The promiscuous phosphomonoestearase activity of Archaeoglobus fulgidus CopA, a thermophilic Cu+ transport ATPase

Article abstract of DOI:10.1016/j.bbamem.2016.04.006

Membrane transport P-type ATPases display two characteristic enzymatic activities: a principal ATPase activity provides the driving force for ion transport across biological membranes, whereas a promiscuous secondary activity catalyzes the hydrolysis of p

Full text of DOI:10.1016/j.bbamem.2016.04.006

The promiscuous phosphomonoestearase activity of Archaeoglobus fulgidus
CopA, a thermophilic Cu+ transport ATPase
Luis M. Bredeston, F. Luis Gonza´lez Flecha
PII:
DOI:
Reference:
S0005-2736(16)30133-X
doi: 10.1016/j.bbamem.2016.04.006
BBAMEM 82202
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BBA - Biomembranes
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Accepted date:
16 November 2015
1 April 2016
13 April 2016
Please cite this article as: Luis M. Bredeston, F. Luis Gonz´alez Flecha, The promiscu-
ous phosphomonoestearase activity of Archaeoglobus fulgidus CopA, a thermophilic Cu+
transport ATPase, BBA - Biomembranes (2016), doi: 10.1016/j.bbamem.2016.04.006
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The promiscuous phosphomonoestearase activity of Archaeoglobus
fulgidus CopA, a thermophilic Cu+ transport ATPase
Luis M. Bredeston and F. Luis González Flecha*
Laboratorio de Biofísica Molecular, Instituto de Química y Fisicoquímica Biológicas,
Departamento de Química Biológica. Facultad de Farmacia y Bioquímica. Universidad de
Buenos Aires – CONICET, Buenos Aires, Argentina
* Corresponding author at: Department of Biological Chemistry, Facultad de Farmacia y
Bioquímica, Universidad de Buenos Aires, Junín 956, 1113 Buenos Aires, Argentina.
E-mail: lgf@qb.ffyb.uba.ar
Abstract
Membrane transport P-type ATPases display two characteristic enzymatic activities: a principal
ATPase activity provides the driving force for ion transport across biological membranes,
whereas a promiscuous secondary activity catalyzes the hydrolysis of phosphate monoesters.
This last activity is usually denoted as the phosphatase activity of P-ATPases. In the present
study we characterize the phosphatase activity of the Cu+-transport ATPase from Archaeglobus
fulgidus (Af-CopA) and compare it with the principal ATPase activity. Our results show that the
phosphatase turnover number was 20 times higher than that corresponding to the ATPase
activity, but it is compensated by a high value of Km, producing a less efficient catalysis for
pNPP. This secondary activity is enhanced by Mg2+ (essential activator) and phospholipids
(non-essential activator), and inhibited by salts and Cu+. Transition state analysis of the
catalyzed and non catalyzed hydrolysis of pNPP indicates that Af-CopA enhances the reaction
rates by a factor of 105 (ΔΔG‡ = 38 kJ/mol) mainly by reducing the enthalpy of activation (ΔΔH‡
= 30 kJ/mol) whereas the entropy of activation is less negative on the enzyme than in solution.
For the ATPase activity the decrease in the enthalpic component of the barrier is higher (ΔΔH‡ =
39 kJ/mol) and the entropic component is small on both the enzyme and in solution. These
results suggest that different mechanisms are involved in the transference of the phosphoryl
group of p-nitrophenyl phosphate and ATP.
Abbreviations
Af-CopA, Cu(I)-transport ATPase from Archaeglobus fulgidus; DDM, n-dodecyl β-D-maltoside;
DTT, dithiothreitol; HAD, haloacid dehalogenase; MOPS, 3-(N-morpholino)propanesulfonic acid;
PC, phosphatidylcholine; PE, phosphatidylethanolamine; Pi, inorganic phosphate; XPL
phospholipid mole fraction in the micellar phase; PMCA, plasma membrane Ca2+-ATPase;

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pNPP, p-nitrophenyl phosphate; PS, phosphatidylserine; SERCA, sarcoplasmic reticulum Ca2+-
ATPase; TM-MBSs, transmembrane metal binding sites.
Keywords
membrane proteins; enzyme catalysis; P-ATPase; principal activity; catalytic proficiency;
enzyme promiscuity
1. Introduction
P1B-ATPases constitutes a group of evolutionarily related integral membrane proteins widely
distributed in all the domains of life (Eukarya, Bacteria, and Archaea), which are involved in the
active transport of heavy metals (Cu+, Zn2+, Cd2+, etc) through cellular membranes [1, 2].
Particular attention has been given to these proteins because of mutations in the human Cu-
ATPase genes, ATP7A and ATP7B, lead to Menkes syndrome and Wilson’s disease
respectively [3, 4]. Archaeoglobus fulgidus CopA (Af-CopA ) is one of the best-studied members
of this group. It can be heterologously expressed in E. coli, purified by affinity chromatography,
and reconstituted in mixed micelles composed of soybean phospholipids and DDM. Following
this procedure a functional protein is obtained which displays characteristic properties of a
thermophilic enzyme with maximal activity at 75ºC [5], high stability against thermal [6] and
chemical [7] denaturation. This behavior seems to not require the unique features of archaeal
lipids (ether linkages and isoprenoid chains). Thus, these micelar preparations have become the
standard ones for structural and functional studies. The protein consists of a single polypeptide
chain of 804 amino acid residues and has eight transmembrane helices, and two large cytosolic
loops comprising the actuator and the catalytic domains, and two metal binding domains in the
N- and C-terminus [2]. Atomic resolution structures were obtained for the actuator [8] and the
catalytic domain in the free [9] and nucleotide bound forms [10] as well as for the C-terminal
metal binding domain [11]. Recently, two crystal structures of a complete P1B Cu+-ATPase from
Legionella pneumophila were resolved at 3.2 Å (PDB 3RFU) [12] and 2.8 Å (PDB 4BBJ) [13]
showing a core structure shared with other P-type ATPases but with a distinctive
transmembrane organization.
Ion transport by Af-CopA begins with the Cu+ transfer from a partnering intracellular
metalochaperone to the transmembrane metal binding sites (TM-MBSs) located on the
transmembrane helices 6, 7 and 8 [14, 15]. Translocation of the bound Cu+ is coupled to the
binding and hydrolysis of ATP following a classical Albers-Post E1/E2 model characteristic of all
P-type ATPases [16, 17]. Upon ATP binding to the E1 conformation, the enzyme is
phosphorylated at the invariant Asp residue in the conserved DKTG motif located at the catalytic
domain, and Cu+ is transiently occluded within the transmembrane region giving the E1P
intermediary. A conformational change is then triggered, forming the E2P intermediary, by
opening the TM-MBs to the extracellular milieu. Finally the ion is released and the enzyme is
dephosphorylated to the E2 conformation that shifts to the conformation E1 in which the TM-MBs
are open toward the cytoplasmic side and is able to initiate a new reaction cycle.
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In addition to the physiologically relevant ATPase activity, some well characterized P-type
ATPases (e.g. the Na+/K+-ATPase [18] the sarcoplasmic reticulum Ca2+-ATPase (SERCA) [19]
and the plasma membrane Ca2+-ATPase (PMCA) [20]) are able to catalyze the hydrolysis of
phosphate monoesters like acetyl-phosphate and p-nitrophenyl phosphate (pNPP) in the
absence of the transported ion. This activity, usually denoted as the “phosphatase” activity of P-
ATPases, was also found in heavy metal P1B-ATPases. We have reported pNPPase activity in
a purified preparation of CopA from Archaeglobus fulgidus in the absence of Cu+ [21]. In
contrast, Hatori et al. showed that another thermophilic Cu+-ATPase, CopA from Thermotoga
maritima, exhibit pNPPase activity but only in the presence of Cu+ [22].
Secondary catalytic activities have been described for many enzymes, and it is believed that
it could be the rule more than the exception [23]. The term enzyme promiscuity was introduced
for denoting these activities other than the activity for which an enzyme evolved and that are not
part of the organism’s physiology [24]. It is believed that contemporary enzymes have evolved
from multi-functional generalist ancestral enzymes, and that natural selection gave rise to their
increased catalytic specificity and efficiency [23]. However, if a physiologically non-relevant
activity is not detrimental to the organism it could persist through the evolution [25].
In the present study we characterize the promiscuous phosphomonoestearase activity of
CopA from Archaeglobus fulgidus using p-nitrophenyl phosphate as substrate, comparing it with
the principal ATPase activity, and explore its regulation by different ligands.
2. Materials and methods
2.1. Reagents
All chemicals used in this work were of analytical grade and purchased mostly from Sigma-
Aldrich Co. p-nitrophenyl phosphate (di Tris salt) was provided by Calbiochem. All the
phospholipids used in this work were purchased from Avanti Polar Lipids and dodecyl maltoside
(DDM) was provided by Anatrace.
2.2. Af-CopA expression and purification
A. fulgidus CopA, cloned into pBADTOPO/His vector, was expressed in E. coli Top10 cells
and purified essentially as reported [5]. The purified protein migrates as a single band in Tris-
tricine SDS-PAGE [26]. Af-CopA concentration was determined using a molar extinction
coefficient of 39700 M-1 cm-1 [7].
2.3. Phosphomonoestearase turnover rate of Af-CopA
The turnover rate for the pNPPase activity of AfCopA was determined as the initial rate of
release of p-nitrophenol from pNPP per mole of enzyme. The phosphatase reaction medium
contained: 0.15 μM purified Af-CopA, 150 μM soybean phospholipids, 200 μM DDM, 50 mM
MOPS-Na (pH 6.1 at working temperature), 60 mM pNPP, 30 mM MgCl2, and 2.5 mM DTT. The
reaction mixture (final volume 250 μl) was incubated at selected temperatures for several time
periods, returned to an ice-bath and mixed with 1 ml of 0.5 M NaOH. Hydrolysis of pNPP was
estimated by measuring the absorption of p-nitrophenol at 410 nm using a molar extinction
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coefficient of 17000 M-1cm-1 [18]. Non-enzymatic hydrolysis of pNPP was determined in the
same reaction medium without Af-CopA.
2.4. ATPase turnover rate of Af-CopA
ATPase turnover rate was determined as the initial rate of release of Pi from ATP per mole of
enzyme. The ATPase reaction medium contained: 0.15 μM purified Af-CopA, 150 μM soybean
phospholipids, 200 μM DDM, 50 mM MOPS-Na (pH 6.1 at working temperature), 3 mM MgCl2,
800 mM NaCl, 3 mM ATP, 0.1 mM CuSO4 and 2.5 mM DTT. Released Pi was determined in
accordance with Lanzetta et al [27]. Non-enzymatic hydrolysis of ATP was determined in the
same reaction medium without Af-CopA.
2.5. Data analysis
At least three independent preparations of purified Af-CopA were evaluated for each
experiment. The equations were fitted to the experimental data using a non-linear regression
procedure implemented on a Excel spreadsheets [28]. The dependent variable was assumed to
be homoscedastic (constant variance), and the independent variable was considered to have
negligible error. The "equation of best-fit" was deemed that which gave the minimal Akaike
index [29] and the least biased fit. Parameters were expressed as the mean ± standard error.
3. Results
3.1. The phosphomonoestearase activity of Af-CopA is more effective but less efficient than the
principal ATPase activity
The first step in characterizing the phosphatase activity of A. fulgidus CopA was to determine
the dependence of the reaction rate on the substrate concentration at 75oC, i.e. the optimal
temperature for the principal ATPase activity [5]. It can be observed in Figure 1A that the
dependence of the turnover rate on pNPP concentration is well described by a hyperbolic
function reaching an asymptotic value (kcat) of 5.9  0.3 s-1 with a Km value for pNPP of 9  1
mM. The principal ATPase activity also show a hyperbolic dependence on the substrate
concentration (Figure 1B), being the best fit parameter values kcat = 0.26  0.01 s-1 and Km =
0.11  0.02 mM.
Figure 1. The substrates curves of Af-CopA. Purified Af-CopA was incubated at 75oC in
the corresponding reaction medium with increasing concentrations of pNPP (A) or ATP (B)
as indicated in the figure. The turnover rate was calculated for each condition and
represented as a function of substrate concentration. Continuous lines are the graphical
representation of a single hyperbolic function fitted to the experimental data with the
parameter values indicated in the text.
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Comparison of the ability of Af-CopA to catalyze the hydrolysis of pNPP respect to ATP
requires the measurement of the spontaneous hydrolysis of both substrates in the absence of
enzyme (knon). These measurements were performed in the same reaction medium used for
measurements the enzyme activities and at the same temperatures. Results of these
experiments are shown in the Supporting Information (Figure S1). It can be observed that the
rate coefficients for the non enzymatic hydrolysis of ATP at 75oC (6.4 ± 0.3 10-6 s-1) is similar to
that corresponding to pNPP (1.20 ± 0.04 10-5 s-1), an observation that can be rationalized taking
into account that both substrates have highly activated leaving groups. Comparing catalyzed
and non catalyzed kinetic constants (Table 1), it can be observed that the rate enhancement
produced by Af-CopA is one order of magnitude higher for the hydrolysis of pNPP than for ATP.
On the other hand, the specificity of Af-CopA for pNPP respect to ATP can be evaluated by
comparing the ratio kcat/Km for both activities (Table 1). Remarkably, despite the higher rate
enhancement observed for the phosphomonoestearase activity, the enzyme is more specific for
ATP. The origin of this higher specificity for the principal substrate is probably due to the
structural complementarity between the ATP molecule and the nucleotide binding domain of the
enzyme [10].
Table 1 – Rate enhancements and catalytic proficiencies of the principal (ATPase) and
promiscuous (pNPPase) activities of Archaeoglobus fulgidus CopA at 75 °C
rate
catalytic
kcat
kcat/Km
knon
enhancement proficiency
kcat/knon
substrate
(s-1)
(M-1 s-1)
(s-1)
(kcat/Km)/knon (M-1)
5.4107
3.8108
1.2010-5 5.0105
6.410-6 4.0104
pNPP
ATP
5.9
642
0.26
2437
Another important parameter for evaluating the power of an enzyme as a catalyst is the
enzyme proficiency [30] defined as the ratio between the second order rate constant for the
enzyme-substrate encounter (kcat/Km) and the rate constant in the absence of enzyme (knon). As
we can observed the catalytic proficiency of Af-CopA is only slightly higher towards its natural
substrate.
Given the temperature dependence of the kinetic constants, we have repeated this analysis
measuring the activities at 60°C (Figure S3 and Table S1). It can be observed that the same
conclusions can be drawn comparing the data at both temperatures.
3.2. Mg2+ is essential for the phosphomonoestearase activity of Af-CopA
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One of the key events in the ATPase reaction cycle is the binding of Mg2+ to the ATP binding
domain. It triggers the phosphorylation, and induces the occlusion of the transported ions inside
the transmembrane domain [10]. Figure 2 shows that the phosphatase activity of Af-CopA
increases with Mg2+ concentration. The best-fit model was a single hyperbolic function without
intercept, indicating that Mg2+ is an essential activator of pNPP hydrolysis by Af-CopA. This
activation was specific for Mg2+, others divalent cations e.g. Mn2+, Co2+, Sr2+, Ba2+ have no effect
on the phosphatase activity.
Figure 2. Activation of Af-CopA catalyzed pNPP hydrolysis by Mg2+. Purified Af-CopA
was incubated at 75oC in the phosphatase reaction medium with increasing concentrations
of Mg2+ as indicated in the figure. The turnover rate was calculated for each condition and
represented as a function of the total Mg2+ concentration. Continuous line is the graphical
representation of a single hyperbolic function fitted to the experimental data. The best-fit
parameter values were kcat = 5.8  0.1 s-1 and K05 = 1.0  0.1 mM.
3.3. Monovalent cations reduce the phosphomonoestearase activity of Af-CopA
It was previously described that Cu+ is an esential activator of the ATPase activity of Af-CopA
[5]. Binding of Cu+ to the specific TM-MBS is required to initiate the ATPase reaction cycle of Af-
CopA, and it is postulated that ion occlusion is coupled to the chemical step in which the -
phosphate of ATP is transferred to Asp 424 [31]. To explore whether the phosphatase activity of
Af-CopA is also regulated by Cu+, the turnover rate was determined in the presence of different
concentrations of this ion (Figure 3). It can be observed that in contrast to that described for the
ATPase activity, the phosphatase activity of Af-CopA decreases hyperbolically with increasing
Cu+ concentration with K0.5 = 0.06 ± 0.01 mM.
Figure 3. Inhibition of Af-CopA catalyzed pNPP hydrolysis by Cu+. Purified Af-CopA
was incubated at 75oC in the phosphatase reaction medium with increasing concentrations
of CuSO4 in the presence of a reducing agent as indicated. The turnover rate was
calculated for each condition and represented as a function of the concentration of Cu+.
The best fit to the experimental data was achieved using a decreasing hyperbolic function.
Continuous line is the graphical representation of this function with the following best-fit
parameter values: (Ao - A∞) = 4.8 ± 0.7 s-1; K0.5 = 0.06 ± 0.01 mM.
On the other hand, it was also described that the ATPase activity of Af-CopA is stimulated by
high concentrations (400-500 mM) of NaCl [5]. To investigate if this characteristic is a general
requirement of Af-CopA function, the effects of increasing NaCl concentrations were assayed on
the phosphatase turnover rate (Figure 4). It can be observed that activity was maximal in the
absence of NaCl and decrease when increasing NaCl concentration, reaching a constant level
at about 0.5 M. To determine if this effect is specific for NaCl, the turnover rate for the
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phosphatase activity of Af-CopA was determined in the presence of 800 mM NaCl, NaNO3 or
KCl. In all the cases the measured activity was smaller than that measured in the control without
added salts (Figure S2).
Figure 4. Dependence of the pNPPase activity of Af-CopA on salt concentration.
Purified Af-CopA was incubated at 75oC in the phosphatase reaction medium with
increasing concentrations of NaCl as indicated in the figure. The turnover rate was
calculated for each condition and represented as a function of the concentration of salt.
3.4. Phospholipids enhance the phosphomonoestearase activity of Af-CopA
It is recognized that phospholipids play an essential role in the biological function of integral
membrane proteins [32]. To evaluate the regulation of the phosphatase activity of Af-CopA by
phospholipids, the enzyme turnover rate of the purified protein reconstituted in detergent
micelles was assessed by increasing the phospholipid mole fraction (XPL) in the micellar phase
(Figure 5). As a first approach we explored this regulation using soybean phospholipids, since
all the available structural and functional information on this enzyme was obtained using this
phospholipid mixture. Given the low critical micellar concentration of both amphiphiles, we
consider that they are exclusively present in the micelles [33].
Figure 5. Phospholipid modulation of Af-CopA pNPPase activity. Purified Af-CopA
was incubated at 75ºC in the phosphatase reaction medium with increasing concentrations
of soybean phospholipids as indicated in the figure. The turnover rate was calculated for
each condition and represented as a function of phospholipid mole fraction in the micelar
phase. Continuous line is the graphical representation of the minimal model described by
Dodes Traian et al. [32] to the experimental data.
It can be observed that the addition of phospholipids produces a significant increase in Af-
CopA phosphomonoestearase turnover rate reaching a maximum at mole fractions about 0.2.
Similar experiments using some pure phospholipids showed that saturated fatty acid PC (16:0)
was the more effective activator (Table 2). Changes in the polar head (PE, PS or PI) or in the
PC fatty acid composition appear to decrease the efficiency of Af-CopA for the hydrolysis of
pNPP. Further studies are needed to explore these effects with archaeal lipids.
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Table 2 – Activation of Af-CopA pNPPase by different phospholipids
Af-CopA phosphatase turnover rate
Phospholipid
at XPL = 0.2 (s-1)
PC (16:0)
8.2
7.6
4.9
5.5
5.1
5.9
PC (14:0)
PC (18:1)
PE (16:0)
PS (16:0)
soybean phospholipids
PC: phosphatidylcholine; PE: phosphatidylethanolamine; PS: phosphatidylserine
3.5. Temperature enhancement of the phosphomonoestearase activity of Af-CopA is lower than
that of the principal ATPase activity
Figure 6 shows temperature dependence of pNPPase turnover rate of purified Af-CopA in the
o
range 30-100 C. It can be observed that the enzyme turnover increases with temperature
o
reaching a maximum between 70-80 C. Further increase in temperature produces a fast
inactivation of the enzyme by protein denaturation impeding the determination of the initial rate
of pNPP hydrolysis (see Figure S3 in the supporting information). This dependence is similar to
that previously reported for the ATPase activity of Af-CopA. However the activation energy
calculated from the Arrhenius plot was 59  3 kJ/mol, about a half of the value reported for the
ATPase activity (103  5 kJ/mol) [5], suggesting that different paths are involved in the
hydrolysis of both substrates by the enzyme.
Figure 6. Dependence of Af-CopA pNPPase activity on temperature. A: Purified Af-
CopA was incubated in the phosphatase reaction medium at the temperatures indicated in
the figure. The turnover rate was calculated for each condition and represented as a
function of temperature. Continuous line is a guide for the eye. B: The Arrhenius plot is
represented for the increasing arm of plot A. The solid line is the graphical representation
of the Arrhenius equation fitted to the experimental data with the activation energy
indicated in the text.
4. Discussion
4.1. Principal and promiscuous activities of Archaeglobus fulgidus Af-CopA
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The catalytic domain of the P-type ATPases share a common fold within the haloacid
dehalogenase (HAD) superfamily, with a set of conserved active-site residues including the
phosphorylatable aspartic acid residue [34]. HAD superfamily members are characterized by a
broader specificity against different classes of substrates (sugars, nucleotides, organic acids,
coenzymes, and small phosphodonors) [35, 36]. Two characteristic enzymatic activities were
described in P-type cation pumps: a principal ATPase activity and a secondary
phosphomonoestearase activity usually denoted as the “phosphatase” activity of these
enzymes. The principal activity provides the driving force for ion transport across cellular
membranes. This activity appear quite specific for ATP, e.g. it was reported that no other
nucleotide triphosphates can be hydrolyzed by the plasma membrane calcium pump [20], and
very low activities were detected with UTP, CTP, GTP and ITP in SERCA [37, 38]. Despite this
specificity for the hydrolysis of phosphoanhydride bonds, P-ATPases are able to catalyze the
hydrolysis of phosphate monoesters like acetyl-phosphate and p-nitrophenyl phosphate [18].
Both activities appear to be independent, since modification of an specific Lys residue in the
catalytic domain with fluorescein isothiocyanate abolish the ATPase, but not the
phosphomonoestearase activity in SERCA [39] and PMCA [40].
Secondary activities resulting from the fortuitous use of active site features to catalyze an
alternative reaction have been extensively characterized for many soluble enzymes [23-25].
These promiscuous activities are not detrimental for efficient catalysis of the primary reaction
[41], and it was postulated that they provide the raw material for the evolution of new enzymes
[23]. Membrane associated enzymes constitute an important group of proteins characterized by
being folded in a heterogeneous water-lipid environment [42]. Despite the essential cell
functions in which they are involved, the relationship between their principal and secondary
catalytic activities is far less characterized than in soluble ones.
It is accepted that catalysis of the secondary reaction is generally less efficient than on the
primary function, with only few exceptions [25, 43]. Comparisons like this are rare for membrane
related enzymes, and thus a clear trend cannot be drawn. For some P-type ATPases the
reported ATPase activities are higher in magnitude than the corresponding phosphatase
activities [19, 44], whereas in others both activities are in the same order of magnitude [20, 45].
Results presented in this work showed that Achaeglobus fulgidus CopA phosphatase turnover
number was 20 times higher than that corresponding to the ATPase activity. This result suggest
that the promiscuous phosphate monoester substrate would be better positioned than ATP
relative to the catalytic machinery of the enzyme [24]. Conversely, the structural
complementarity between the nucleotide bind domain of AfCopA and ATP, results in a smaller
Km value for the natural substrate. These features make Af-CopA an interesting system to
explore the relationship between principal and promiscuous activities.
It is well established that turnover numbers provide a measure of the maximal capacity of an
enzyme to catalyze the transformation of substrate into product. However enzymes usually work
in the presence of sub-saturating substrate concentrations under physiological conditions, thus
allowing the reaction rate to respond to changes in substrate concentration during cell function.
In these conditions, the reaction rate is governed by the second order constant for the enzyme-
substrate encounter (kcat/Km), a quantity denoted as the catalytic efficiency of the enzyme. It was
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proposed that enzyme evolution proceed via optimizing the catalytic efficiency [46] allowing the
catalyzed reaction to occur on biologically relevant timescales [43]. Promiscuous substrates
usually bind weakly to the enzyme resulting in high Km values [24]. This is certainly the case for
Af-CopA with a Km for pNPP in the order of 10 mM. This high value of Km compensates the high
turnover rate, producing a less efficient catalysis for pNPP. In addition, the low values of kcat/Km
for both activities -compare the values in Table I with 109 M-1s-1 corresponding to the so-called
perfect enzymes [47]- indicates that the capture of the substrate is far to be diffusion limited.
The catalytic effectiveness of a given enzyme can be expressed in terms of the factor by
which it enhances the rate of a reaction [30]. Comparisons of the rates of the enzyme catalyzed
reactions with the non-catalyzed background reaction gives an indication of the strength of
interactions in the enzyme active site [30]. For this comparison, uncatalyzed and catalyzed
reactions have to be measured in the same reaction medium and at the same temperature.
o
Being Af-CopA a thermophilic enzyme with maximum activity at 75 C, direct measurement of
the non-enzymatic substrate hydrolysis is possible, avoiding the large extrapolation usually
required for doing this comparison in mesophilic enzymes [30, 48].
Our results show that the first order kinetic coefficients for the non catalyzed hydrolysis of
pNPP and ATP are quite similar. Both substrates have highly activated leaving groups with pKa
values of 7.14 for p-nitrophenol [49] and 6.4 for ADP [50]. Despite these similarities, the
temperature dependence of the rate coefficients is different for the non catalyzed hydrolysis of
ATP and pNPP, giving different enthalpic and entropic contributions to the free energy barrier
(see the discussion in section 4.3).
In this work we show that the rate enhancement (kcat/knon) is one order of magnitude higher
for the phosphomonoestearase activity of Af-CopA (Table 1). Nevertheless, the rate
enhancement values obtained for both Af-CopA activities are low when compared with the
previously reported values for soluble phosphate monoester hydrolases at 25oC [36, 51]. These
low values result from the large differences in the rates of the non-catalyzed reactions rather
than the catalyzed reactions, as was previously reported for many soluble enzymes.
Enzyme effectiveness as a catalyst can be also expressed as the ratio between the catalytic
efficiency (kcat/Km) and the first order rate constant for the uncatalyzed reaction knon under the
same conditions [30]. This ratio is called “catalytic proficiency” and gives a measure of the
enzyme ability to reduce the activation barrier for the reaction of the substrate free in solution
[30]. The catalytic proficiency of Af-CopA to hydrolyze ATP and pNPP are both similar and low
when comparing with mesophilic phosphohydrolases [51]. Again, these differences arise from
differences in the rates of the uncatalyzed reactions rather than the catalyzed reactions. The
inverse of the enzyme proficiency has been show to provide an lower limit for the association
constant of the enzyme with the altered substrate in the transition state [52]. For Af-CopA these
constants are in the nanomolar range indicating a weak interaction in the transition states.
Low proficiencies are common in thermophilic soluble enzymes, indicating that enzymes
working in warm environments do not need to enhance to much the rate of the reaction they
catalyze [47]. As was mentioned, P-ATPases use the energy of ATP hydrolysis to actively
transport ions across biological membranes. A high proficiency of these enzymes could result in
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transporting ions at a higher rate than required for maintaining cellular homeostasis. If this was
the case, the enzyme could uncouple the enzyme activity to the transport. However, it was
demonstrated that uncoupling ATP hydrolysis and transport results in heat production, and
normally occurs in situations were the cell need to generate heat to maintain its temperature
[53]. Certainly, this is not the case for a thermophilic organism living in its natural environment.
In this way, our results allow to extend the hypothesis drawn by Wolfenden [47] postulating that
in a warm environment, like that of the primordial biology, some metal-interacting hydrophobic
proteins even with a modest enzyme activity would be enough to generate ionic gradients
across cell membranes.
4.2. Regulation of the phosphatase activity of Archaeglobus fulgidus CopA
In this work we demonstrate that Mg2+ is an essential activator for the phosphate monoesters
hydrolase activity of Af-CopA, as was also reported for the principal ATPase activity [5]. We find
that the enhancement of the pNPPase activity of Af-CopA by Mg2+ can not be produced by other
divalent cations. It was proposed that divalent metal ions can play several catalytic functions in
phosphoryl transfer enzymes including a) activation of substrate by polarization of the
phosphoryl group, b) activation of the nucleophile (water or an active site residue) by lowering
the pKa of OH groups, c) offsetting negative charge on the oxygen of the leaving group [54]. For
the ATPase activity of Af-CopA, Tsua and Toyoshima propose a possible scenario where Mg2+
dissociates from ATP during the catalytic cycle and binds to Asp 618 in the P-domain, a
coordination residue full conserved in all P-ATPases (Figure 7) [10]. Thermal movements of the
P domain would allow a change in ATP conformation with Mg2+ bridging the γ-phosphate, Asp
424 and Asp 618 in a similar way as was observed for SERCA [55]. In this form the γ-phosphate
of ATP is fixed by hydrogen bonds allowing its approximation to Asp 424 with the correct
geometry. Then Mg2+ promotes charge rearrangements on the γ-phosphate and the carboxyl
group of Asp 424 facilitating the formation of a covalent bond [10]. Phosphorylation of P-
ATPases by pNPP has been previously reported [56], so that it is possible to postulate -as a first
approximation- a similar role for Mg2+ in the phosphoryl transfer step. Furthermore,
computational studies from Kamerlin et al. demonstrate that requirement of Mg2+ for phosphate
ester hydrolysis may be a characteristic of an associative mechanism via a pentavalent
phosphrous intermediate [57], in this case including coordination with the p-nitrophenyl leaving
group and the Asp 424 nucleophile.
Figure 7. The catalytic site Af-CopA. Detailed view of the interactions of ATP with the
contours of the binding pocket in the ATPBD. ATP and interacting residues are shown in a
ball and stick representation with atoms colored by atom-type: C, cyan; O, red; N, blue, H,
white. Mg2+ is represented by a pink sphere. The picture is rendered with VMD 1.9.1 [58].
The principal ATPase activity of Af-CopA is also enhanced by Cu+ and Na+ [5]. Conversely,
our results show that the promiscuous phosphatase activity is inhibited by these monovalent
cations. The effect of NaCl appears to be a generalized non specific effect of salts. It is known
that some charges at the surface of proteins are shielded at high salt concentration. Our results
11

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show that charge shielding produces opposing effects on both enzymatic activities, suggesting
that some charged residues would favor the phosphatase activity whereas ATP hydrolysis
would be favored by the absence of these charges. This observation suggests that different
mechanisms are involved in the transfer of the phosphoryl group of pNPP and ATP. On the
other hand, our experiments demonstrated that Cu+ is not necessary for pNPP hydrolysis thus
suggesting that Cu+ is not transported during pNPP hydrolysis cycle. These results are in
accordance with a previous mutagenesis study where replacing the residues involved in binding
of Cu+ produce the lack of ATPase activity but does not abolish the phosphatase activity [59].
We also observe that high Cu+ concentration inhibits the phosphatase activity of Af-CopA. This
effect can not be identified as a salt effect because it occurs at lower concentrations, thus a
specific effect of Cu+ should be operating. A possible explanation is that the conformation of Af-
CopA induced by Cu+ (i.e. E1-Cu+), is less or not able to catalyze the hydrolysis of phosphate
monoesters. A similar hypothesis was drawn for SERCA for which the phosphatase activity was
postulated to be specific of the E2 conformation [39].
Phospholipids also produce an increase in the phosphatase activity of Af-CopA, but in this
case the activation is not essential since a significant activity (near half of the maximal activity)
was measured on the delipidated preparation of this enzyme. This is an important difference
respect the principal activity which is very small -about 10 fold lower- in the absence of added
phospholipids (Cattoni DI, PhD Thesis, Universidad de Buenos Aires). The modulation by
phospholipids is highly cooperative (being the cooperativity factor [32] c = 54 ± 8), reaching the
maximal activity when phospholipids constitute only the 10% of the amphiphiles in the
reconstitution medium. It was demonstrated that preferential interaction of phospholipids with
the transmembrane helices induces conformational changes in the transmembrane domain of
membrane proteins [32]. This change would be translated to all the conformations of the ATP
binding domain along the catalytic cycle giving a more active enzyme. The relatively low
enhancement by phospholipids suggests that the promiscuous phosphatase activity is mainly a
property of the cytoplasmic catalytic domain of Af-CopA.
4.3. Transition state analysis of the principal and promiscuous activities of Archaeglobus
fulgidus CopA
It is well established that the large catalytic efficiency of an enzyme can be accomplished by
interacting differentially with the substrate in the ground and activated states. However, the
mechanism through which enzymes reduce the energetic barrier is still not clear [46]. To explore
the origins of the catalysis of pNPP hydrolysis by AfCopA, we analyze the variation in the kinetic
coefficients kcat and knon with the temperature in terms of a Kramers-type model [60, 61] (Table
3):
‡
‡
‡
ΔG
R T
ΔH
R T
ΔS
k =
e

e
e
o
T
o
T
R
12

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where  represents the k value at the reference temperature for a barrier-less process [62],  is
the viscosity of the medium at a reference temperature (0) and at the incubation temperature
(T), and ΔG‡, ΔH‡, and ΔS‡ are the activation free energy, enthalpy and entropy, respectively.
In this analysis the reference temperature was selected as 75°C and the initial parameter values
were takes as those calculated using the classical transition state theory. It is worth noting that
the viscosity effect included in the Kramers model is relevant in this work because of the wide
temperature range used in the experiments.
Table 3 – Thermodynamic activation parameters at 75 ºC for the non catalyzed, the
principal (ATPase) and promiscuous (pNPPase) activities of Archaeoglobus fulgidus
CopA
ATP non
ATP Af-CopA
0.26 ± 0.01
89.6 ± 0.1
pNPP non
pNPP Af-CopA
5.9 ± 0.3
k (s-1)
(6.4 ± 0.3) 10-6
(1.20 ± 0.04) 10-5
‡ (kJ/mol)
120.4 ± 0.2
118.4 ± 0.1
80.5 ± 0.1
ΔG
ΔH
‡ (kJ/mol)
‡ (kJ/mol)
126 ± 9
87 ± 3
79 ± 3
49 ± 4
6 ± 9
1 ± 3
- 30.8
- 39
- 40 ± 3
- 32 ± 3
- 37.9
- 30
TΔS
ΔΔG
ΔΔH
‡ (kJ/mol)
‡ (kJ/mol)
‡ (kJ/mol)
-
-
-
-
-
-
- 5
8
TΔΔS
It can be observed that the free energy barrier for the non catalyzed hydrolysis of both
substrates is similar, with a dominant enthalpic term which is significantly higher for the
hydrolysis of ATP. Conversely, the activation entropy is close to zero for the ATP hydrolysis but
negative for the hydrolysis of pNPP. Near-zero activation entropies have been described in
solvolysis reactions not involving a molecule of solvent in the transition state [63], On the other
hand, negative activation entropies values have been previously reported for the non catalyzed
hydrolysis of phosphate monoesters and indicate that the degrees of freedom of the
phosphomonoester and water are decreased in the transition state respect to the initial non
reacting state. These differences suggest that, beyond the above mentioned similarities, both
substrates undergo non enzymatic hydrolysis through different mechanisms.
Comparing ΔH‡ values for kcat with those corresponding to the non enzymatic reactions, it is
evident that Af-CopA act on both substrates by lowering the enthalpy of activation. Full
comparison between the catalyzed and non catalyzed hydrolysis of pNPP indicates that Af-
CopA enhances the reaction rates by a factor of 105 (ΔΔG‡ = 38 kJ/mol) mainly by reducing the
enthalpy of activation (ΔΔH‡ = 30 kJ/mol). For the ATPase activity the decrease in the enthalpic
13

ACCEPTED MANUSCRIPT
component of the barrier is higher (ΔΔH‡ = 39 kJ/mol). Activation entropies are only slightly
different on the enzyme than in solution for both activities. The enthalpic basis of rate
enhancements have been interpreted in terms of new electrostatic and hydrogen bonds as
responsible for binding the altered substrate in the transition state [64]. These mechanisms are
commonly found in soluble enzymes [47]. Our results suggest that this evolutive feature also
holds for membrane related enzymes.
5. Concluding remarks
In this work we characterize the phosphatase activity of CopA from Archaeglobus fulgidus.
This activity seems to be a property of the catalytic domain E2 conformation of Af-CopA that
shows distinctive features compared with the principal ATPase activity. Our results show that
Af-CopA is more efficient for capturing ATP, but the promiscuous phosphate monoester
substrate would be better placed at the catalytic site than ATP. Requirement of Mg2+ suggest an
associative mechanism for phosphoryl transfer and the salt inhibition allow to propose that
charged residues play an important role for the phosphatase activity whereas ATP hydrolysis
would be favored by the absence of charges. Despite these differences in the mechanism,
catalysis is driven in both cases by an enthalpy lowering mechanism, and this effect is more
prominent for the principal activity allowing a better response against changes in temperature.
Acknowledgments
The authors are grateful to José M Argüello (Worcester Polytechnic Institute) for his advice
and encouragement, and for kindly providing the plasmids used in this study. We also thank to
Lurdes Sabeckis and Jeremias Incicco for critical reading of the manuscript, and to Lurdes
Sabeckis for the artful rendering of Figure 7.
This work was supported by grants from Agencia Nacional de Promoción Científica y
Tecnológica (PICT2010-1876 and PICT2013-1691) and Universidad de Buenos Aires (UBACyT
20020130100460BA).
Appendix A. Supplementary Data
Supplementary data to this article can be found online
14

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References
[1] K.B. Axelsen, M.G. Palmgren, Evolution of substrate specificities in the P-type ATPase
superfamily, J Mol Evol, 46 (1998) 84-101.
[2] J.M. Argüello, E. Eren, M. González-Guerrero, The structure and function of heavy metal
transport P1B-ATPases, Biometals, 20 (2007) 233-248.
[3] P.C. Bull, D.W. Cox, Wilson disease and Menkes disease: new handles on heavy-metal
transport, Trends Genet, 10 (1994) 246-252.
[4] S. Lutsenko, M.J. Petris, Function and regulation of the mammalian copper-transporting
ATPases: insights from biochemical and cell biological approaches, J Membr Biol, 191
(2003) 1-12.
[5] A.K. Mandal, W.D. Cheung, J.M. Argüello, Characterization of a thermophilic P-type Ag+/Cu+-
ATPase from the extremophile Archaeoglobus fulgidus, J Biol Chem, 277 (2002) 7201-
7208.
[6] D.I. Cattoni, F.L. González Flecha, J.M. Argüello, Thermal stability of CopA, a polytopic
membrane protein from the hyperthermophile Archaeoglobus fulgidus, Arch Biochem
Biophys, 471 (2008) 198-206.
[7] E.A. Roman, J.M. Argüello, F.L. González Flecha, Reversible unfolding of a thermophilic
membrane protein in phospholipid/detergent mixed micelles, J Mol Biol, 397 (2010) 550-
559.
[8] M.H. Sazinsky, S. Agarwal, J.M. Argüello, A.C. Rosenzweig, Structure of the actuator
domain from the Archaeoglobus fulgidus Cu(+)-ATPase, Biochemistry, 45 (2006) 9949-
9955.
[9] M.H. Sazinsky, A.K. Mandal, J.M. Argüello, A.C. Rosenzweig, Structure of the ATP binding
domain from the Archaeoglobus fulgidus Cu+-ATPase, J Biol Chem, 281 (2006) 11161-
11166.
[10] T. Tsuda, C. Toyoshima, Nucleotide recognition by CopA, a Cu+-transporting P-type
ATPase, EMBO J, 28 (2009) 1782-1791.
[11] S. Agarwal, D. Hong, N.K. Desai, M.H. Sazinsky, J.M. Argüello, A.C. Rosenzweig, Structure
and interactions of the C-terminal metal binding domain of Archaeoglobus fulgidus CopA,
Proteins, 78 (2010) 2450-2458.
[12] P. Gourdon, X.Y. Liu, T. Skjorringe, J.P. Morth, L.B. Møller, B.P. Pedersen, P. Nissen,
Crystal structure of a copper-transporting PIB-type ATPase, Nature, 475 (2011) 59-64.
15

ACCEPTED MANUSCRIPT
[13] M. Andersson, D. Mattle, O. Sitsel, T. Klymchuk, A.M. Nielsen, L.B. Moller, S.H. White, P.
Nissen, P. Gourdon, Copper-transporting P-type ATPases use a unique ion-release
pathway, Nat Struct Mol Biol, 21 (2014) 43-48.
[14] M. González-Guerrero, J.M. Argüello, Mechanism of Cu+-transporting ATPases: soluble
Cu+ chaperones directly transfer Cu+ to transmembrane transport sites, Proc Natl Acad Sci
U S A, 105 (2008) 5992-5997.
[15] T. Padilla-Benavides, C.J. McCann, J.M. Argüello, The mechanism of Cu+ transport
ATPases: interaction with Cu+ chaperones and the role of transient metal-binding sites, J
Biol Chem, 288 (2013) 69-78.
[16] D. Lewis, R. Pilankatta, G. Inesi, G. Bartolommei, M.R. Moncelli, F. Tadini-Buoninsegni,
Distinctive features of catalytic and transport mechanisms in mammalian Ca2+ (SERCA)
and Cu+ (ATP7A/B) ATPases, J Biol Chem, 287 (2012) 32717-33227.
[17] G. Inesi, R. Pilankatta, F. Tadini-Buoninsegni, Biochemical characterization of P-type
copper ATPases, Biochem J, 463 (2014) 167-176.
[18] P.J. Garrahan, M.I. Pouchan, A.F. Rega, Potassium activated phosphatase from human red
blood cells. The mechanism of potassium activation, J Physiol, 202 (1969) 305-327.
[19] G. Inesi, p-nitrophenyl phosphate hydrolysis and calcium ion transport in fragmented
sarcoplasmic reticulum, Science, 171 (1971) 901-903.
[20] A.F. Rega, D.E. Richards, P.J. Garrahan, Calcium ion-dependent p-nitrophenyl phosphate
phosphatase activity and calcium ion-dependent adenosine triphosphatase activity from
human erythrocyte membranes, Biochem J, 136 (1973) 185-194.
[21] Y. Yang, A.K. Mandal, L.M. Bredeston, F.L. González Flecha, J.M. Argüello, Activation of
Archaeoglobus fulgidus Cu(+)-ATPase CopA by cysteine, Biochim Biophys Acta, 1768
(2007) 495-501.
[22] Y. Hatori, E. Majima, T. Tsuda, C. Toyoshima, Domain organization and movements in
heavy metal ion pumps: papain digestion of CopA, a Cu+-transporting ATPase, J Biol
Chem, 282 (2007) 25213-25221.
[23] P.J. O'Brien, D. Herschlag, Catalytic promiscuity and the evolution of new enzymatic
activities, Chem Biol, 6 (1999) R91-R105.
[24] O. Khersonsky, D.S. Tawfik, Enzyme promiscuity: a mechanistic and evolutionary
perspective, Annu Rev Biochem, 79 (2010) 471-505.
[25] A. Babtie, N. Tokuriki, F. Hollfelder, What makes an enzyme promiscuous?, Curr Opin
Chem Biol, 14 (2010) 200-207.
[26] H. Schagger, Tricine-SDS-PAGE, Nat Protoc, 1 (2006) 16-22.
[27] P.A. Lanzetta, L.J. Alvarez, P.S. Reinach, O.A. Candia, An improved assay for nanomole
amounts of inorganic phosphate, Anal Biochem, 100 (1979) 95-97.
16

ACCEPTED MANUSCRIPT
[28] G. Kemmer, S. Keller, Nonlinear least-squares data fitting in Excel spreadsheets, Nat
Protoc, 5 (2010) 267-281.
[29] H. Akaike, A Bayesian Analysis of the Minimum AIC Procedure, in: E. Parzen, K. Tanabe,
G. Kitagawa (Eds.) Selected Papers of Hirotugu Akaike, Springer New York, 1998, pp.
275-280.
[30] R. Wolfenden, Benchmark reaction rates, the stability of biological molecules in water, and
the evolution of catalytic power in enzymes, Annu Rev Biochem, 80 (2011) 645-667.
[31] A.K. Mandal, Y. Yang, T.M. Kertesz, J.M. Argüello, Identification of the transmembrane
metal binding site in Cu+-transporting PIB-type ATPases, J Biol Chem, 279 (2004) 54802-
54807.
[32] M.M. Dodes Traian, D.I. Cattoni, V. Levi, F.L. González Flecha, A two-stage model for lipid
modulation of the activity of integral membrane proteins, PLoS One, 7 (2012) e39255.
[33] V. Levi, J.P. Rossi, M.M. Echarte, P.R. Castello, F.L. Gonzalez Flecha, Thermal stability of
the plasma membrane calcium pump. Quantitative analysis of its dependence on lipid-
protein interactions, J Membr Biol, 173 (2000) 215-225.
[34] L. Aravind, M.Y. Galperin, E.V. Koonin, The catalytic domain of the P-type ATPase has the
haloacid dehalogenase fold, Trends Biochem Sci, 23 (1998) 127-129.
[35] C. Pandya, J.D. Farelli, D. Dunaway-Mariano, K.N. Allen, Enzyme Promiscuity: Engine of
Evolutionary Innovation, J Biol Chem, 289 (2014) 30229-30236.
[36] H. Huang, C. Pandya, C. Liu, N.F. Al-Obaidi, M. Wang, L. Zheng, S. Toews Keating, M.
Aono, J.D. Love, B. Evans, R.D. Seidel, B.S. Hillerich, S.J. Garforth, S.C. Almo, P.S.
Mariano, D. Dunaway-Mariano, K.N. Allen, J.D. Farelli, Panoramic view of a superfamily of
phosphatases through substrate profiling, Proc Natl Acad Sci U S A, 112 (2015) 1974-
1983.
[37] J. Nakamura, G. Tajima, C. Sato, Substrate regulation of calcium binding in Ca2+-ATPase
molecules of the sarcoplasmic reticulum. II. Effect of CTP, GTP, ITP, and UTP, J Biol
Chem, 277 (2002) 24191-24196.
[38] G. Inesi, Active transport of calcium ion in sarcoplasmic membranes, Annu Rev Biophys
Bioeng, 1 (1972) 191-210.
[39] G. Barrientos, M. Taylor, C. Hidalgo, J.D. Cavieres, TNP-8N3-ADP photoinactivation of the
phosphatase activity of FITC-modified SERCA, Ann N Y Acad Sci, 986 (2003) 315-317.
[40] C. Donnet, A.J. Caride, S. Talgham, J.P. Rossi, Chemical modification reveals involvement
of different sites for nucleotide analogues in the phosphatase activity of the red cell
calcium pump, J Membr Biol, 163 (1998) 217-224.
[41] A.C. Babtie, S. Bandyopadhyay, L.F. Olguin, F. Hollfelder, Efficient catalytic promiscuity for
chemically distinct reactions, Angew Chem Int Ed Engl, 48 (2009) 3692-3694.
17

ACCEPTED MANUSCRIPT
[42] E.A. Roman, F.L. González Flecha, Kinetics and thermodynamics of membrane protein
folding, Biomolecules, 4 (2014) 354-373.
[43] M.F. Mohamed, F. Hollfelder, Efficient, crosswise catalytic promiscuity among enzymes that
catalyze phosphoryl transfer, Biochim Biophys Acta, 1834 (2013) 417-424.
[44] A. Ferreira-Pereira, M. Alves-Ferreira, P.C. de Carvalho-Alves, p-nitrophenylphosphatase
activity of plasma membrane H(+)-ATPase from yeast. Implications for the regulation of
the catalytic cycle by H+, J Biol Chem, 269 (1994) 12074-12079.
[45] H. Homareda, M. Ushimaru, Stimulation of p-nitrophenylphosphatase activity of Na+/K+-
ATPase by NaCl with oligomycin or ATP, FEBS J, 272 (2005) 673-684.
[46] A. Warshel, J. Florian, Computer simulations of enzyme catalysis: finding out what has
been optimized by evolution, Proc Natl Acad Sci U S A, 95 (1998) 5950-5955.
[47] R. Wolfenden, Massive Thermal Acceleration of the Emergence of Primordial Chemistry,
the Incidence of Spontaneous Mutation, and the Evolution of Enzymes, J Biol Chem, 289
(2014) 30198-30204.
[48] R. Wolfenden, Degrees of difficulty of water-consuming reactions in the absence of
enzymes, Chem Rev, 106 (2006) 3379-3396.
[49] P.K. Grzyska, P.G. Czyryca, J. Purcell, A.C. Hengge, Transition state differences in
hydrolysis reactions of alkyl versus aryl phosphate monoester monoanions, J Am Chem
Soc, 125 (2003) 13106-13111.
[50] S.J. Admiraal, D. Herschlag, Mapping the transition state for ATP hydrolysis: implications
for enzymatic catalysis, Chem Biol, 2 (1995) 729-739.
[51] C. Lad, N.H. Williams, R. Wolfenden, The rate of hydrolysis of phosphomonoester dianions
and the exceptional catalytic proficiencies of protein and inositol phosphatases, Proc Natl
Acad Sci U S A, 100 (2003) 5607-5610.
[52] R. Wolfenden, Analog approaches to the structure of the transition state in enzyme
reactions, Acc Chem Res, 5 (1972) 10-18.
[53] L. de Meis, Role of the sarcoplasmic reticulum Ca2+-ATPase on heat production and
thermogenesis, Biosci Rep, 21 (2001) 113-137.
[54] N.H. Williams, B. Takasaki, M. Wall, J. Chin, Structure and Nuclease Activity of Simple
Dinuclear Metal Complexes: Quantitative Dissection of the Role of Metal Ions, Acc Chem
Res, 32 (1999) 485-493.
[55] M. Picard, A.-M.L. Jensen, T.L.M. Sørensen, P. Champeil, J.V. Møller, P. Nissen, Ca2+
versus Mg2+ coordination at the nucleotide-binding site of the sarcoplasmic reticulum Ca2+-
ATPase, J Mol Biol, 368 (2007) 1-7.
[56] A. Yamazaki, S. Kaya, T. Tsuda, Y. Araki, Y. Hayashi, K. Taniguchi, An extra
phosphorylation of Na+,K+-ATPase by paranitrophenylphosphate (pNPP): evidence for the
oligomeric nature of the enzyme, J Biochem, 116 (1994) 1360-1369.
18

ACCEPTED MANUSCRIPT
[57] S.C. Kamerlin, J. Wilkie, The role of metal ions in phosphate ester hydrolysis, Org Biomol
Chem, 5 (2007) 2098-2108.
[58] W. Humphrey, A. Dalke, K. Schulten, VMD: visual molecular dynamics, J Mol Graph, 14
(1996) 33-38.
[59] M. González-Guerrero, E. Eren, S. Rawat, T.L. Stemmler, J.M. Argüello, Structure of the
two transmembrane Cu+ transport sites of the Cu+ -ATPases, J Biol Chem, 283 (2008)
29753-29759.
[60] H.A. Kramers, Brownian motion in a field of force and the diffusion model of chemical
reactions, Physica, 7 (1940) 284-304.
[61] K.S. Siddiqui, S.A. Bokhari, A.J. Afzal, S. Singh, A novel thermodynamic relationship based
on Kramers Theory for studying enzyme kinetics under high viscosity, IUBMB Life, 56
(2004) 403-407.
[62] S.B. Kaufman, F.L. González-Flecha, R.M. González-Lebrero, Opposing effects of Na+ and
K+ on the thermal stability of Na+,K(+)-ATPase, J Phys Chem B, 116 (2012) 3421-3429.
[63] L.L. Schaleger, F.A. Long, Entropies of Activation and Mechanisms of Reactions in
Solution, Adv Phys Org Chem, 1 (1963) 1-33.
[64] R. Wolfenden, M. Snider, C. Ridgway, B. Miller, The Temperature Dependence of Enzyme
Rate Enhancements, J Am Chem Soc, 121 (1999) 7419-7420.
Graphical Abstract
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Fig. 1
Fig. 2
Fig. 3
20

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Fig. 4
Fig. 5
Fig. 6
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Fig. 7
Highlights


The promiscuous phosphomonoestearase activity of AfCopA is characterized and
compared with the principal ATPase activity.
The enzyme is more efficient for capturing ATP, but pNPP would be better placed at the
catalytic site.



Both activities are enhanced by Mg2+ (essential) and phospholipids (non-essential),
Salts and Cu+ have opposite effects on the ATPase and phosphatase activities.
Catalysis of both substrates is driven by an enthalpy lowering mechanism.
22