From the Department of Chemistry, Texas A&M University,
College Station, Texas 77843
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INTRODUCTION |
The bacterial phosphotriesterase from Pseudomonas
diminuta catalyzes the hydrolysis of a wide range of
organophosphate nerve agents with high efficiency (1, 2). Paraoxon, the
best characterized substrate for the zinc-substituted
phosphotriesterase, is hydrolyzed with a
kcat/Km of 4 × 107 M
1 s
1 and a
kcat of 2100 s
1. The active site
of this enzyme contains a coupled binuclear metal center, which is
absolutely essential for catalytic activity (1). The native enzyme
contains two Zn2+ ions, but these metal ions can be
replaced with Co2+, Ni2+, Mn2+, or
Cd2+ with retention of full catalytic activity. From
chemical, kinetic, and genetic studies, it has been demonstrated that
the reaction proceeds via an Sn2-like
associative mechanism in which a metal-bound hydroxide ion attacks the
electrophilic phosphorus center of the substrate (2-16). The role of
one of the two metal ions within the active site is thought to involve
the activation of the hydrolytic water molecule, whereas the companion
metal ion is most likely involved in the polarization of the phosphoryl
oxygen bond of the substrate to increase the electrophilicity of the
substrate for nucleophilic attack (16). Not surprisingly, the
substrate-binding site pocket consists predominantly of hydrophobic
residues (12) that can readily accommodate a variety of nonpolar
organophosphate triesters, which explains, in part, the relatively
broad substrate specificity of this enzyme.
Phosphodiesters are chemically more resistant to hydrolysis than are
phosphotriesters. For example, it has been estimated that
ethyl-4-nitrophenyl phosphate (Structure
1, compound I) spontaneously
hydrolyzes with kobs
10
10
s
1 at 25 °C and pH 8 (17), whereas under the same
reaction conditions, the base-catalyzed hydrolysis of paraoxon,
diethyl-4-nitrophenyl phosphate (Structure 1, compound II),
proceeds with a rate constant of ~10
7 s
1
(1). The exceptional chemical stability of the phosphodiester backbone
in molecules such as RNA and DNA facilitates the conservation of
genetic information. An array of model compounds that promote the
catalytic hydrolysis of phosphodiester bonds has been designed and
synthesized (17-23). Nearly all of the model compounds constructed to
date utilize metal ions as essential cofactors to activate the
substrate and nucleophile while holding the reactants together in close
proximity (17-21). However, the enzymatic hydrolysis of the
phosphodiester bond is typically much faster than the small molecule
mimics. Many enzymes are known to catalyze the hydrolysis of the
phosphodiester bond, with kcat values ranging
from 10
2 to 103 s
1. This class
of enzymes includes, for example, the hammerhead ribozyme, which
catalyzes the site-specific hydrolysis of a phosphodiester bond with
kcat ~ 1 min
1 (24); ribonuclease
A (kcat = 1400 s
1 for UpA) (25);
ribonuclease T1 (kcat = 350 s
1 for
GpC) (26); EcoRI endonuclease (kcat = 0.9 s
1 for GpAATTC) (27); and staphylococcal nuclease
(kcat = 150 s
1 for calf thymus
DNA) (28).

structure 1. Compounds I and II.
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The existence of phosphotriesters in nature is decidedly quite rare,
and thus, it is still unclear as to the origin of the metabolic
pressure for the selection and enhancement of the enormous catalytic
activity exhibited by the bacterial phosphotriesterase from P. diminuta. Since the catalytic machinery required for the hydrolysis of phosphodiesters is apparently similar to the binuclear metal center found within the active site of phosphotriesterase, we
anticipated that phosphotriesterase would possess an inherent ability
to hydrolyze phosphodiester substrates. However, the active site of
phosphotriesterase is filled predominantly with hydrophobic residues,
and thus, this site may not be suitable for the accommodation of the
negatively charged phosphodiesters. In this paper, we provide direct
experimental evidence to support the conclusion that the bacterial
phosphotriesterase has a significant amount of phosphodiesterase activity. Moreover, the rate enhancement can be intensified by the
incorporation of a positive charge within the active site, either
through site-directed mutagenesis or the addition of alkylamines to the
aqueous medium.
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MATERIALS AND METHODS |
General--
The bacterial phosphotriesterase and the
metal-substituted derivatives were isolated as described previously
(29). The site-directed mutagenesis of the phosphotriesterase variants
(M317A, M317H, M317K, and M317R) was carried out as described
previously (8, 9, 30). Cell growth and purification of the mutant
enzymes were performed according to published procedures (29). All of the chemicals used in these experiments were purchased from Sigma, Aldrich, Fisher, or U. S. Biochemical Corp. Biochemical supplies were
purchased from Promega, Amersham Pharmacia Biotech, Bio 101, Inc.,
Perkin-Elmer, or Hoffmann-La Roche. The synthesis of oligonucleotides and DNA sequencing reactions were carried out in the Gene Technology Laboratory of the Biology Department at Texas A&M University.
Synthesis of Phosphodiesters--
Compound I was
synthesized according to the procedure of Hendry and Sargeson (17).
Compounds III-VI (Structure 2) were synthesized by a similar
procedure with the following modifications. Ethyl dichlorophosphate
(12.3 mmol, 1.46 ml) was mixed with triethylamine (12.3 mmol, 1.72 ml)
in dry ethyl ether (10 ml). One equivalent of the substituted phenol,
dissolved in dry ethyl ether (20 ml), was added dropwise to this
solution over a period of 30 min at 0 °C. The mixture was stirred
for 1 h; triethylamine (1.72 ml) was added to the mixture; and the
stirring was continued for an additional 15 min. Finally, water (5 ml)
was added, and the mixture was stirred for 30 min. The product was
extracted three times with 30-ml portions of a solution of 0.2 M aqueous triethanolamine hydrochloride, and then the water
was removed in vacuo. The resulting white solid was
resuspended in tetrahydrofuran, and then the insoluble salt was removed
by filtration. The solution was dried over anhydrous MgSO4,
and the solvent was removed. The resulting crude product was dissolved
in dichloromethane and subsequently washed with 30 ml of 2 N hydrochloric acid to remove the phosphomonoester impurity. The organic layer was dried over anhydrous MgSO4,
and the solvent was evaporated in vacuo to obtain the
desired phosphodiester. The structures of compounds
I-VI were confirmed by 1H and
13C NMR spectroscopy.
 structure 2. Compounds III-VI.
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Kinetic Measurements--
Spectroscopic determinations were made
using a Gilford 260 UV-visible spectrophotometer. The reactions were
followed by monitoring the appearance of the substituted phenol from
the hydrolysis of the phosphodiester (1-20 mM) upon the
addition of the phosphotriesterase at 25 °C. The
pKa values, extinction coefficients at pH 9.0, and
max values of the leaving group phenols were obtained from the literature (3). Extinction coefficients at different pH values
were calculated using Equation 1,
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(Eq. 1)
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where o is the extinction coefficient of the
substituted phenolate anion. Stock solutions of the various amines were
made at 2.5-5.0 M concentrations, and pH values were
adjusted using hydrochloric acid or sodium hydroxide.
Data Analysis--
The values of kcat and
Km were determined by fitting the kinetic data to
Equation 2,
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(Eq. 2)
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where v is the initial velocity,
kcat is the maximum velocity,
Km is the Michaelis constant, and A is
the substrate concentration. The pH-rate profiles were analyzed by
fitting the data to Equations 3-5 to obtain the pK
values.
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(Eq. 3)
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(Eq. 4)
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(Eq. 5)
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The Brønsted plots were fit to straight lines, and the values were obtained directly from the slopes of these lines. The kinetic data from the experiment where the phosphodiester concentration was varied at different levels of methylamine were fit to Equation 6,
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(Eq. 6)
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where A is the concentration of phosphodiester,
B is the concentration of amine, Km is
the Michaelis constant of the phosphodiester in the absence of added
amine, Vmax is the maximum velocity in the
absence of added amine, Ka is the equilibrium
constant for the formation of an enzyme-amine complex, and and are the ratios of Km (or KA) and
Vmax in the presence and absence of added
methylamine, respectively.
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RESULTS |
Enzymatic Hydrolysis of Ethyl-4-nitrophenyl Phosphate (Compound
I)--
Compound I was tested as a substrate for the
cobalt-substituted bacterial phosphotriesterase. The rate of hydrolysis of compound I catalyzed by the phosphotriesterase was very slow (kcat = 0.06 ± 0.01 s 1
and kcat/Km = 1.6 ± 0.3 M 1 s 1 at pH 9.0) compared with
that of paraoxon (kcat = 8600 s 1
and kcat/Km = 4.3 × 107 M 1 s 1 at pH
9.0), but was >108 times faster than the uncatalyzed
reaction under similar reaction conditions. The phosphotriesterase used
for this study was purified by gel filtration. The phosphodiesterase
and phosphotriesterase activities for compounds I and
II were measured for each fraction during the purification
in addition to the absorbance readings at 280 nm. A plot of these
activities versus the fraction number is presented in Fig.
1 and serves to illustrate the
coincidence of the two activities during the purification. Four mutant
enzymes were also prepared in a preliminary attempt to enhance the
overall rate of phosphodiesterase activity exhibited by the native
enzyme. Of the four mutant enzymes, M317K catalyzed the hydrolysis of the phosphodiester test substrate (compound I) with the highest kinetic constants (kcat = 0.34 ± 0.03 s 1 and
kcat/Km = 11 ± 1 M 1 s 1 at pH 9.0). The kinetic
constants for the other mutants (M317A, M317R, and M317H) are listed in
Table I.

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Fig. 1.
Gel filtration chromatography of the
phosphotriesterase. , A280; ,
phosphotriester hydrolysis activity; , phosphodiester hydrolysis
activity. The phosphodiesterase activity was normalized to adjust for
the relative specific catalytic activities. Additional details are
given under "Results."
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Table I
Kinetic constants for the mutant enzymes with phosphodiester and
paraoxon
Conditions were pH 9.0 and 25 °C for the cobalt-substituted
phosphotriesterase. The data were fit to Equation 2, and the standard
errors for each of the kinetic constants reported below were <25% of
the stated values.
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The slow hydrolysis rate of compound I was accelerated up to
200-fold when various alkylamines (0.1-2.0 M) were added to the reaction mixture. The kinetic constants for these rate enhancements are summarized in Table II.
In general, a greater rate acceleration was observed for the wild-type
enzyme when amines with shorter and/or branched alkyl chains were
added. Secondary amines showed a larger enhancement than primary
amines. The greatest rate enhancement was observed with 2.0 M dimethylamine (kcat = 0.23 ± 0.01 s 1 and
kcat/Km = 260 ± 30 M 1 s 1 at pH 9.0). For the M317A
mutant enzyme, amines with longer alkyl chains yielded greater
enhancements (Table III).
t-Pentylamine was the best activator for this mutant enzyme
(kcat = 3.8 ± 0.1 s 1 and
kcat/Km = 210 ± 10 M 1 s 1 at pH 9.0). No
enhancement of the phosphodiesterase activity of the wild-type
phosphotriesterase was observed in the presence of Ca2+ or
Mg2+ at concentrations of <100 mM.
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Table II
Effect of added amines on phosphodiester (compound I)
hydrolysis by phosphotriesterase
These assays were conducted at 25 °C and pH 9.0. The data were fit
to Equation 2. The standard errors for each of the kinetic constants
reported below were <20% of the stated values.
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Table III
Effect of added amines on the phosphodiester hydrolysis by the
M317A mutant enzyme
These assays were conducted at 25 °C and pH 9.0. The data were fit
to Equation 2. The standard errors reported for each of the kinetic
constants were <10% of the stated values.
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pH-rate Profiles--
The pH-rate profiles of
log(V/Km) versus pH were made
without any amine added (Fig.
2A) and also with either
guanidine (Fig. 2B) or dimethylamine (Fig. 2C) in
the reaction mixtures. In general, the reactions were very slow and the
Km values were very large when no amine was added,
and thus, only V/Km values could be
determined with sufficient confidence. In the pH profile without added
amine, it can be seen that protonation of a single group accelerates
the catalytic reaction. The pKa value of this
functional group could not be determined from the experimental data
obtained over the pH range of 6.4-9.6. When guanidine was added,
protonation of a single group diminished the reaction rate. From a
least-square fit of the data to Equation 3, the pKa
value of this group was determined to be 6.5 ± 0.2. Two ionizable
groups are involved in the enzymatic reaction in the presence of
dimethylamine. Fitting of the data to Equation 5 yielded
pKa values of 5.8 ± 0.2 and 9.1 ± 0.1 for these two groups. The pH-rate profiles for phosphodiester
hydrolysis by the M317H and M317R mutant enzymes were also obtained.
The profiles of log(V/Km)
versus pH for both enzymes show a drop in activity above pH
~7 (data not shown). From a fit of these data to Equation 4,
pKa values of 7.7 ± 0.1 and 7.0 ± 0.1 were obtained for the M317H and M317R mutants, respectively.

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Fig. 2.
The pH-rate profiles of the phosphodiester
hydrolysis catalyzed by the phosphotriesterase. A, in
the absence of added amine; B, in the presence of 0.4 M guanidine; C, in the presence of 1.0 M dimethylamine. Additional details are given under
"Results."
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Variation of Metal Substitution--
The effects of substitution
of the binuclear metal center of the wild-type phosphotriesterase with
Co2+, Zn2+, and Cd2+ at pH 7 and 9 are presented in Table IV. The kinetic
constants for paraoxon (compound II) hydrolysis are
presented for comparison. The Co2+-substituted enzyme shows
the highest kcat value for phosphodiester (compound I) hydrolysis as well as for paraoxon (compound II) hydrolysis. The relative magnitude of the
kcat values (Co2+ > Cd2+ > Zn2+) at pH 9 is similar to that of
paraoxon hydrolysis.
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Table IV
Hydrolysis of phosphodiester (compound I) and paraoxon by
the metal-substituted enzymes
These assays were conducted at 25 °C. The data were fit to Equation 2. The standard errors are <25% of the stated values.
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Variation of Amine Concentration--
The added amine and
phosphodiester concentrations were varied together in order to
elucidate the kinetic mechanism for the enhancement reaction. The
overall reaction rate was increased with increasing amounts of
methylamine (Fig. 3), and these data were
fit to Equation 5. The values of kcat and
Km are 0.06 ± 0.01 s 1 and
38 ± 10 mM, respectively. The and values are
0.03 ± 0.02 and 7 ± 2, respectively. These values
demonstrate that the addition of the amine decreases
Km and increases kcat.

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Fig. 3.
Double-reciprocal plot of the
phosphodiester hydrolysis catalyzed by the phosphotriesterase in the
presence of 0 ( ), 0.063 ( ), 0.125 ( ), 0.25 ( ), 0.50 ( ),
and 1.0 ( ) M methylamine. The data were fit to
Equation 6. Additional details are given under "Results."
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Brønsted Plots for the Hydrolysis of Phosphodiesters--
Kinetic
constants were determined for compounds I and
III-VI in the presence and absence of trimethylamine. Trimethylamine was selected because primary and secondary amines form
imine or iminium adducts with the carbonyl groups of compounds III and V. In the absence of added amine, only
V/Km values could be determined because
the Km values were too large to be accurately
measured. The Brønsted plots (Fig. 4)
show a linear relationship between the pKa values of
the leaving group and log(Vmax) or
log(V/Km). The values are 1.3 ± 0.3 and 1.7 ± 0.1 for log V/Km in the
absence and presence of added amine, respectively. The value for
Vmax in the presence of added amine is
1.9 ± 0.4.

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Fig. 4.
Brønsted plots for the hydrolysis of
phosphodiester compounds I and III-VI. Upper
panel, log(V/Km)
versus pKa of the leaving group in the
absence ( ) and presence ( ) of trimethylamine; lower
panel, log(Vmax) versus
pKa of the leaving group in the presence of
trimethylamine. Additional details are given under "Results."
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DISCUSSION |
Hydrolysis of Phosphodiesters--
The full negative charge within
the phosphodiester substrate is thought to be primarily responsible for
the slow rate of catalytic hydrolysis of these compounds by the
phosphotriesterase. The active site of the phosphotriesterase is
largely hydrophobic (12), and thus, it would not be expected to
accommodate the negative charge on the substrate very well.
Furthermore, the nucleophile in the active site, i.e.
metal-bound hydroxide ion, may not be able to attack the anionic
substrate effectively. As a result, the phosphodiesters have diminished
kcat and elevated Km values.
To enhance the hydrolytic reaction rate, cations were added to the
reaction medium in an attempt to neutralize the negative charge of the
substrate. Alkylamines were selected because they are cationic in the
pH range used for this investigation, and the size and hydrophobicity
could easily be controlled by varying the number and the length of the
alkyl chains. For the wild-type enzyme, faster reaction rates were
observed when amines with shorter alkyl chains were added, whereas the
M317A mutant enzyme preferred amines with longer alkyl chains. Met-317
is located within the pro-S-binding pocket of the enzyme
active site (12). It was thought that by mutating the methionine
residue to a smaller alanine residue, larger amines could be
accommodated more effectively. Amines with branched alkyl chains
accelerated the reaction faster than amines with straight chains, and
secondary amines were more effective than primary amines in enhancing
the reaction. The reasons for these preferences are not yet clear. The
other three mutant enzymes, M317H, M317K, and M317R, were designed to
incorporate a cationic group within the enzyme active site without
adding amines from the external medium. The rate enhancements were not very large, and the pH-rate profiles showed a pattern similar to that
of the wild-type enzyme without added amine. It appears that these
mutant enzymes catalyze the phosphodiesterase reaction with little help
from the substituted cationic residues, with the possible exception of
M317K, which showed a 7-fold enhancement of
kcat/Km. The small rate
enhancement by these mutations could, in part, be attributed to the
distance between Met-317 and a substrate analogue bound in the enzyme
active site (Fig. 5). The distance
between the thiomethyl group of the side chain of Met-317 and the
phosphorus center of the bound substrate analogue is 8.2 Å (12). When
this methionine residue is replaced with other amino acid residues with
cationic side chains (arginine and lysine), the distance between the
positive charge of the new side chains and the negative charge of a
bound phosphodiester substrate was estimated to be ~6 Å using
Insight II (Biosym Technologies) to model the approximate locations of
the substrate and mutated amino acid.

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Fig. 5.
Active-site structure of the
phosphotriesterase with the substrate analogue diethylphenyl
phosphonate (12). The distance between the terminal methyl group
of the side chain of methionine 317 and the phosphorus atom of the
bound substrate analogue is 8.2 Å. DEP, diethylphenyl
phosphonate.
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Many previous attempts have been made to construct mutant enzymes with
altered substrate specificity. For example, the arginine residue of
aspartate aminotransferase that ion pairs with the side chain
carboxylate of the substrate was mutated to aspartate in an attempt to
change the substrate specificity of this enzyme from aspartate to the
cationic amino acids (lysine and arginine). Rate enhancements of
9-16-fold of the kcat/Km
values for lysine and arginine were observed (31). In a subsequent study using the same enzyme, the
kcat/Km value for the transamination of phenylalanine was increased 1500-fold after mutation
of six active-site residues (32).
pH-rate Profiles--
The pH profile for diester hydrolysis in the
absence of added amine shows that a particular group needs to be
protonated for catalytic activity. This group could be either the
P-O group of the phosphodiester substrate or the
metal-bound hydroxide ion. The protonation of the substrate would
accelerate the reaction by neutralizing the negative charge of the
substrate and making it appear more like a phosphotriester. On the
other hand, protonation of the metal-bound hydroxide would reduce the
nucleophilicity of the attacking group, but charge repulsion would be
reduced, and the overall reaction rate may be enhanced. The pH-rate
profile in the presence of 1.0 M dimethylamine displays a
bell-shaped curve, suggesting that there are two ionizable groups
involved in the catalytic reaction. One of these may be the metal-bound water (pKa = 5.8), whereas the other may be
dimethylamine (pKa = 9.1). The reaction rate
decreases at low pH, where the bound hydroxide is protonated, and also
at high pH, where the amine itself is deprotonated. The pH profile in
the presence of 0.4 M guanidine shows a somewhat different
behavior. The rate increases as the pH increases, until it is saturated
at pH ~8. The pKa of the group is 6.5, and from
this value and the shape of the profile, it is thought that this group
is the metal-bound water molecule (16). Unlike the case with
dimethylamine, the rate does not decrease at high pH because
guanidinium cation cannot be deprotonated below pH 10.
The pH profiles of the M317H and M317R mutant enzymes for
phosphodiester hydrolysis were also measured. The patterns of these profiles were similar to the one obtained with the wild-type enzyme reaction without added amine. The M317R profile shows a loss of activity at high pH, whereas the profile of the wild-type enzyme in the
presence of 0.4 M guanidine shows a loss of activity at low
pH. It appears that the guanidine group of the M317R mutant enzyme is
not properly located for effective charge neutralization, and the
reaction proceeds without significant assistance from the cationic
residue. The relatively small 2-fold enhancement of the
kcat/Km value supports this
view.
Kinetic Mechanism of Phosphodiester Hydrolysis--
Kinetic
measurements were made at six different methylamine concentrations in
an effort to elucidate the kinetic mechanism of the amine-assisted
enzymatic reaction. The data and the least-square fit are shown in Fig.
3. Equation 5, to which the kinetic data were fit, was derived from the
mechanism shown in Scheme 1.
In this mechanism, the amine forms a complex with the enzyme, and
then the substrate adds to form an enzyme-amine-substrate complex. This
ternary complex is then hydrolyzed prior to the release of the
products. When [B] = 0, the reaction mechanism becomes a
simple Michaelis-Menten-type reaction with kcat = 0.06 s 1 and Km = 38 mM.
The amine-assisted pathway has a faster kcat
( = 7) and a smaller Km ( = 0.03) than the
non-assisted pathway. An alternative mechanism in which a
substrate-amine complex (AB) is formed prior to binding to
the active site did not fit the experimental data.
Brønsted Plots--
The Brønsted plots for the dependence of
log(V/Km) on the pKa
of the leaving group in the absence and presence of trimethylamine have
lg values of 1.3 and 1.7, respectively (Fig. 4). The
large negative lg values strongly suggest that the
chemical step is the rate-limiting step, and the transition state of
the reaction has considerable product-like character. In the presence
of trimethylamine, the plot of log(Vmax)
versus pKa of the leaving groups has a
slope of 1.9 and was slightly more negative than the slope of the
log(V/Km) versus
pKa plot of the same reaction ( 1.7).
Mechanism of Phosphodiester Hydrolysis--
The data
presented in this paper demonstrate that the catalytic machinery that
has evolved for the hydrolysis of phosphotriesters can be exploited to
effectively hydrolyze phosphodiester substrates. The biggest obstacle
the phosphotriesterase has in recognition of phosphodiester substrates
is in accommodation of the negative charge and the loss of binding
energy derived from the missing alkyl substituent. These factors can,
in part, be overcome through chemical rescue via the addition of
alkylamines from the external medium. Attempts to accomplish the same
aim through site-directed mutagenesis were less successful. The most
likely cause for this failure is the imprecise placement of the
positive charge from the mutant residue. Therefore, further work is
ongoing to construct and characterize mutant enzymes that can more
effectively catalyze the hydrolysis of phosphodiester bonds. A working
model for phosphodiester hydrolysis as catalyzed by the bacterial
phosphotriesterase is presented in Fig.
6. The phosphoryl oxygen bond is
polarized by the metal ions, and the negative charge is neutralized by
the added amine. The suggested mechanism is similar to that of the phosphotriester hydrolysis (16), where the metal ions polarize the
phosphoryl oxygen bond and the metal-bound hydroxide attacks the
phosphorus atom.
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FOOTNOTES |
*
This work was supported in part by National Institutes of
Health Grant GM 33894 and by the Advanced Technology Program from the
State of Texas.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed. E-mail:
raushel{at}tamu.edu.
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