Characterization of acetyl-CoA synthetase kinetics and ATP-binding

Article abstract of DOI:10.1016/j.bbagen.2019.03.017

Background: The superfamily of adenylating enzymes is a large family of enzymes broadly distributed from bacteria to humans. Acetyl-CoA synthetase (Acs), member of this family, is a metabolic enzyme with an essential role in Escherichia coli (E. coli) acetate metabolism, whose catalytic activity is regulated by acetylation/deacetylation in vivo. Methods: In this study, the kinetics and thermodynamic parameters of deacetylated and acetylated E. coli Acs were studied for the adenylating step. Moreover, the role of the T264, K270, D500 and K609 residues in catalysis and ATP-binding was also determined by Isothermal titration calorimetry. Results: The results showed that native Acs enzyme binds ATP in an endothermic way. The dissociation constant has been determined and ATP-binding showed no significant differences between acetylated and deacetylated enzyme, although k_cat was much higher for the deacetylated enzyme. However, K609 lysine mutation resulted in an increase in ATP-Acs-affinity and in a total loss of enzymatic activity, while T264 and D500 mutant proteins showed a total loss of ATP-binding ability and a decrease in catalytic activity. K609 site-specified acetylation induced a change in Acs conformation which resulted in an exothermic and more energetic ATP-binding. Conclusions: The differences in ATP-binding could explain the broadly conserved inactivation of Acs when K609 is acetylated. General Significance: The results presented in this study demonstrate the importance of the selected residues in Acs ATP-binding and represent an advance in our understanding of the adenylation step of the superfamily of adenylating enzymes and of their acetylation/deacetylation regulation.

Full text of DOI:10.1016/j.bbagen.2019.03.017

Accepted Manuscript
Characterization of acetyl-CoA synthetase kinetics and ATP-
binding
Julia Gallego-Jara, Gema Lozano Terol, Ana Écija Conesa,
Barbara Zambelli, Manuel Cánovas Díaz, Teresa de Diego Puente
PII:
S0304-4165(19)30073-X
DOI:
Reference:
https://doi.org/10.1016/j.bbagen.2019.03.017
BBAGEN 29331
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BBA - General Subjects
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Revised date:
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1 February 2019
20 March 2019
25 March 2019
Please cite this article as: J. Gallego-Jara, G.L. Terol, A.É. Conesa, et al., Characterization
of acetyl-CoA synthetase kinetics and ATP-binding, BBA - General Subjects,
https://doi.org/10.1016/j.bbagen.2019.03.017
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Characterization of acetyl-CoA synthetase kinetics and ATP-binding
Julia Gallego-Jara1*, Gema Lozano Terol1*, Ana Écija Conesa1, Barbara Zambelli2,
Manuel Cánovas Díaz1 and Teresa de Diego Puente1.
1Department of Biochemistry and Molecular Biology and Immunology (B), Faculty of
Chemistry, University of Murcia, Campus of Espinardo, Regional Campus of
International Excellence ‘‘Campus Mare Nostrum’’, P.O. Box 4021, Murcia E-30100,
Spain.
2Laboratory of Bioinorganic Chemistry, Department of Pharmacy and Biotechnology,
University of Bologna, Via Giuseppe Fanin 40, I-40127 Bologna, Italy.
*Corresponding author
Email: julia.gallego@um.es and gema.lozano@um.es; Tel.: +34868887397; Fax:
+34968364148.

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Abstract
Background
The superfamily of adenylating enzymes is a large family of enzymes broadly distributed
from bacteria to humans. Acetyl-CoA synthetase (Acs), member of this family, is a
metabolic enzyme with an essential role in Escherichia coli (E. coli) acetate metabolism,
whose catalytic activity is regulated by acetylation/deacetylation in vivo.
Methods
In this study, the kinetics and thermodynamic parameters of deacetylated and acetylated
E. coli Acs were studied for the adenylating step. Moreover, the role of the T264, K270,
D500 and K609 residues in catalysis and ATP-binding was also determined by
Isothermal titration calorimetry.
Results
The results showed that native Acs enzyme binds ATP in an endothermic way. The
dissociation constant has been determined and ATP-binding showed no significant
differences between acetylated and deacetylated enzyme, although kcat was much higher
for the deacetylated enzyme. However, K609 lysine mutation resulted in an increase in
ATP-Acs-affinity and in a total loss of enzymatic activity, while T264 and D500 mutant
proteins showed a total loss of ATP-binding ability and a decrease in catalytic activity.
K609 site-specified acetylation induced a change in Acs conformation which resulted in
an exothermic and more energetic ATP-binding.
Conclusions
The differences in ATP-binding could explain the broadly conserved inactivation of Acs
when K609 is acetylated.
General Significance
The results presented in this study demonstrate the importance of the selected residues
in Acs ATP-binding and represent an advance in our understanding of the adenylation
step of the superfamily of adenylating enzymes and of their acetylation/deacetylation
regulation.
Keywords: Escherichia coli; Acetyl-CoA synthetase; ITC; ATP; lysine acetylation.

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1. Introduction
The ANL superfamily of adenylating enzymes contains acyl- and aryl-CoA
synthetases, firefly luciferase, and the adenylation domains of the modular non-
ribosomal peptide synthetases (NRPSs). ANL enzymes are broadly distributed in
prokaryotes and eukaryotes and share a sequence identity of
̴ 20 %. Although ANL
enzymes show differences in their respective catalytic mechanisms, all of them are
structurally homologous and share a global mechanism of two-step catalysis: a first
adenylation reaction and a diverse set of second partial reactions [1]. Accordingly, acyl-
and aryl-CoA synthetases catalyse the activation of a wide variety of carboxylates into
the acyl- aryl-CoA derivatives in two independent reactions, as was described in 1956
by Paul Berg [2]. In the first step the carboxylate is activated into a carboxyl-AMP
intermediate. Subsequently, a CoASH molecule reacts with the adenylate intermediate
to lead to the acyl- aryl-CoA and AMP. Carboxylic substrates can vary from acetate (2C)
to long fatty acids such as myristic acid (14C). Acyl-CoA formation has been widely
studied for 60 years [2] and a ping-pong kinetic mechanism has been established for
many of these synthetases [3–7] (Figure 1A).
Figure 1. (A) Acyl- and aryl-CoA synthetases ping-pong mechanism. (B)
Crystallographic structure of Salmonella enterica acetyl-CoA synthetase (PDB ID:
1PG4). The N- domain is colored in green, C-terminal domain is colored in yellow and
A8 loop is shown in blue.
The structure of the ANL superfamily of adenylating enzymes is formed by a long
non-catalytic N-terminal domain and a short C-terminal domain containing the main
catalytic residues (Figure 1B). The substrates are positioned at the interface of the two
domains. All ANL enzymes are flexible proteins, and were found in two different
conformations that differ for the position of the C-terminal domain that rotates of ~ 140 ᵒ
[1]: an adenylating or closed conformation carries out the first catalytic reaction, while a
second open conformation (called thioester-forming conformation for acyl-CoA
synthases) is responsible for the final catalytic step. Proteins belonging from the ANL
superfamily contain ten highly conserved motifs (A1-A10). Several conserved residues
have been shown to be involved in ATP-binding in the structures crystalized in the
presence of ATP [8–16]: an aspartic acid (D500 in E. coli Acs) from A7 domain interacts
through hydrogen bonds with the ATP ribose molecule [9,11,14,16], an arginine (R519
in E. coli Acs) from A8 domain binds the β-phosphate of the PPi [8–11,14], a lysine (K609

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in E. coli Acs) from A10 domain coordinates ATP pyrophosphate, together with a
conserved phosphate binding loop (P-loop) (S/T)(S/T/G)G(S/T)TGxPK (residues 264-
272 in E. coli Acs) from A3 domain, which binds the beta and gamma phosphates of
pyrophosphate [8–13,15,16].
Acetyl-CoA synthetase is an important metabolic enzyme for prokaryote and
eukaryote organisms. Its physiological role is to activate acetate to acetyl-CoA to be used
in several metabolic routes. Prokaryotic organisms, such as E. coli, can consume acetate
from the environment as carbon source, converting it to acetyl-CoA by two independent
routes, one catalysed by Acs [17,18]. The enzyme, as other AMP-forming synthetases,
is regulated by reversible acetylation of the conserved lysine in A10 domain (K609 in E.
coli Acs) [19–24]. Acetylation inhibits the catalytic activity of the first partial reaction
(adenylation), leaving the second step unaffected, and it is reversed by a sirtuin
deacetylase [25,26].
The present study aims to characterize E. coli Acs kinetics and thermodynamics.
A structural model for the adenylating conformation, not available so far, was predicted
using other ANL enzyme structures as templates. From the Acs model, four amino acids
were selected to study their role in catalysis and ATP-binding: T264, K270, D500 and
K609. The results evidenced to the important role of these residues in the catalytic ability
and ATP-binding of Acs enzyme.
2. Methods
2.1 Plasmids construction and incorporation of N-(Ɛ)-acetyl-lysine into Acs
at K609
To overexpress Acs protein (uniprot code P27550), the corresponding ASKA plasmid
was used (ASKAacs) [27]. Overexpression plasmids of the single amino acid mutants of
Acs (T264A, K270A, D500A and K609A) were obtained by site-directed mutagenesis
from ASKAacs plasmid (ASKAacsT264A, ASKAacsK270A, ASKAacsD500A, ASKAacsK609A). To
purify CobB deacetylase protein, the gene cobB of E. coli BW25113 was PCR-amplified
and cloned into pBAD24-MBP [28]. To overexpress Acs protein acetylated on K609
(K609Kac), incorporation of N-(Ɛ)-acetyl-lysine was carried out using the genetic code
expansion concept. A pRSF-Duet-1 modified plasmid for site-specific incorporation of
acetyl-L-lysine (pRSF-Duet-1- acetyl-lysyl-tRNA-synthetase AcKRS3/MbtRNACUA),
kindly supplied by Prof. Michael Lammers (University of Cologne) was employed [29,30].

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acs gene containing an amber stop codon at the 609 position was constructed by site-
directed mutagenesis from ASKAacs plasmid. His6-acsK609ambercodon gene was
PCR-amplified and cloned into pRSF-Duet-1- acetyl-lysyl-tRNA-synthetase
AcKRS3/MbtRNACUA plasmid (pRSFacsK609ac). All molecular biology enzymes used were
purchased from Thermo Fisher Scientific. The strains, plasmids and primers used are
listed in Supplementary Table S1.
2.2 Proteins overexpression and purification
Chemically competent E. coli BL21 (DE3) strains were transformed by heat shock
at 42 °C with overexpressing plasmids. Cultures were grown overnight at 30 °C with
orbital shaking (200 rpm). The culture medium was lysogen broth (LB) (10 g L-1 tryptone,
5 g L-1 yeast extract and 5 g L-1 NaCl). Expression was induced with 0.1 mM Isopropyl
β-D-1-thiogalactopyranoside (IPTG) when the culture optical density at 600 nm (OD600
)
reached 0.5-0.6. To obtain site-specifically acetylated Acs (Acs K609ac), overexpression
cultures were supplemented with 10 mM L-acetyl Lysine and 20 mM nicotinamide (Sigma
Aldrich) after IPTG induction. Cell pellets were harvested by centrifugation (20 min; 6000
x g) and resuspended in binding buffer (50 mM potassium phosphate, 500 mM NaCl, 25
mM imidazole, pH 8). Cells were disrupted by three passages of the crude extract
through a French pressure cell system at 20,000 psi. Following the removal of cell debris
by centrifugation at 18000 x g for 30 min at 4 °C, the supernatant was applied onto a Ni
(II)-loaded 5 mL His-Trap HP column (GE Heathcare) previously equilibrated in binding
buffer. Protein was eluted using a linear gradient of imidazole from 0 to 500 mM at a flow
rate of 5 mL min-1. The protein buffer was then changed to buffer A (50 mM Tris-HCl, 2
mM dithiothreitol (DTT), pH 7.4) using a HiPrep™ 26/10 desalting column (GE
Heathcare) at a flow rate of 5 mL min-1. The final Acs fractions were then concentrated
and further purified using a Superdex 75 10/300 GL gel filtration column (GE Heathcare)
equilibrated with buffer B (100 mM Tris-HCl, 150 mM NaCl, 1 mM TCEP, 3 mM MgCl2,
pH 7.4) at a flow rate of 0.5 mL min−1. CobB sirtuin was purified using an amylose resin
(New England Biolabs) following a previously reported protocol [28].
2.3 Protein deacetylation
Selected proteins were deacetylated with CobB sirtuin. The reactions were carried
out with a 1:50 ratio (Acs:CobB) in the presence of 2 mM NAD+ at 30 ᵒC for 8 hours [28].
To separate Acs and CobB proteins, reactions were loaded onto a 5 ml Hi-trap Q HP
(GE Heathcare) column anionic exchange column, at a flow rate of 5 mL min-1 previously

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equilibrated with buffer A. A step gradient procedure was used to separate the proteins
with increasing ionic strength from 0 to 1 M NaCl.
2.4 Liquid chromatography–mass spectrometry assay (LC-MS)
To evaluate acetylation level of Acs, purified Acs before and after (deacetylated
Acs, deacAcs) CobB incubation, and Acs K609ac, were analysed by LC-MS. Samples
were alkylated by incubation with 100 mM iodoacetamide (IAA) for 30 min at room
temperature in the dark. Proteins were digested with 0.5–1 μg of proteomics grade
Trypsin Gold (Promega) for 3 h at 37 ˚C. The reaction was stopped by the addition of
0.1% formic acid, and the samples were dried using a vacuum evaporator. Tryptic
peptides generated from the samples were separated and analysed by LC-MS. An
Agilent 1100 (Agilent Technologies) was equipped with a Zorbax SB-C18 HPLC column
(Agilent Technologies) and connected to an Agilent Ion Trap XCT Plus mass
spectrometer (Agilent Technologies) that had an electrospray (ESI) interface. Two
mobile phases were used: phase A was composed of water/acetonitrile/ formic acid
(94.9:5:0.1, v/v), and phase B consisted of water/acetonitrile/formic acid (10:89.9:0.1,
v/v). The digested peptides were resuspended in 20 μl of phase A and eluted using a
linear gradient of 0–80% phase B for 180 min at a flow rate of 10 μl min-1. The mass
spectrometer was operated in positive mode with a capillary spray voltage of 3500 V, at
a scan speed of 8100 (m/z)/sec from 50 to 2200 m/z, with a target mass of 1000 m/z,
and three scans were averaged. The nebulizer gas pressure was set at 15 psi, and the
drying gas flowed at 5 L min-1 at a temperature of 350˚C. MS/MS data were collected in
an automated data-dependent mode (AutoMS mode). Data processing was performed
with the Data Analysis program for LC/MSD Trap Version 3.3 (Bruker Daltonik) and the
Spectrum Mill MS Proteomics Workbench (Agilent Technologies) [60,61]. After
automated validation of the results, the identified proteins and the sequences of the
digested peptides were compiled. Peptides with a score threshold of 8 and a percentage-
scored peak intensity higher than 70% were considered valid.
2.5 Circular dichroism
Circular dichroism spectroscopy (CD) was performed on a PiStar-180
spectrophotometer (Applied Photophysics, U.K.) equipped with a N2 purge and a Peltier
system for temperature control. Far-UV CD spectra (190-240 nm) were obtained at 20
°C from protein samples (0.15 mg mL-1) in 50 mM potassium phosphate buffer pH 7.4
containing 20 mM NaCl). The spectra were obtained in a 0.1 cm path length cuvette,

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reading every nm. Data were converted to molar ellipticity units by using the mean
residue mass of 110.40 kDa.
2.6 Kinetics characterization
The acetyl-CoA synthetase assay was based on the coupled assay reported by
Williamson and Corkey [31]. AMP production was detected via a coupled enzyme assay
in which myokinase (MK), pyruvate kinase (PK) and lactate dehydrogenase (LDH)
couple AMP production to NADH oxidation. Standard acetyl-CoA synthetase assays (0.2
mL) were performed at 37 °C in 50 mM potassium phosphate buffer at pH 7.5 containing
3.0 mM PEP (phosphoenolpyruvate), 5 units MK, 1 unit PK, 1.5 units LDH, 5 mM MgCl2,
2.5 mM ATP, 1.5 mM CoA, 0.1 mM NADH, 5 mM acetate and 1 mM DTT. The reaction
was started by the addition of Acs. All reactions were performed in triplicate. Specific
activity was calculated using the extinction coefficient of NADH (6.22 mM-1 cm-1) and was
based on the oxidation of two molecules of NADH for each AMP molecule released. One
unit of Acs activity is defined as 1 μmole of acetyl-CoA formed per minute at pH 7.5 and
37 °C. Obtained milliunits of absorbance per minute at 340 nm (Synergy H1 Hybrid Multi-
Mode Reader, Biotek), were converted to units of absorbance per minute by means of
the PathCheck Sensor feature. Substrate concentrations were varied from 0 to 0.5 mM
for ATP and 0 to 2 mM for acetate. Pseudo-first-order kinetic parameters were
determined using Prism v6 (GraphPad) analytical software and standard deviations were
determined from the three replicates. Statistical multiple comparison one-way ANOVA
has been carried out for kinetic parameters to know if the differences between
parameters were or not significatively different. Acs inhibition assays were carried out
employing a pyrophosphate detection kit (Sigma Aldrich) following the commercial
protocol.
2.7 Isothemal titration calorimetry assays
To determine the thermodynamic parameters of interaction between Acs proteins
and ATP, isothermal titration calorimetry (ITC) assays were carried out using a VP-ITC
micro-calorimeter (MicroCal). Protein was inserted in the cell at a concentration of 70
µM, while the ligand concentration in the syringe was at 800 µM. Titrations were
performed at 25 °C with 30 injections of 10-µL each, separated by 5 min, into the 2 ml
sample cell. Data were fitted to a one-binding-site model using Peaq-ITC software
(Malvern). Each binding isotherm was measured at least in duplicate and standard
deviations were determined from the two replicates. Statistical multiple comparison one-

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way ANOVA has been carried out for thermodynamic parameters to know if the
differences between parameters were or not significatively different.
3. Results
3.1 Acs structural model
To ascertain the 3D disposition of the residues involved in the adenylating step
performed by E. coli Acs, structural models were built using SWISS-MODEL software
[32], using ten ANL structures in the adenylating conformation as templates. The quality
parameters of the models are summarized in Supplementary Table S2. On the basis of
the QMEAN and GMQE values [33], the model generated with the acyl-CoA synthetase
from Saccharomyces cerevisiae (PDB ID: 1RY2) as template was selected [8]. The
model showed a z-score QMEAN of -2.55 and was compared to 1RY2 template through
structural alignment. The structures were essentially identical (Figure 2A2) and
conserved lysine from A10 motif was located at the same position (Figure 2A3).
Figure 2. E. coli Acs model generated using medium chain acyl-CoA synthetase from
human mitochondria. (A1) Selected residues (K609 in blue, T264 in green, K270 in
yellow, and D500 in orange) surrounding ATP (red) ligand. (A2) Model (light blue) aligned
with target structure (dark blue). (A3) Conserved A10 lysines, K609 from model (blue)
and K557 from target (yellow) and ATP ligand (red). (B) Conserved ANL family motifs
A1-A10 (ATP substrate is in red). Figure was made using PyMOL Molecular Graphics
System, Version 2.0 Schrödinger, LLC.
3.2 Effects of single amino acid substitutions on Acs structure
To identify the conserved residues in the ANL sequences, the E. coli Acs sequence
was aligned with the sequences of ANL proteins from different organisms whose
structures had been previously determined in the closed conformation (these structures
were also used to construct Acs model): acetyl-CoA synthetase from Sacharomyces
cerevisiae, medium chain acyl-CoA synthetase from human mitochondria, 4-
coumarate:CoA ligase from Nicotiana tabacum, malonyl CoA synthetase (MatB) from
Rhodopseudomonas palustris, acyl-CoA synthase (FadD32) from Mycobacterium
smegtatis, 2-succinylbenzoate-CoA ligase (MenE) from Bacillus subtilis and D-alanyl
carrier protein ligase (DltA) from and Bacillus cereus (Figure 3) [8–12,16,26,34,35].
Identity percentages were 48%, 26 %, 23 %, 23 %, 25 %, 20% and 27 %, respectively.
From protein alignment, 3D model and previous literature [8–12,16,26,34,35], four

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residues were selected from the Acs E. coli sequence to study their role in Acs catalytic
mechanism: threonine 264 (T264) and lysine 270 (K270) from A3 motif P-loop, aspartic
acid 500 (D500) from A7 motif and lysine 609 (K609) from A10 motif (Figure 3).
Figure 3. Protein alignment. Selected residues to single substitutions (yellow box) are
shown.
The selected residues were mutated to alanine with site-directed mutagenesis,
producing single Acs mutants, which were deacetylated in vitro by CobB after
purification: Acs T264A, Acs K270A, Acs D500A and Acs K609A. The proteins were
analysed by CD in order to investigate the effect of the substitutions and acetylation on
the conformation of Acs. The α-helical content was also determined using the software
Bestsel [36,37] (Table 1). As shown in Figure 4, the spectra for the single mutants were
very similar to the spectrum of the native Acs, indicating the absence of major structural
changes induced by mutations. Quantitative analysis of α-helical content indicated a
relative amount from 12.2 to 13.8 %, also suggesting that the Acs structure was not
affected by the mutations. On the other hand, site-specific K609 acetylation produced a
protein (Acs K609ac) with a significant increase of the α-helical content (22.3%),
suggesting an impact of this protein modification onto the Acs protein structure.
Figure 4. Far UV spectra of proteins. The color code is indicated in the figure.
Table 1. α-helical content of Acs proteins.
3.3 Effects of single amino acid substitutions on Acs activity
Acs protein purified from E. coli BL21 (DE3) showed 14 acetylated Lys (K11, K106, K115,
K123, K130, K207, K221, K408, K416, K585, K604, K609, K617, K640), as verified using
liquid chromatography mass spectrometry (LC-MS/MS). Wild-type and mutants were
incubated with CobB and NAD+, and LC-MS/MS showed that eight acetylated Lys (K106,
K123, K130, K207, K221, K585, K604 and K617) were maintained, while K11, K115,
K408, K416, K609 and K640 were deacetylated by CobB, suggesting that they are site-
specific substrates of CobB deacetylase (Table 2). In vitro acetylation of K609 to produce

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K609ac followed by LC-MS/MS, produced six acetylated Lys (K123, K130, K408, K604,
K609 and K617).
Table 2. Acs LC-MS/MS assay. Site-specific lysine deacetylation of Acs by CobB is
shown in bold. Acetylated lysines not detected previously are shown in italic.
Kinetics characterization of Acs and its mutants was carried out using ATP and
acetate substrates. Michaelis-Menten behaviors were observed for all the proteins and
substrates. Kinetic parameters are summarized in Table 3.
Deacetylated Acs showed a KM value of 73 and 35 µM for ATP and acetate,
respectively. Acetylation did not affect to the KM values (76.32 and 41 µM for Acs without
deacetylation), while it strongly affected the kcat values, being 6900 and 4600 min-1 for
ATP and acetate in the deacetylated form, and 52.23 and 38.26 min-1 for the acetylated
form. T246A and D500A mutations decreased both KM and kcat values: specificity
constants (kcat/KM) for T264A mutant dropped of 36 and 43-fold for ATP and acetate
substrates respectively, while kcat/KM for D500A mutant protein decreased 3200 and
2500-fold for ATP and acetate, respectively. Surprisingly, mutation of K270 produced an
increase of 8 times for ATP and 6 times for acetate as compared to the native Acs, while
the KM values were similar to native Acs. Finally, K609A and K609Kac proteins did not
show any activity.
Table 3. Kinetic parameters for Acs and mutant proteins with ATP and acetate as
substrates.
3.4 Effects of single amino acid substitutions on ATP binding by Acs
The thermodynamics of ATP-binding to Acs proteins were studied using ITC.
Titration of ATP onto acetylated and deacetylated proteins produced endothermic peaks
indicative of the occurrence of protein-ligand interaction (Figure 6). A fitting of the binding
isotherms indicates that one ATP molecule binds per protein monomer, with a
dissociation constant of 30 µM (deacAcs, Figure 6A) and 50 µM (Acs, Figure 6B). The
binding is entropy-driven, showing a positive favourable ∆S (T∆S = 86.3 kJ mol-1 K-1 for
deacAcs and 31.6 kJ mol-1 K-1 for Acs) and a positive unfavourable enthalpy (∆H = 10 kJ
mol-1 for deacAcs and 7 kJ mol-1 for Acs) (Table 4). No-binding was observed when

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acetate was titrated into a sample cell containing Acs alone (Supplementary Figure S1),
indicating that Acs interacts with its organic acid substrate only in the ATP-bound form.
K609A (Figure 6C) and K270A (Figure 6D) Acs mutants bind ATP with an
endothermic reaction, as observed for the native protein. The K609A mutant (Figure 6C)
binds ATP with the highest affinity, with a Kd of 3.55 µM, one order of magnitude lower
than the one measured for the native protein, the highest value of ΔH 16.5 kJ mol-1 and
T∆S = 47.7 kJ mol-1 K-1 (Table 4). The K270A mutant, on the other hand, showed a
similar affinity to the native protein with Kd of 20 µM, and lower values of ΔH = 2.7 kJ
mol-1 and T∆S = 29.6 kJ mol-1 K-1. On the other hand, D500A (Figure 6E) and T264A
(Figure 6F) mutations produced Acs variants unable to bind ATP.
Figure 6. ATP titrations onto deacAcs (A), Acs (B), K609A (C), K270A (D), T264A (E)
and D500A (F), followed by ITC. Raw data of heat flow as a function of time (top panels)
and integrated data after subtraction of the heat of dilution (bottom panels) are reported.
The solid lines represent the best-fit curves to the data, using a single-set-of-site model.
Differently from the deacetylated proteins, injections of ATP onto K609ac solution
produced an exothermic response. A fit of the binding isotherm indicated that ATP binds
K609ac protein with higher affinity, as compared to the deacetylated proteins, with Kd of
2.07 µM, a negative favorable ∆H = -131 kJ mol-1 and a negative unfavorable T∆S = -
98.4 kJ mol-1 K-1 (Table 4).
Figure 7. ATP-binding to K609ac Acs protein followed using ITC. Raw data representing
heat flow over time are reported (top panel) together with integrated head data after
subtraction of the heat of dilution (bottom panel). The solid line represents the best-fit of
the data, using a one-set-of-sites binding model.
Table 4. Thermodynamic parameters of ATP-binding for Acs wt and mutant proteins. Kd:
dissociation constant, ∆H: enthalpy change, ∆S: entropy change.
3.5 Acs inhibition by cAMP
S. enterica Acs inhibition by cAMP has been recently reported [38]. To verify if E.
coli Acs was similarly inhibited by cAMP and AMP, enzymatic reactions were carried out
in the presence of 1 mM cAMP or AMP. cAMP was able to inhibit Acs, while no inhibition

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was observed when AMP was present (Figure 8). To determine the inhibition constant
(ki) of cAMP, apparent KM were determined at different cAMP concentrations from 0 to 1
mM (0, 50, 500 and 1000 µM) and plotted versus inhibitor concentration (Figure 8A). The
calculated value of ki was 232 µM. Moreover, a double-reciprocal plot (1/v versus 1 /
[ATP]) at different cAMP concentrations was built (Figure 8B), suggesting the occurrence
of a competitive inhibition mechanism, as previously described for S. enterica Acs [38].
Figure 8. Analysis of CobB inhibition by cAMP. (A) Acs apparent KM at different cAMP
concentrations. (B) Double-reciprocal plot (1/v versus 1/ ATP]) of Acs activity at different
cAMP concentrations: 0 ( ), 0.5 ( ), 0.75( ) and 1 ( • ) µM. (C) Acs activity (%) after
CobB deacetylation in the absence or in the present of ATP, cAMP, CoA or acetate. (D)
Acs activity (%) in the absence and in the presence of AMP 1mM. Presented data are
average of triplicates. Standard deviations were always less than 10%
To ascertain whether the presence of substrates or cAMP inhibitor affected Acs
deacetylation by CobB, as observed for S. enterica Acs, deacetylation assays using Acs
as substrate were carried out in the presence of 1 mM ATP, cAMP, CoA or acetate. Acs
activity was measured after deacetylation, and the activity was compared with a control
assay without substrate or inhibitor (Figure 8C). Acs deacetylation by CobB is partially
inhibited in the presence of ATP and cAMP, while it is not affected by the presence of
acetate or CoA.
Acs-cAMP binding was studied by ITC for all the proteins. Calorimetric assays were
carried out under the same conditions employed for ATP-binding studies, but no-binding
was observed under the conditions tested (data not shown).
4. Discussion
ANL enzymes belong to a superfamily of proteins essential for the metabolism of
all organisms, from bacteria to humans. Acs protein has a principal role in E. coli acetate
metabolism, catalysing one of the acetate uptake pathways. E. coli is the most used
microorganism in biotechnology, and acetate excretion limits growth rates and
bioprocess yields [39,40]. Thus, the understanding of the acetate metabolism is
important to optimize the biotechnological systems based on E. coli metabolism.
ANL dynamic conformation is an atypical characteristic of this family of enzymes.
Several ANL enzymes have been resolved in the thioester-forming or open conformation
[41–45], but less information is available about the adenylating or closed conformation.

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To our knowledge, ten proteins have been crystalized in this conformation [8–16,35]. In
this study we used these structures as templates to predict models of E. coli Acs protein
in the closed conformation. The model with least z-score was selected and was aligned
with the template structure to locate conserved positions. This allowed us to select four
residues which were located around the ATP substrate (Figure 2A1), two of them (T264
and K270) belonging to P-loop from A3 motif, which has a similar sequence than those
from ATPases and GTPases [46] and has been found wrapping ATP pyrophosphate in
several ANL structures [8–11,13,14]. In addition, D500 is a 100% conserved ANL residue
from the A7 motif involved in ATP-binding through ribose sugar moiety [9,11,14,16] and
K609 lies in the A10 motif and is involved in adenylating catalytic step being found to
interact with the α-phosphate [11,13,16,47,48], the β-phosphate [8,9] or both of them
[10] of the ATP substrate. The role of K609 residue has been widely studied for several
ANL enzymes showing a great decrease or a null activity when this residue is mutated
[7,13,25,28,45,49–51]. Thus, Reger et al. reported a total loss of activity of Salmonella
enterica Acs when this residue was mutated by alanine [45]. The acetylation of the A10
conserved lysine has been described as a regulator mechanism in many ANL enzymes
[19,21,35,52,53]. Thus, in E. coli, Acs activity is regulated in vivo by
acetylation/deacetylation, which constitutes an acetate metabolism regulation system
[28,54]. Acs is acetylated by PatZ acetyltransferase (also known as Pka or YfiQ) in
several lysines, although, to date, K609 is the only one responsible for Acs activity
regulation. K609 acetylation induces Acs inhibition [24], which is reversed by CobB
sirtuin [28]. In this study 14 acetylated lysines were observed in Acs sequence and six
of these lysines were identified as CobB substrates (K11, K115, K408, K416, K609 and
K640). This result agrees with our previous work which demonstrated that CobB
deacetylates several acetylated Lys in Acs sequence, not only K609. Moreover, LC-MS
assay identified six lysines acetylated in Acs sequence that had not been observed
previously (K11, K115, K123, K408, K416 and K640) [55–60].
Acs kinetics were determined for the Acs native enzyme and for the single amino
acid selected mutants. Native Acs kinetic parameters were similar to those reported for
other ANL enzymes, with a KM in the range of 20-100 µM. However, kcat values were
much higher than those previously reported [6,9,12,13,15,16,26,45,49,61]. ANL kcat
determined to date are between 10-2000 min-1, whereas our Acs kcat was 6900 and 4600
min-1 for ATP and acetate, respectively. These high values could be due to protein
deacetylation after purification. Kinetic parameters measured for Acs without
deacetylation showed no change of enzyme specificity (KM), but a significant reduction

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of activity (kcat). This result suggests that acetylation decreases the amount of active
enzyme through enzyme inhibition, although it does not affect substrates specificity. Acs
deacetylation increased kcat values more than a 100-fold. Thus, protein deacetylation is
essential to study Acs activity and single amino acids substitutions, although, to our
knowledge, this is the first kinetic study of an ANL enzyme previously deacetylated to
ensure that all the protein is catalytically active.
The kinetic parameters of mutant proteins showed differences with respect to
native Acs. Acs D500A protein showed a 3.69 and 45-fold increase for ATP and acetate
KM, respectively. The catalytic response of an ANL enzyme to this conserved aspartic
acid mutation has been previously reported. Thus, a 75-fold and 500-fold increase in the
KM values for ATP were observed for DltA enzyme from B. cereus and 4CBL (4-
chlorobenzoate: Coenzyme A ligase) from Alcaligenes sp, respectively, when this
residue was mutated [7,9]. Wu et al., suggest that, although the conserved aspartic is
not near acyl- binding pocket, its role may be important for the second step of the 4CBL
protein [7]. T264 replacement by alanine induced an important change in Acs catalytic
response. KM values were 4.6 and 10 times higher than native values for ATP and
acetate, respectively. The catalytic role of T264 (first residue in P-loop) has previously
been studied and similar catalytic effects were described [7,11]. KM values for K270A
mutant were very similar to those observed for native Acs; however, kcat values were
much different. K270A mutant showed a surprising increase in the kcat constants (almost
10-fold). To our knowledge, the catalytic role of this residue has not been studied to date.
From our results we suggest that this residue may have an important catalytic role in Acs
reaction that had not been shown before.
Acs proteins (Acs and deacAcs) showed very similar thermodynamic parameters,
suggesting that in vivo Acs acetylation does not affect ATP-binding. The values of
dissociation constants (30 and 50 µM) are similar to those determined for the
Rhodopseudomonas palustris (R. palustris) malonyl-CoA synthetase (MatB) protein (5.6
µM) [12] and Mycobacterium smegtatis (M. smegtatis) FadD32 protein (35 µM) [14], and
were lower than that reported for S. enterica Acs (322 µM) [38]. Kd values measured by
ITC are in the same order of magnitude as the ATP KM determined in this study, 73 and
76.32 µM, respectively. Acs K270A mutant protein showed a Kd for ATP-binding very
similar to native Acs, as was observed when ATP KM were compared. K609A mutant
protein showed a larger enthalpy of reaction and a lower dissociation constant (3.55 µM).
A similar behavior was observed by Crosby et al. (2012) for the R. palustris MatB protein

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[12]: MatB, a protein of ANL family, showed a lower affinity and binding enthalpy as
compared to its mutant in the A10 conserved lysine [12].
To study the effect of K609 acetylation in ATP-binding, an Acs version specifically
acetylated in K609 was constructed and purified. Circular dichroism results showed that
Acs K609ac had a different conformational fold from the others Acs proteins, as well as
a changed behaviour for ATP-binding, with a higher affinity and opposite binding
signatures ΔH and ΔS. This result again points out the importance of this residue in Acs
catalytic mechanism which is reflected in the enzyme inhibition by K609 acetylation. The
diverse ATP-binding mode of the K609ac protein reflects a change in the catalytic
reaction: in particular, the higher affinity of this protein variant for ATP. It is important to
note that, according to MS results, both acetylated and deacetylated molecules of Acs
co-exist in the protein preparation. Thus, MS spectrometry showed K609 in the
acetylated and deacetylated states for Acs protein. This observation could reflect that
Acs showed a behavior very similar to deacetylated Acs and different from K609ac
protein.
Competitive inhibition of S. enterica Acs by cAMP has been recently demonstrated
[38], so we decided to study E. coli Acs inhibition. An inhibition constant of 232 µM was
established. This value was very similar to those determined for S. enterica Acs (185
µM) and intracellular cAMP concentration calculated for E. coli grown in the absence of
glucose [62,63]. Acs-cAMP binding was studied by ITC. However, no-binding was
observed under the conditions tested, indicating a low binding affinity which is coherent
to the ki measured for cAMP, which is almost 10 times higher than the dissociation
constant for ATP. The presence of cAMP and ATP hindered CobB deacetylation. This
result was also observed for S. enterica Acs in the presence of cAMP and the authors
suggested that Acs-CobB binding could induce a different Acs conformation and the
presence of cAMP, which might hinder the formation of this conformation. Further
investigation is necessary to confirm this suggestion, which would suppose a new level
of regulation of Acs deacetylation by CobB depending on cAMP and ATP intracellular
concentrations.
In conclusion, this work studies for the first time the E. coli Acs ATP-binding
parameters and the role of four important conserved residues. Thus, the effect of K609
mutation on ATP-binding observed in this study opens a new research field for
understanding how acetylation induces enzyme inhibition.

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5. Acknowledgements
Dr. Michael Lammers (Institute of Biochemistry, Synthetic and Structural Biochemistry,
University of Greifswald, Greifswald, Germany) is acknowledged for pRSF-Duet-1-
acetyl-lysyl-tRNA-synthetase AcKRS3/MbtRNACUA plasmid transference. Dr. Alejandro
Torrecillas (CAID, University of Murcia) is acknowledged for technical support.
6. Funding
This work was supported by grants from the MICINN BIO2014-54411-C2-1-R, which
includes ERDF European cofounding, and the Seneca Foundation CARM (19236/PI/14).
7. CRediT author statement
Julia Gallego-Jara: conceptualization, methodology, investigation, writing-original draft.
Gema Lozano Terol: methodology, investigation. Ana Écija Conesa: methodology,
investigation. Barbara Zambelli: conceptualization, methodology, investigation, writing –
review and editing, supervision and visualization. Manuel Cánovas Díaz: writing – review
and editing, project administration, funding acquisition. Teresa de Diego Puente:
conceptualization, investigation, writing – review and editing, supervision, project
administration, funding acquisition.
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Highlights:
-
-
-
-
-
E. coli Acs enzyme binds ATP in an endothermic way.
Acs K609 mutant showed an increase in ATP affinity and a total loss of activity.
T264 and D500 mutant proteins showed a total loss of ATP-binding ability.
K609 acetylation induced a change in Acs conformation.
K609 acetylation resulted in an exothermic and more energetic ATP-binding.

Figure 1

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Figure 6