFPD analysis during hydrolysis (left side of the
cube: black bars, Tris buffer; red, wild-type; blue, V346A)
Detoxifi-cation was monitored by residual acetylcholinesterase inhibition by
IMP-pNP (right side of the cube: green bars, Tris buffer; pink,
wild-type; khaki, V346A) The time axis (minutes) is drawn on a
logarith-mic scale PON1 wild-type and V346A concentration is
0.03 mgÆmL)1; 50 m M Tris, pH 8; 1 m M CaCl2; 25 C.