Transition States and Control of Substrate Preference in the Promiscuous Phosphatase PP1

Article abstract of DOI:10.1021/acs.biochem.7b00441

Catalytically promiscuous enzymes are an attractive frontier for biochemistry, because enzyme promiscuities not only plausibly explain enzyme evolution through the mechanism of gene duplication but also could provide an efficient route to changing the catalytic function of proteins by mimicking this evolutionary process. PP1γ is an effectively promiscuous phosphatase for the hydrolysis of both monoanionic and dianionic phosphate ester-based substrates. In addition to its native phosphate monoester substrate, PP1γ catalyzes the hydrolysis of aryl methylphosphonates, fluorophosphate esters, phosphorothioate esters, and phosphodiesters, with second-order rate accelerations that fall within the narrow range of 10¹¹-10¹³. In contrast to the different transition states in the uncatalyzed hydrolysis reactions of these substrates, PP1γ catalyzes their hydrolysis through similar transition states. PP1γ does not catalyze the hydrolysis of a sulfate ester, which is unexpected. The PP1γ active site is tolerant of variations in the geometry of bound ligands, which permit the effective catalysis even of substrates whose steric requirements may result in perturbations to the positioning of the transferring group, both in the initial enzyme-substrate complex and in the transition state. The conservative mutation of arginine 221 to lysine results in a mutant that is a more effective catalyst toward monoanionic substrates. The surprising conversion of substrate preference lends support to the notion that mutations following gene duplication can result in an altered enzyme with different catalytic capabilities and preferences and may provide a pathway for the evolution of new enzymes.

Full text of DOI:10.1021/acs.biochem.7b00441

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Article
Transition States and Control of Substrate
Preference in the Promiscuous Phosphatase PP1
YUAN CHU, Nicholas H Williams, and Alvan C. Hengge
Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.7b00441 • Publication Date (Web): 05 Jul 2017
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Transition States and Control of Substrate Preference in the Promiscuous Phosphatase
PP1
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Yuan Chu,a Nicholas H. Williams*b and Alvan C. Hengge*a
aDepartment of Chemistry and Biochemistry, Utah State University, Logan, Utah 84322-0300
bCentre for Chemical Biology, Department of Chemistry, University of Sheffield, Sheffield, U.K.,
S3 7HF
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Abstract
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Catalytically promiscuous enzymes are an attractive frontier for biochemistry, because enzyme
promiscuities not only plausibly explain enzyme evolution through the mechanism of gene
duplication, but could also provide an efficient route to changing the catalytic function of
proteins by mimicking this evolutionary process. PP1γ is an effectively promiscuous
phosphatase for the hydrolysis of both monoanionic and dianionic phosphate-ester based
substrates. In addition to its native phosphate monoester substrate, PP1γ catalyzes the hydrolysis
of aryl methylphosphonates, fluorophosphate esters, phosphorothioate esters, and
phosphodiesters, with second-order rate accelerations that fall within the narrow range of 1011 to
1013. In contrast to the different transition states in the uncatalyzed hydrolysis reactions of these
substrates, PP1γ catalyzes their hydrolysis through similar transition states. PP1γ does not
catalyze the hydrolysis of a sulfate ester, which is unexpected. The PP1γ active site is tolerant of
variations in the geometry of bound ligands, which permit the effective catalysis even of
substrates whose steric requirements may result in perturbations to the positioning of the
transferring group, both in the initial enzyme-substrate complex and in the transition state. The
conservative mutation of arginine 221 to lysine results in a mutant that is a more effective
catalyst toward monoanionic substrates. The surprising conversion of substrate preference lends
support to the notion that mutations following gene duplication can result in an altered enzyme
with different catalytic capabilities and preferences, and may provide a pathway for the evolution
of new enzymes.
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Introduction
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Catalytic promiscuity describes the ability of an enzyme to catalyze chemically distinct
reactions. This can involve forming and cleaving different type of bonds, or using more than one
mechanistic pathway for the same overall bond making and breaking process. A related concept
is substrate promiscuity, in which substrates with varying structures undergo the same chemical
reaction with similar transition states. In both cases, the catalytic efficiencies (kcat/KM) are often
significantly lower than the native reaction.
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In recent years the promiscuity of enzymes has drawn considerable attention for the
insights offered into enzyme evolution, and for potential applications in enzyme engineering and
biocatalysis. It has been proposed that promiscuity might provide a starting point for divergent
evolution after gene duplication, based upon the fact that many enzymes have remarkably broad
substrate specificities.1,2 Observations of catalytic promiscuity in the alkaline phosphatase (AP)
superfamily identified close evolutionary relationships between AP members where the
promiscuous activity of one member is the native function of another.3 Promiscuity has been
utilized to engineer enzymes with altered activities. These results show that single or few point
mutations can substantially improve the ability of enzymes to carry out new reactions.4-10
In contrast to the growing number of examples of catalytic promiscuity, the detailed
study of the origins of the promiscuity is deficient.11 The illustration of effects contributing to
catalytic promiscuity, such as the mechanistic basis, structures of active sites, and the
identification of the critical properties of substrates, are needed for a deep understanding of
promiscuity, and eventually, for predicting and utilizing protein evolution pathways.
Protein phosphatase-1 (PP1), a member of the phosphoprotein phosphatase (PPP) gene
family, is a metalloenzyme that utilizes a dinuclear metal center to catalyze the hydrolysis of
phosphate esters using a metal-coordinated hydroxide nucleophile. Highly conserved residues
bind the metal ions within approximately 3 Å of each other in octahedral coordination with a
bridging hydroxyl and bridging acetate oxygen. Other cationic residues (histidine and arginine)
line the active site which lies near the enzyme surface12 (Figure 1).
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Figure 1. Active site of PP1 showing residues implicated in coordination of the metal
ions at the active site and in substrate binding and catalysis.
Besides its native phosphate monoesterase activity PP1 exhibits activity for aryl
methylphosphonate substrates.13 In this work, PP1 is shown to also possess substrate
promiscuity for fluorophosphate esters, phosphorothioate esters, and phosphodiesters (Figure 2,
structures 2, 3 and 5). These substrates vary in their intrinsic reactivity, the size and charge of
the transferring group, and in the mechanisms and transition states for their uncatalyzed
hydrolysis. Linear free energy relationships and kinetic isotope effects were used to probe the
transition states of the PP1-catalyzed reactions compared to the uncatalyzed hydrolysis reactions.
Two active site arginine residues, R96 and R221, are implied by crystal structures to
hydrogen bond to the substrate, and probably provide transition state stabilization. These were
each mutated to lysine, and the effects on the respective substrate activities were assessed.
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Figure 2. Compound classes used as substrates for PP1 catalysis. Phenyl substituents
are defined in Table 1.
Table 1. Phenolic leaving groups used in PP1-catalyzed hydrolysis reactions with their pKa
values obtained by titration.13 Data for cyano phenols are literature values.14
Phenyl substituent (X)
pKa
7.01
7.54
7.95
8.14
8.61
9.30
8.82
9.97
4-NO2
4-Cl, 3-NO2
4-CN
a
b
c
d
e
f
3-NO2
3-CN
4-Cl
3-Cl
H-
g
h
Experimental section
General Experimental Details. All chemical reagents and solvents were commercial
products and were used as received unless otherwise noted. Thiophosphoryl chloride was
distilled under nitrogen. Pyridine was distilled from calcium hydride. Acetonitrile was dried over
anhydrous K2CO3 for 24h followed by distillation.
Synthesis of substrates. 4-Nitrophenyl phosphate monoester bis(cyclohexylammonium)
salt (1a) was prepared and purified as previously reported.15 The aryl phosphorothioate
bis(cyclohexylammonium) salts (2a – h) were prepared and purified as previously reported.16, 17
The potassium salts of aryl fluorophosphate monoesters (3a - f) were prepared by a modification
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of a literature method from 2,4-dinitrofluorobenzene and the appropriate aryl phosphoric acids in
the presence of triethylamine.18, 19 3a and 3d-f are previously reported compounds, and NMR
spectra were in agreement with those previously reported. 3b and 3c are new compounds. A
typical procedure, described for the potassium salt of phenyl fluorophosphate monoester, was as
follows. The bis(cyclohexylammonium) salt of phenyl phosphate was dissolved in a minimum
amount of water. The solution was cooled, then strongly acidified with sulfuric acid and
extracted with ether. The ethereal extract was combined and evaporated, leaving phenyl
phosphoric acid as a thick syrup. The crude phenyl phosphoric acid (10 mmol, 1.74 g), 2,4-
dinitrofluorobenzene (12 mmol, 2.23 g) and triethylamine (23 mmol, 3.2 mL) were stirred in dry
acetonitrile (10 mL) at room temperature for 24 h. The solvent was evaporated, the product was
dissolved in 25 mL water and treated with amberlite IR120 (H) in an ice bath and then filtered.
The filtrate was washed with diethyl ether, then neutralized with potassium carbonate. The crude
yellow product was obtained after lyophilization. The pure product was recrystallized from
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methanol/acetone/diethyl ether. 4-cyanophenyl fluorophosphate potassium salt (3b): H NMR
(300MHz, D2O) δ/ppm 7.68 (2H, d, J = 6.2 Hz), 7.21 (2H, d, J = 8.6 Hz); 31P NMR (122MHz,
D2O) δ/ppm -11.28 (d, JPF = 946 Hz); 19F NMR (283MHz, D2O) δ/ppm -75.41 (d, JPF = 946 Hz).
1
3-cyanophenyl fluorophosphate potassium salt (3c): H NMR (300MHz, D2O) δ/ppm 7.53-7.33
(4H, m); 31P NMR (122MHz, D2O) δ/ppm -10.76 (d, JPF = 946 Hz); 19F NMR (283MHz, D2O)
δ/ppm -75.93 (d, JPF = 946 Hz)
4-nitrophenyl methylphosphonate sodium salt (4a). The sodium salt of 4-nitrophenyl
methylphosphonate was prepared according to a literature method.13
Methyl 4-nitrophenyl phosphate diester (5a). The sodium salt of methyl 4-
nitrophenylphosphate diester was prepared using a modification of a literature procedure.20 4-
Nitrophenol (3.0 g, 21.6 mmol) in dry pyridine (10 mL) was added dropwise to a solution of
phosphoryl chloride (2 mL, 21.5 mmol) in dry pyridine (25 mL). After addition was finished the
mixture was stirred for another 45 min at room temperature. Methanol (1.6 mL, 40 mmol) was
added slowly to the mixture, the reaction mixture then refluxed for 2 h. The reaction solution was
filtered to remove pyridinium hydrochloride salt. The filtrate was added to 20 mL of cold water.
The resulting solution was titrated down to pH 4-5 using HCl and extracted with
dichloromethane. Removal of the solvent by rotary evaporation yielded the neutral form of
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methyl 4-nitrophenyl phosphate diester. The crude product was re-dissolved in 20 mL of water
and titrated to pH 9 with sodium carbonate. The pale yellow product was obtained after
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lyophilization. The pure product was obtained by recrystallization from acetone/methanol. H
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NMR (300MHz, D2O) δ/ppm 8.15 (2H, d, J = 8.9 Hz), 7.23 (2H, d, J = 8.6 Hz), 3.58 (3H, d,
JPOCH = 11.4 Hz); 31P NMR (122MHz, D2O) δ/ppm -3.03.
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Expression and purification of PP1 wild type. Four isoforms of the PP1 catalytic
subunit are found in mammals, and all are highly homologous and differ only in the C-terminal
domain.21 This work used the PP1γ isoform, and will be referred to as PP1 in this paper.
Expression in E. coli DH5α harboring the plasmid pCWOri+ and purification were accomplished
by a slight modification of the previously described method. 22 Briefly, the plasmid pCWOri+
was used to transform competent E. coli DH5α cells. Single colonies from E. coli DH5α cells
were taken from cells plated on LB/ampicillin plates and used to inoculate 20 mL cultures in
LB/ampicillin media containing 1.0 mM MnCl2, which were grown overnight at 37ºC. These
were then used to inoculate 2 liter cultures (LB/ampicillin media containing 1.0 mM MnCl2) and
grown at 37ºC until the absorbance at 600 nm reached a value of about 0.6. IPTG was then added
to a concentration of 1.0 mM, and the culture was grown at room temperature for 22 hours. The
bacteria were harvested by centrifugation at 8000 rpm and 4ºC for 30 min. Typical yields of cell
pellet were in the range 8-10 gram per liter culture. The next steps were carried out at 4ºC. The
resulting pellet (10 g) was resuspended in 25 mL of buffer A (0.1 mM EGTA, 25 mM triethanol
amine, 1 mM MnCl2, 0.1% v/v 2-mercaptpethanol, 5% v/v glycerol, 3% by mass Brij 35, pH 7.5)
containing protease inhibitors (2 mM benzamidine and 200 μg/mL each of aprotinin, pepstatin,
and leupeptin). The cells were lysed by sonication and spun down at 45,000 rpm for 30 min. The
supernatant was filtered through a 0.45 μM PES filter to remove residual debris followed by a
three-step chromatographic purification that included a Heparin hi-trap column, a Q sepharose
column, and a SP sepharose column. After each column, aliquots of each fraction were taken to
access the progress of the purification by SDS-PAGE and pNPP activity. Enzyme-containing
fractions were concentrated and stored at -80ºC. Typical yield of purified PP1 was 15 mg per
liter of culture. The buffer for PP1 stock solution contained 0.1 mM EGTA, 25 mM triethanol
amine, 1 mM MnCl2, 0.1% v/v 2-mercaptoethanol, 30% v/v glycerol, 3% by mass Brij 35 at pH
7.5.
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Generation of Mutants. Primers used for each specific mutation were as follows: PP1F
5’-GGAGGTCATATGGCGGATTTAG-3’; PP1R 5’-
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CATCGATAAGCTTGCATGCCTGCAGCTCGAC-3’; R96K 5’-
GGGGACTATGTGGACAAGGGAAAGCAG-3’; R221K 5’-
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GGCTGGGGTGAAAATGACAAAGGAGTG. The underlined bases indicate the changes in
sequence from the wild type PP1. Bases shown in bold and italic indicate the restriction sites
NdeI (PP1F) and HindIII (PP1R). Site-directed mutagenesis was performed using a two-step
extension overlap PCR method.23 The first PCR amplification using the PP1F primer and 3’
mutant primer created the DNA fragment running from the beginning of the PP1γ cDNA to the
mutated sequence of interest. This procedure was also performed with the PP1R primer and 5’
mutant primer to give the other section running from the beginning of the mutated sequence to
the end of PP1γ cDNA. The PCR conditions used were 95ºC for 1 min for 1 cycle, followed by
30 cycles consisting of 95ºC for 30 seconds, 50ºC for 30 seconds, 72ºC for 1 minute; followed
by 72ºC for one 3 min cycle. The resulting PCR product was purified and applied to the second
PCR amplification with the PP1F primer and PP1R primer to give the full length mutated PP1γ
cDNA. The PCR conditions for the second round amplification were, for the first 5 cycles, 95ºC
for 30 seconds, 45ºC for 40 seconds 72ºC for 90 seconds; and for the remaining 35 cycles, 95ºC
for 30 seconds, 58ºC for 30 seconds, and 72ºC for 1 minute. The PCR product was purified
using the Qiagen MinElute gel extraction kit and subjected to digestion with NdeI and HindIII.
This fragment was ligated into the pCWOri+ vector, which was previously digested with
NdeIHindIII and purified by agarose gel electrophoresis. The construct was then used to
transform competent DH5α cells. The mutant plasmid DNA was isolated from the culture using
Qiagen Miniprep DNA purification kit and sent for sequencing. The mutant of PP1 was
expressed and purified as described for PP1 wild type.
Kinetic measurements. For the reactions producing 4-nitrophenol, reactions were
followed by measuring the absorbance of the product at 400 nm in a Spectramax plate reader at
25 °C. All of the reactions were performed in buffered solutions (50 mM Tris, 50 mM Bis-Tris,
100 mM NaOAc, 0.05 mM MnCl2). Enzymatic reactions were quenched by adding 1.5 M
sodium carbonate bringing the final pH to ≥ 10. The literature value of the extinction coefficient
for the 4-nitrophenolate anion of 18300 cm-1 M-1 was used.24 Initial rates (less than 5% of the
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reaction) were determined at a range of substrate concentrations and used to construct Michaelis-
Menten plots. Values for Vmax and KM were obtained by fitting the data to the Michaelis-Menten
equation using a nonlinear least-squares fit (Kaleidagraph, Synergy Software). Values of kcat
were obtained by dividing Vmax by the enzyme concentration. For reactions with other leaving
groups, reactions were followed by reverse phase analytical HPLC (Agilent Eclipse XDB-C18
column, 4.6 × 150 mm, 5 μm). The substituent phenol products were eluted using an isocratic
gradient of methanol and water. The composition of the eluting solution and detection
wavelength were altered for each phenol to reach the optimum separation and retention time.
Table S1 in Supporting Information gives the detection wavelength and retention times for each
phenol (b – h in Table 1). A concentration calibration curve for each substituent phenol was
constructed using concentration ranges of 5 – 300 μM. The concentrations of phenol product
from the enzyme assay were determined by integration of the peak area at this wavelength and
interpolation of the calibration curve. The literature pKa for each substituent phenol was used in
the Brønsted plot.25
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Inhibition measurements. The rates of PP1-catalyzed pNPP hydrolysis in the presence
of the inhibitors were determined by measuring the formation of the 4-nitrophenolate product at
400 nm as described above. Inhibitor concentrations were in the range between 1/5-fold to 3-fold
times Ki. The initial rates were fitted to Lineweaver–Burk plots to determine the type of
inhibition. All inhibitors produced lines intersecting on the y-axis, consistent with competitive
inhibition. A sample plot is provided in the Supporting Information. The resulting apparent
kcatapp/KMapp values were plotted against the inhibitor concentration ([I]) and fitted to Equation 1
to obtain the inhibition constant (Ki). For inhibition experiments with magnesium and aluminum
fluorides, the AlFx and MgFx species were generated in situ from aluminum or magnesium
chloride and sodium fluoride. Control experiments showed that neither aluminum or magnesium
chloride alone, nor sodium fluoride, are inhibitory at concentrations below 50 mM. These
inhibition experiments included from 2- to 6-fold excess sodium fluoride stoichiometry relative
to Al or Mg. Previous inhibition experiments with other enzymes suggest that the inhibitory ion
has the MF3 stoichiometry.26-28
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Isotope effect measurements. The 18O kinetic isotope effects with 2a were measured by
the remote label method, using the nitrogen atom in the nitro group as a reporter for isotopic
fractionation in the labeled oxygen positions. Figure 3 shows the positions at which KIEs were
measured. The experimental procedures used were the same used to measure KIEs in other
phosphoryl transfer reactions in which the leaving group is 4-nitrophenol29 and as previously
reported for 1a and 4a.13 The details of the data analysis for the reactions of 2a are given in the
Supporting Information.
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a
O
18
k
nonbridge
a
S
P
O
b
18
k
O
bridge
NO2
15k
Figure 3. The substrate pNPPT (2a) with the positions indicated at which isotope effects were
measured.
Test for sulfatase activity. Both control and enzymatic reactions were assayed by
measuring the absorbance of the 4-nitrophenolate product at 400 nm at 25 °C. Both were
performed in buffered solutions (50 mM Tris, 50 mM Bis-Tris, 100 mM NaOAc, 0.05 mM
MnCl2, 3 mM DTT, pH 7.0). The concentration of para-nitrophenyl sulfate potassium salt was
9.3 mM, and PP1 concentrations up to 134.6 μM were used. A control reaction was set up
identically but without PP1. After 15 hours, the changes in absorbance at 400 nm for both
reactions were less than 0.003 absorbance units. The test for the PP1 R96K and R221K mutants
led to the same observation. Assuming a lower limit for product detection based on a change of
0.005 absorption units, ~ 0.7 μM of product could have been detected under these conditions.
Results
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Hydrolysis of different substrate classes by PP1 wild type. PP1 performs multiple turnovers
with each phosphate substrate examined, and saturation kinetics was observed for all substrates
except p-nitrophenyl sulfate, 6. No enzymatic activity with the sulfate ester was detected for
either wild type PP1 or mutants. The pH dependencies of kcat/KM for substrates 2a, 3a, and 5a
were measured and show a bell-shaped profile indicative of catalysis by both acidic and basic
residues (Figure 4). For the pH range sampled the protonation states of the substrates do not
change. The data were fitted to Equation 2, derived on the assumption that the active ionic form
requires one acidic (defined by Ka1) and one basic residue (defined by Ka2). The derived pKa
values and values of (kcat/KM)lim are shown in first three columns in Table 2. The previously
reported pH profiles for substrates 1a and 4a are also shown in Figure 4.
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!"#
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!
!
!
!
!
!
!
!"#
!"#
=
Equation 2
!
!
!
!
!
!
! ! ! ! !
!
! !
!
!
!
!
!
3.5
1a pNPP
2a pNPTP
3a pNPFP
4a pNPMP
5a diester
3
2.5
2
1.5
1
0.5
0
4
5
6
7
8
9
10
11
pH
Figure 4. pH-rate profiles of kcat/KM for the hydrolysis of five different substrate types catalyzed
by PP1: 2a (■), 3a (●) and 5a (▲) are from this work; 1a (●) and 4a (□) have been previously
reported.13 Solid lines are from fits to Equation 2.
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Table 2. The kinetic pKa values for the wild type PP1-catalyzed hydrolysis of substrates 1a-5a at
25 ºC derived from fits to Equation 2. Substrates 2a, 3a and 5a are from this work. Data for
substrates 1a and 4a were previously reported.13
7
8
9
lim
kcat/KM
(M-1 s-1)
kw
(kcat/KMlim)/
substrates
pKa1
pKa2
(M-1 s-1)
kw
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
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30
31
32
33
34
35
36
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60
6.0±0.2 7.2±0.2 1850±500 2.3 x 10-11
8.0 x 1013
1.7 x 1011
5 x 1011
1a (pNPP)
2a (pNPTP)
6.0±0.1 7.4±0.1
6.5±0.1 8.2±0.1
6.0±0.2 7.2±0.2
31±4
1.8 x 10-10
2 x 10-10
105±15
240±30
3a (pNPFP)
1.8 x 10-11
1.3 x 1013
8.2 x 1012
4a. (pNPMP)
5a (pNP diester)
7.2±0.2 7.5±0.2 0.98±0.4 1.2 x 10-13
Comparisons with uncatalyzed hydrolysis and rate accelerations. The fifth column
of Table 2 (kw) lists the second-order rate constants for the uncatalyzed reactions with water of
substrates 1a, 2a, 4a, and 5a. These were obtained from literature information as follows. For 1a,
the pseudo-first order rate constant of 1.3 x 10-9 s-1 for the water reaction at 25ºC was calculated
from reported activation parameters30 and divided by 55M to obtain the second order rate
constant. Similarly, for 2a, the pseudo first order rate constant of 9.8 x 10-9 s-1 at 25ºC was
calculated from reported activation parameters16 and divided by 55M. The second order rate
constant shown for 4a has been reported previously at 30ºC and 60 °C,31 and the Eyring equation
was used to predict the rate constant at 25 °C. For 5a, an estimate of 1.3 x 10-11 s-1 was obtained
for the reaction of bis-4-nitrophenyl diester with water by using the reported temperature
dependence of the spontaneous hydrolysis of the bis-2,4-dinitrophenyl diester and LFER data for
symmetrical diaryl phosphate diesters at 100 °C. An Eyring plot for the bis-2,4-dinitrophenyl
diester hydrolysis was constructed, and the assumption made that the bis-4-nitrophenyl diester
will have the same intercept (effectively assuming that the entropy of activation is the same for
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the two compounds). The LFER data at 100 °C were used to generate a second point for this
compound, and the two points used to estimate the rate of hydrolysis at 25 °C. This number is
statistically corrected for the two leaving groups (the effect of the non-leaving group on
phosphate diester hydrolysis is small, unlike the effect in phosphate triesters)32 and for the
concentration of water to give the value in Table 2. The rate constant for the water reaction of
fluorophosphate ester 3a has been measured at 80 °C (4 x 10-7 s-1), but without activation
parameters (N. H. Williams and M. Sasi, unpublished data). However, this reaction can be
compared with the second order rate constants for reaction with hydroxide at this temperature
(0.058 M-1 s-1), to give a ratio of 7 x 10-6:1. If the enthalpy of activation is at least as large as for
the specific base catalyzed reaction, then the ratio at 25 °C will be at least as small. As the
second order rate constant for reaction with hydroxide has also been measured at 42 °C,36 it can
be estimated as 3 x 10-5 M-1 s-1 at 25 °C. Combining this with the ratio of reactivity at 80 °C leads
to the estimate shown in Table 2 as an upper limit.
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The ratio of kcat/KMlim to kw gives the second order rate acceleration provided by PP1.
These vary from 1.7 x 1011 to 8.0 x 1013, which are in the range of values observed for other
catalytically promiscuous enzymes, such as BcPMH.33 It is noted that even with a reduction in
the rate for 4a to account for the 5ºC difference in temperature, the rate acceleration for this
substrate will still fall within the bounds of the other substrates.
LFER and KIE measurements. The leaving group dependency of the PP1-catalyzed
hydrolysis reactions were used to obtain the Brønsted slope (βLG) for the log of (kcat/KM) versus
pKa of the leaving phenolate at pH 7.0 for the series of phosphorothioate (2) and fluorophosphate
(3) esters using the aryl leaving groups in Table 1. The diester substrate (5) reacted too slowly to
obtain LFER data. The results are compared with previous data from phosphate monoesters and
methyl phosphonate esters. Figure 5 shows the Brønsted plots for the substrate classes 1-4. The
slopes are collected in Table 3 and shown with the values for the corresponding nonenzymatic
hydrolysis reactions. The Brønsted slopes for all of the PP1-catalyzed reactions lie within the
range -0.25 to -0.40, while those for the nonenzymatic reactions are more variable. Insufficient
data exists to compare the Brønsted slopes of substrate classes 1-4 for their water and hydroxide
reactions. However, data for both nucleophiles on phosphate diesters show that the βLG
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decreases modestly (~ 0.2 units) for the stronger nucleophile, although exact comparison is not
possible due to differences in reaction conditions and in the diesters themselves.34
7
8
9
Table 4 gives the KIEs for the PP1-catalyzed reactions of substrates 1a, 2a, and 4a.
Syntheses for the needed double-labeled forms of 3a could not be carried out with sufficient
isotopic purity for KIE determinations. All KIEs were measured by the competitive method, in
which a mixture of the isotopic isomers is present in solution. Measurement of KIEs using the
competitive method yields the isotope effect on (V/K), or, the isotope effect on the rate-limiting
step for the part of the overall reaction sequence up to and including the first irreversible step.
For PP1-catalyzed reactions this will be P-O bond fission, and thus, the isotope effects will
reflect this chemical step even if a subsequent step such as product release is rate-limiting in the
overall kinetic mechanism.
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4
1 phosphate
2 phosphorothioate
3 fluorophosphate
3.5
4 phosphonate
3
2.5
2
1.5
1
0.5
0
6.5
7
7.5
8
8.5
9
9.5
10
10.5
pK
a
Figure 5. Brønsted plot for the PP1-catalyzed hydrolysis of four substrate classes: aryl
phosphorothioate monoesters 2 (■) and aryl fluorophosphate monoesters 3 (●) are from this
work; aryl phosphate monoester dianions 1 (●) and aryl phosphonate monoesters 4 (□) are from
previous work.13
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Table 3. Brønsted βLG values for the PP1-catalyzed and the nonenzymatic hydrolysis reactions
of substrate types 1-4 with either water or hydroxide.
6
7
8
9
Substrate
βLG for PP1-catalyzed reaction
βLG for nonenzymatic hydrolysis
class
1
-0.32 13
-0.25 ± 0.02
-0.40 ± 0.07
-0.30 13
-1.23 24 (water)
-1.1 35 (water)
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25
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32
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35
36
37
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52
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60
2
3
4
-0.63 36 (hydroxide)
-0.69 13 (hydroxide)
Table 4. KIE data for uncatalyzed and PP1-catalyzed reactions. New data from this study for
substrate 2a are in bold.
Results for 2a (pNPTP)
Results for 1a (pNPP)
Results for 4a (pNPMP)
Dianion
Dianion
PP1-
Monoanion
PP1-
H2O
PP1-
H2O attack,
catalyzed
50ºC 35, 37
OH attack,
catalyzed 13
25ºC 13
attack,
catalyzed13
95ºC 24, 38
18
k
1.0135 (13) 0.9717 (18) 0.9994 (5) 0.9959 (4)
1.0015 (4)
1.0098 (4)
1.0006 (3)
0.9973 (4)
1.0129 (3)
1.0010 (1)
nonbridge
18
k
1.0237 (7)
1.0027 (1)
1.0161 (5) 1.0189 (5) 1.0170 (3)
1.0001 (1) 1.0028 (2) 1.0010 (2)
bridge
15k
Inhibition. Table 5 reports the inhibition constants for the species that were tested as inhibitors
of PP1 as described in the Experimental Section. Competitive inhibition was found in all cases.
For the metal fluoride species, fits of the data to Equation 1 assumed that the concentration of
inhibitory species equaled the concentration of Mg or Al. Since not all of the metal will be in the
correct inhibitory complex state with fluoride, these fits give an underestimation of the inhibition
constants of the putative MF3 species. Magnesium chloride and sodium fluoride alone were very
weak inhibitors, with estimated inhibition constants of ~70 mM (MgCl2) and ~150 mM (NaF).
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Table 5. Inhibition of the PP1-catalyzed hydrolysis of 1a (pNPP) by inhibitors at pH 7.0.
Inhibition constants for MgFx and AlFx were calculated as described in the Experimental section.
6
7
8
Inhibitor
Geometry
Ki (mM)
Inhibitor
Geometry
Ki (mM)
9
10
11
12
13
14
15
16
17
18
19
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22
23
24
25
26
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31
32
33
34
35
36
37
38
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O
O S O
O
O
O
Tetrahedral
1.2 ± 0.2
Tetrahedral
36 ± 2
P
HO
O
S
O S O
O
O
O
Tetrahedral
3.0 ± 0.2
Tetrahedral
Tetrahedral
Tetrahedral
22 ± 2
30 ± 2
40 ± 9
P
F
O
-
Octahedral
Tetrahedral
5.6 ± 0.8
8 ± 2
PF6
O
O Se O
O
-
BF4
O
MgFX
AlFX
planar
planar
0.83 ± 0.06
O2N
O S O
O
0.032 ± 0.003
Hydrolysis of substrates 1a-5a by PP1 mutants. The R221K and R96K mutants and
the double mutant were tested for the hydrolysis of the nitrophenyl substrates 1a-5a. Reduced
rates of some mutants did not permit a full pH-rate profile to be obtained, so catalysis of each
mutant compared with native PP1 at pH 7.0, shown in Table 6. The R96K mutant was less
active than R221K mutant for all substrate classes. The R221K mutant was found to be a more
efficient enzyme for the hydrolysis of the monoanionic substrates 3a and 4a than wild type PP1.
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Table 6. Catalytic efficiencies (kcat/KM, M-1s-1) for the hydrolysis of substrates 1a-5a at pH 7.0
by wild type and arginine mutants of PP1.
6
7
8
Double
WT
R96K
R221K
mutant
kcat/KM,
(M-1s-1)
18±2
9
10
11
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Substrates
kcat/KM,
(M-1s-1)
869±3
20±3
kcat/KM,
(M-1s-1)
60±8
kcat/KM,
(M-1s-1)
315±31
1.9±0.2
157±8
kcat (s-1)
KM(mM)
1a
2a
3a
4a
5a
3.2±0.1
0.029±0.001
0.76±0.03
1.12±0.07
0.016±0.001
3.6±0.3
1.5±0.1
9.2±0.4
13±1
0.8±0.1
25±12
11±2
---a
83±7
17±8
86±8
115±10
1.8±0.4
5.7±0.7
---a
9.2±0.9
1.7±0.3 0.35±0.08
a. the activity is too slow to be accurately measured.
Discussion
The transition states of the PP1-catalyzed reactions are more similar than the
transition states of the uncatalyzed reactions. The substrate classes may be grouped into two
types by charge: the dianionic 1 and 2, and the monoanionic 3-6. PP1 efficiently catalyzes the
hydrolysis of five of the six substrate classes. These esters also differ in the size of the group
undergoing transfer from the ester group to water, and have pronounced differences in the
mechanism and transition states of their uncatalyzed hydrolysis reactions. The uncatalyzed
reactions of phosphate monoester dianions (1) are concerted and have a loose transition state
with extensive bond fission to the leaving group and little bond formation to the nucleophile.24, 30,
37-40 Phosphorothioates (2) react via an SN1 (DN+AN) mechanism in which a (thio)metaphosphate
intermediate is formed.16, 35, 41 Sulfate monoesters (6) hydrolyze by loose transition states similar
to those for the hydrolysis of phosphate monoester dianions.42 In contrast, the reaction
mechanisms of substrates 3-5 are concerted, with a tighter transition state with less bond fission
to the leaving group and significant bond formation to the nucleophile. 13, 20, 36, 39 Of interest is
whether the transition states of the PP1-catalyzed reactions retain these differences.
Linear free energy relationships (LFER) were applied to characterize transition states for
the PP1-catalyzed hydrolysis of aryl phosphorothioates 2 and aryl fluorophosphate monoesters 3.
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The Brønsted coefficient βLG is defined as the slope of rate constants versus leaving group pKa,
reflecting the extent of bond cleavage in transition states. The βLG values for the four substrate
classes for which such data could be obtained show that their values for the enzymatic reactions
fall within a much narrower range (-0.25 to -0.40) than the uncatalyzed reactions (-0.63 to -1.23).
This suggests that, in contrast to the uncatalyzed reactions, the PP1-catalyzed reactions share
more similar transition states. The reduced βLG values are consistent with the proposal that H125
carries out general acid catalysis, as previously proposed based on structural data and
comparisons of the active site with related metallophosphatases.12, 43
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Like the βLG values, the KIEs for the three substrate classes measured are more similar to
one another than the same data for their uncatalyzed reactions. Like the βLG, the KIEs in the
leaving group for PP1-catalyzed reaction of 2a are diminished from their values in the
uncatalyzed hydrolysis, as expected from general acid catalysis. The magnitude of 15(V/K)
reflects negative charge developed on the leaving group in the transition state. For 2a, the 15(V/K)
of unity indicates that leaving group neutralization is complete, and is more effective than with
substrates 1a and 4a. Why general acid catalysis should be more effective with 2a is not clear,
but the magnitude of the Brønsted βLG slope for 2a is also slightly smaller than the other
substrate types. The leaving group 18(V/K)bridge is affected by P-O bond fission and by
protonation. With the nitrophenyl leaving group, this KIE may be as large as 1.035 when there is
no inverse contribution from protonation.29 The 18(V/K)bridge in the PP1-catalyzed hydrolysis of 2a
is similar to previously obtained values for reactions of 1a in which the transition state is
characterized by extensive P-O bond fission and concurrent protonation of the leaving group.29
In the uncatalyzed hydrolysis of 2a, the magnitude for 18kbridge implies a large degree of P-O bond
fission in the transition state, and 15k demonstrates that the leaving group departs as the charged
anion. In the PP1-catalyzed hydrolysis reaction of 2a the magnitude of 18(V/K)bridge is reduced,
consistent with general acid protonation of the leaving group, hypothesized to be carried out by
histidine 125. This KIE is also similar to those of substrates 1a and 4a, supporting the notion
from the Brønsted βLG data that the transition state is similar in each enzymatic reaction.
The other oxygen KIE, 18(V/K)nonbridge, is significantly inverse, in contrast to the normal
KIE for the solution hydrolysis. This same switch to an inverse 18(V/K)nonbridge has been noted
previously in the alkaline phosphatase-catalyzed reaction of 2a, and ascribed to an inverse effect
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arising from coordination of the thiophosphoryl group to the metal center.17, 35 Both LFER and
KIE data indicate that the PP1-catalyzed hydrolysis reactions for all substrate types have similar
transition states, with extensive P-O bond fission involving general acid protonation of the
leaving group.
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We conclude that, while the uncatalyzed hydrolysis reactions of these substrates proceed
through quite different transition states, the transition states of their PP1-catalyzed reactions are
more similar. Interactions between substrates and the active site are different than those with
water in solution. These interactions will affect vibrational modes as well as characteristics of
the transition state, and similar strong active site interactions across the substrate classes are
likely responsible for the observed similarities in the LFER and KIE data for the PP1-catalyzed
reactions. The notion that PP1 is able to modify transition states is consistent with recent
computational analyses of factors that contribute to promiscuity, although other such enzymes,
such as alkaline phosphatase, appear to accommodate variable transition states in the same active
site.44
PP1-catalyzed hydrolysis of phosphorothioate monoesters, fluorophosphate
monoesters and phosphonate monoesters follow the same catalytic mechanism proposed for
phosphate monoester hydrolysis. The pH-rate profiles for the substrates (1a – 5a) are bell-
shaped and largely coincide with a similar pH optimum, indicating the same active site and
enzymatic mechanism is followed. The deduced kinetic pKa values obtained from fits to
Equation 2 are listed in Table 2. Based on the crystal structure of PP1 and related Ser/Thr
phosphatases, a metal-coordinated water molecule is proposed to be the nucleophile and the
deprotonation of this species is likely represented by pKa1.12 Although the values for pKa1
obtained for esters 2a and 3a differ slightly (6.0 and 6.5, Table 2), plotting kcat for 2a and 3a
yields identical values for pKa1 (pKa1 = 6.5 ± 0.1) of the ES complex. These pKa values are
consistent with the assignment of the lower pKa to a metal-bound water.45 The fitted kinetic pKa1
value for substrate 5a is about 1 pKa unit higher than the other substrate classes. The residue
H125 is plausibly assigned to act as a general acid, responsible for the basic limb of the pH-rate
profile. The differences in pKa values from the pH-rate profiles for the fluorophosphate and the
methyl phosphate diester, compared to the other substrates, are greater than experimental
uncertainty but are of uncertain origin. It is possible that small local differences in solvation or
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side chain interactions, resulting from differences in sterics, charge, and polarizability of these
substrates are responsible. These are monoanionic substrates, but the differences cannot be due
to charge alone, as the monoanionic methyl phosphonate exhibits values for pKa1 and pKa2 that
match the natural dianionic monoester substrate.
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Binding of substrates and inhibitors is affected more by charge than size. The values
for KM for substrates 1a-5a at pH 7.0 are shown in Table 6. The dianionic substrates 1a and 2a
have the best affinity, as expected for a phosphatase for which dianionic monoesters are the
natural substrates. The lower KM of the phosphorothioate may reflect the previously noted
preference of Mn2+ to coordinate sulfur ligands.46, 47 The higher KM values for monoanionic
substrates 3a, 4a, and 5a, and the similarity of the KM for 3a and 5a, indicates that loss of a
negative charge has a larger effect on binding to PP1 than the addition of steric bulk by addition
of a methyl or methoxyl in place of a nonbridging oxygen atom.
To further investigate the relative influences of size, geometry, and charge on affinity for
the active site of PP1, a series of small molecule ionic inhibitors with various geometries were
examined. The left side of the Table 5 compares the affinity of inorganic phosphate to a series of
fluoride-based inhibitors. Phosphate monoesters undergo reaction by a concerted mechanism
with trigonal bipyramidal transition state. Of the tested inhibitors, only the metal fluorides are
able to assume this geometry. As phosphate analogues, the metallic fluorides AlFX and MgFX
have been shown to be useful chemical probes for structural and mechanistic studies in
phosphoryl transfer reaction because they are able to mimic the transition state for phosphoryl
transfer when x = 3. The Al-F bond has a similar length as the P-O bond (1.5-1.6Å). It is
believed that aluminofluorides can adopt tetrahedral, trigonal bipyramidal, and octahedral
geometries in a protein active site.48, 49 Magnesium fluoride complexes with trigonal bipyramidal
and octahedral geometries have been found in active sites of proteins in X-ray structures.26, 50
Here, while neither magnesium, aluminum, or fluoride ion alone are effective inhibitors, strong
inhibition is observed in combination. This indicates that the inhibitory species are magnesium
fluoride and aluminum fluoride complexes formed in situ. It is not certain what geometry these
metallic fluoride inhibitors adopt in the active site of PP1, but based on precedents with other
phosphoryl transfer enzymes, a trigonal bipyramidal geometry with three fluorides in equatorial
positions is most likely. 19F NMR has been used to successfully characterize MgF3 complexes at
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an enzymatic active site 26 but was unsuccessful here due to the presence of paramagnetic Mn2+
ions.
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The simple fluoride salt NaF shows negligible inhibition of PP1. In contrast, fluoride has
been found to significantly inhibit the activity of bovine spleen purple acid phosphatase (PAP), a
member of a related class of metallophosphatases with a dinuclear metal center, with Ki ranging
between 2 mM to 3 μM depending upon the pH and metal in the active site.51 Crystal structures
show fluoride displaces the hydroxide bridging the two metal ions in PAP.52
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Unexpectedly, the octahedral hexafluorophosphate anion is also a competitive inhibitor
with Ki = 5.6 mM, lower than that for tetrafluoroborate. To the best of our knowledge this is the
first example of an octahedral anion inhibitor of a phosphatase.
Together, the results indicate that the binding of anions to the active site of PP1 is more
affected by charge than geometry. The ability of the PP1 active site to accommodate ions of
differing conformations but similar charge is consistent with its ability to catalyze reactions of
differing substrates, and to modify their transition states.
PP1 has high, and similar, catalytic efficiencies towards the substrate classes 1-5.
The five substrate types (Figure 2) hydrolyzed by PP1 have differences in their charge, steric
requirements, and in the transition states for their respective uncatalyzed hydrolysis reactions.
The kcat/KMlim for five substrates (1a – 5a) obtained from the pH profiles (Table 2) allows a
comparison of catalytic efficiencies among substrates at optimum pH. For wild type PP1,
catalytic efficiency decreases in the order 1a>4a ~ 3a>2a ~ 5a. The difference in kcat/KMlim from
the fastest to the slowest substrate is only ~2000-fold. Catalytic efficiencies for the monoanionic
substrates 3a and 4a are superior to that for 2a, although 2a shares the dianionic charge of
phosphate monoesters that are the natural substrates for PP1.
Due to slower rates, the full pH-rate profiles needed to fit kcat/KMlim for the five substrates
catalyzed by the mutants were not obtainable. However, kcat/KM data at pH 7.0 was obtained for
all three mutants for the five substrate types, and show the same trends as the wild type (Table 6).
The pH 7.0 data for substrates 1a-5a show the same trends in catalytic efficiency as the limiting
(kcat/KMlim). Variations in the molecular properties of the substrates affect kcat more than KM.
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PP1 is a less efficient catalyst for the hydrolysis of phosphorothioate esters 2 than for
phosphate esters 1. Reduced catalytic effectiveness for phosphorothioates has also been reported
in alkaline phosphatase17, 35, 53, 54 and in protein-tyrosine phosphatases.23, 55, 56 This is opposite
from the effect of sulfur substitution on the uncatalyzed reaction; phosphorothioate monoesters
undergo hydrolysis faster than their phosphate ester counterparts. The reduced enzymatic rates
have been attributed to reduction in transition state stabilization, resulting from poorer affinity of
the enzyme for the transition state of the phosphorothioate substrate. Sulfur substitution has
several consequences on charge distribution and geometry; compared to oxygens in the
nonbridging positions, sulfur has greater negative charge and less double bond character 57, 58 as
well as a larger Van der Waals radius and longer bond length (1.94 Å versus 1.57Å).
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In the fluorophosphate substrates, the substitution of a non-bridging oxygen by fluoride
retains the size and hydrogen bond donor/acceptor properties of the phosphoryl group in the
natural monoester substrate. The kcat at pH 7.0 for 3a is only ~4-fold lower than 1a; the higher
KM results in an overall catalytic efficiency that is about an order of magnitude lower than 1a.
The methyl substitution (4a) for a nonbridging oxygen atom maintains a similar bond length
around the reaction center but removes hydrogen bond donor-acceptor properties and reduces the
negative charge. The kcat for 4a is only 3-fold lower than 1a at pH 7.0, while the overall catalytic
efficiency is about an order of magnitude lower. For the fluorophosphate and
methylphosphonate substrates the kcat and KM values are similar to one another. The methoxyl
substitution (5a) on a nonbridging oxygen atom converts the substrate into a diester, which will
have the largest steric requirement of all of the substrates tested. The kcat is most strongly
affected by this substitution, lower by ~200-fold compared to 1a and kcat/KM is more than 400-
fold reduced.
The second order rate constants for the reactions of these compounds with water are all
very low, reflecting the fact that these hydrolysis reactions have high kinetic barriers. This
constant for the fluorophosphate ester has not been determined and is difficult to obtain due to
domination by the hydroxide rate, but is probably similar to the other monoanionic substrates.
The ratio of the enzymatic catalytic efficiency (kcat/KMlim) to the second-order rate constant for the
reaction with water (kw in Table 2) gives the second-order rate enhancement, a measure of the
degree to which PP1 reduces the activation barrier for the hydrolysis reactions. These values are
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all high and vary over a narrow range of only ~2 orders of magnitude, and correspond to
reductions in activation energy of from 16 to 19 kcal mol-1. This demonstrates that PP1 is an
effective catalyst for all of these substrates despite the variations in size and charge of the
transferring group. For comparison, the promiscuous BcPMH, assigned as a member of the
alkaline phosphatase superfamily, exhibits second-order rate accelerations ranging from 107 to as
high as 1019 for a range of phosphate monoester, diester, and triester substrates, as well as sulfate
monoesters, corresponding to decreases in the energy of activation between 14.4 and 27.2 kcal
mol−1.33 The hydrolase BcPMH uses a formyl glycine nucleophile and a single metal ion,
features commonly found in sulfatases. The larger and more variable rate accelerations of
BcPMH suggest that its active site provides the potential for additional effects that synergize
with the reactivity of the metal ion. The different active site of PP1 provides a smaller, and more
uniform, stabilization of catalysis toward different substrate classes.
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A simple Arg to Lys mutant of PP1 exhibits catalytic efficiency toward monoanionic
substrates that are superior to wild type. The structure of PP1 suggests that residues R96 and
R221 hydrogen bond with the substrate oxygens and probably contribute to substrate binding and
stabilization of the transition state. In a previous report, the R96E mutant exhibited a 700-fold
lower catalytic efficiency toward the physiological substrate phosphorylated phosphorylase a.43
A smaller effect on KM indicates that this residue is more critical for catalysis than substrate
recognition. The R96A mutant exhibited a large decrease in kcat (>400-fold) but no significant
change in KM.59 The R221S mutant exhibited a large reduction in kcat (≈200-fold),59 but in contrast
to the R96A mutant, also showed a significant reduction in affinity for the phosphorylase a
substrate (~10-fold increase in KM).
The catalytic promiscuity of members of the alkaline phosphatase family has been
attributed, in part, to the ability of certain side chains in the active site reposition, in order to
accommodate more sterically demanding substrates.60, 61 The relatively similar catalytic
proficiencies for the substrate classes may result from a similar factor in PP1. Inspection of the
crystal structure and hypothetical binding mode (Figure 1) suggests R96 and R221 as candidates,
as neither has a direct catalytic role but may donate a hydrogen bond. We investigated the
mutations of R96 and R221 to lysine, with the hypothesis that this change would maintain
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hydrogen bond donating ability while providing additional space for the more sterically
demanding substrates. Based on potential enzyme-substrate interactions suggested from the
crystal structure, it was anticipated that the R96K mutant would be more effective in this regard.
The kinetic parameters kcat and kcat/KM were obtained for the nitrophenyl esters at pH 7 for the
R96K and R221K mutants, and for the double mutant.
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Table 6 shows the catalytic efficiencies at pH 7.0 for the wild type PP1 and the arginine
mutants for substrates 1a-5a. Table S2 shows the full set of kcat and KM data. Both the R96K
and R221K mutations have minor effects on KM and primarily affect kcat, consistent with R96A
and R96E results. Most interestingly, the mutant R221K exhibits kcat and kcat/KM values superior
to the native enzyme for the monoanionic substrates 3a and 4a. For the monoanionic diester 5a
the values are similar. This is suggestive of a more important role for R221 in stabilization of
the transition state than in binding substrate. Precedent for mutations in catalytically
promiscuous enzymes resulting in altered preferences for different substrates is found in the
alkaline phosphatase and the related phosphate monoesterase PafA, where mutations alter the
monoesterase/diesterase preferences.61, 62
p-Nitrophenyl sulfate is not a substrate for PP1. The fact that p-nitrophenyl sulfate
(pNPS) is not a substrate is surprising in light of the promiscuity of PP1 for monoanionic
phosphorus-based substrates. Sulfate and phosphate monoesters share tetrahedral geometry with
comparable bond angles and lengths. The uncatalyzed hydrolysis reactions of sulfate and
phosphate monoesters occur with similar rate constants and with similar transition states. This
similarity between phosphoryl and sulfuryl transfer reactions is reflected in the ability of several
other promiscuous enzymes, including alkaline phosphatase, the sulfatase PAS, and the
aforementioned BcPMH that carry out both activities.63 The inability of PP1 to catalyze pNPS
hydrolysis is not due to an inability to bind to the active site. Inhibition experiments demonstrate
that 6a binds to the active site, albeit weakly, as a competitive inhibitor (Ki = 40 mM). The
significantly higher Ki compared to the monoanionic phosphoester substrates is in keeping with
the weaker affinity of sulfate as a metal ligand compared with phosphate. However, even at high
substrate concentrations and long reaction times, no activity with pNPS was observed. The
reason for this discrimination is not clear.
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Inhibition experiments show that sulfate and sulfate analogues are weak competitive
inhibitors of the phosphate monoesterase activity of PP1, with inhibition constants (Ki) ranging
22 mM to 40 mM (Table 5). Even with the same molecular geometry, changing the center atom
from phosphorus to sulfur or selenium increases the Ki by an order of magnitude.
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Conclusions
PP1 is an effective catalyst for the hydrolysis of both monoanionic and dianionic
phosphate-ester based substrates 1-5. The second-order rate accelerations are significant and
similar across the range of monoanionic and dianionic substrates, ranging from 1011 to 1013. The
transition states for the PP1-catalyzed reactions are more similar than the transition states for the
uncatalyzed hydrolysis reactions. Thus, PP1 catalyzes their hydrolysis by transition states that
are controlled by the active site environment more than by the intrinsic nature of the substrates.
The reason for the inability of PP1 to catalyze the hydrolysis of a sulfate ester is unclear, and
unexpected, since the charge and transition state of this substrate are well within the range of
those of the phosphorus-based substrates whose hydrolysis are effectively catalyzed.
Inhibition experiments suggest that the PP1 active site is tolerant of variations in the
geometry of bound ligands. This characteristic may assist the effective catalysis of substrates
whose steric requirements result in perturbations to the positioning of the transferring group both
in the initial enzyme-substrate complex, and in the transition state.
The mutation of arginine 221 to lysine results in a mutant that, relative to the native
enzyme, has reduced catalytic efficiency toward dianionic substrates but somewhat higher
efficiency for the monoanionic substrates. The surprising result in substrate preference from a
single, conservative mutation lends support to the notion that modest mutations can result in an
enzyme with different catalytic capabilities and preferences, and may, following subsequent
mutations, provide a pathway for the evolution of new enzymes.
Mutation of arginine 96 to lysine resulted in a reduction in activity for all substrates, but
was more deleterious on the activity of dianionic substrates. The comparative mutagenesis
results of the two active site arginines in PP1 echoes the observation that lysine, rather than
arginine, is found in the active sites of enzymes that hydrolyze monoanionic versus dianionic
substrates. Except for enzymes that use a dinuclear metal center to bind the substrate, arginine is
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commonly found in active sites of phosphomonoesterases (PTPs, alkaline phosphatases, acid
(histidine) phosphatases), while lysines are typically found in the active sites of enzymes that
hydrolyze monoanionic phosphate diesters or sulfate esters.
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Supporting Information
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Formulas used for calculation of kinetic isotope effects; conditions used for enzymatic activity
assays using HPLC; sample Michaelis-Menten plot of kinetic and inhibition data; kinetic data for
the reactions of substrates 1a – 5a by native PP1 and mutants at pH 7.0.
The Supporting Information is available free of charge on the ACS Publications website.
Acknowledgment
This work was supported by a grant from the National Institutes of Health to A.C.H.
(GM47297) and by a grant from the Biology and Biotechnology Science Research Council to
N.H.W. (C18734).
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