“Pinching” the ammonia tunnel of CTP synthase unveils coordinated catalytic and allosteric-dependent control of ammonia passage

Article abstract of DOI:10.1016/j.bbagen.2018.08.008

Molecular gates within enzymes often play important roles in synchronizing catalytic events. We explored the role of a gate in cytidine-5′-triphosphate synthase (CTPS) from Escherichia coli. This glutamine amidotransferase catalyzes the biosynthesis of CTP from UTP using either L-glutamine or exogenous NH₃ as a substrate. Glutamine is hydrolyzed in the glutaminase domain, with GTP acting as a positive allosteric effector, and the nascent NH₃ passes through a gate located at the end of a ~25-? tunnel before entering the synthase domain where CTP is generated. Substitution of the gate residue Val 60 by Ala, Cys, Asp, Trp, or Phe using site-directed mutagenesis and subsequent kinetic analyses revealed that V60-substitution impacts glutaminase activity, nucleotide binding, salt-dependent inhibition, and inter-domain NH₃ transport. Surprisingly, the increase in steric bulk present in V60F perturbed the local structure consistent with “pinching” the tunnel, thereby revealing processes that synchronize the transfer of NH₃ from the glutaminase domain to the synthase domain. V60F had a slightly reduced coupling efficiency at maximal glutaminase activity that was ameliorated by slowing down the glutamine hydrolysis reaction, consistent with a “bottleneck” effect. The inability of V60F to use exogenous NH₃ was overcome in the presence of GTP, and more so if CTPS was covalently modified by 6-diazo-5-oxo-L-norleucine. Use of NH₂OH by V60F as an alternative bulkier substrate occurred most efficiently when it was concomitant with the glutaminase reaction. Thus, the glutaminase activity and GTP-dependent activation act in concert to open the NH₃ gate of CTPS to mediate inter-domain NH₃ transport.

Full text of DOI:10.1016/j.bbagen.2018.08.008

BBA-GeneralSubjects1862(2018)2714–2727
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BBA - General Subjects
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“Pinching” the ammonia tunnel of CTP synthase unveils coordinated
catalytic and allosteric-dependent control of ammonia passage
⁎
Gregory D. McCluskeya, Stephen L. Bearnea,b,
a
Department of Biochemistry and Molecular Biology, Dalhousie University, Halifax, NS B3H 4R2, Canada
Department of Chemistry, Dalhousie University, Halifax, NS B3H 4R2, Canada
b
A R T I C L E I N F O
A B S T R A C T
Keywords:
CTP synthase
Mutagenesis
Molecular gate
Ammonia tunnel
Kinetics, catalytic synchronization
Molecular gates within enzymes often play important roles in synchronizing catalytic events. We explored the
role of a gate in cytidine-5′-triphosphate synthase (CTPS) from Escherichia coli. This glutamine amidotransferase
catalyzes the biosynthesis of CTP from UTP using either L-glutamine or exogenous NH3 as a substrate. Glutamine
is hydrolyzed in the glutaminase domain, with GTP acting as a positive allosteric effector, and the nascent NH3
passes through a gate located at the end of a ~25-Å tunnel before entering the synthase domain where CTP is
generated. Substitution of the gate residue Val 60 by Ala, Cys, Asp, Trp, or Phe using site-directed mutagenesis
and subsequent kinetic analyses revealed that V60-substitution impacts glutaminase activity, nucleotide binding,
salt-dependent inhibition, and inter-domain NH3 transport. Surprisingly, the increase in steric bulk present in
V60F perturbed the local structure consistent with “pinching” the tunnel, thereby revealing processes that
synchronize the transfer of NH3 from the glutaminase domain to the synthase domain. V60F had a slightly
reduced coupling efficiency at maximal glutaminase activity that was ameliorated by slowing down the gluta-
mine hydrolysis reaction, consistent with a “bottleneck” effect. The inability of V60F to use exogenous NH3 was
overcome in the presence of GTP, and more so if CTPS was covalently modified by 6-diazo-5-oxo-L-norleucine.
Use of NH2OH by V60F as an alternative bulkier substrate occurred most efficiently when it was concomitant
with the glutaminase reaction. Thus, the glutaminase activity and GTP-dependent activation act in concert to
open the NH3 gate of CTPS to mediate inter-domain NH3 transport.
1. Introduction
(Gln), contain tunnels to facilitate efficient transfer of the NH3 from its
site of production to its site of utilization [3–8]. By and large, the
Multi-domain enzymes often catalyze several different chemical
reactions, and the chemistry at each site must be coordinated to limit
the premature release of substrates and/or reaction intermediates.
Indeed, many proteins contain tunnels and gates that are often highly
dynamic structures that play an important role synchronizing catalytic
events occurring at different locations during enzyme catalysis [1, 2].
Engineering of such protein gates and tunnels offers an attractive ap-
proach for rationally modifying the activity of enzymes, but requires a
detailed understanding of how the opening and closing of such gates or
tunnels are governed by ligand binding and/or catalytic events. The
glutaminase active sites are structurally similar among the subfamily
members owing to the shared chemistry of the catalyzed step; however,
the domains that catalyze the “downstream” amination (synthase) ac-
tivity are unique to specific enzymes [9, 10]. As such, these enzymes
furnish excellent models for understanding the evolution [11] and
mechanism [12–17] of synchronization between multiple, and often
distant, active sites.
Cytidine-5′-triphosphate (CTP) synthase (CTPS, EC 6.3.4.2) affords
a particularly interesting example of a multi-domain, gated-amido-
transferase, which must coordinate the hydrolysis of Gln in the C-
terminal glutaminase (or GATase) domain [18] with the delivery of the
resulting nascent NH3 to the N-terminal synthase (or amidoligase) do-
main where it reacts with 4-phospho-UTP [19–21] to form CTP [22]
class
I (or triad) subfamily of the glutamine-dependent amido-
transferases, which catalyze the amination of ATP-activated substrates
using the nascent NH3 generated from the hydrolysis of L-glutamine
Abbreviations: CD, circular dichroism; CPS, carbamoyl phosphate synthase; CTPS, CTP synthase; Gln, L-glutamine; dH, mean hydrodynamic diameter; DON, 6-diazo-
5-oxo-L-norleucine; DLS, dynamic light scattering; EcCTPS, CTPS from Escherichia coli; GlmS, glucosamine-6-phosphate synthase; MD, molecular dynamics; NadE,
glutamine-dependent NAD+ synthetase; NTP, nucleoside-5′-triphosphate; N4-OH-CTP, N4-hydroxy-CTP; SD, standard deviation
⁎
Corresponding author at: Department of Biochemistry and Molecular Biology, Dalhousie University, Halifax, NS B3H 4R2, Canada.
E-mail address: sbearne@dal.ca (S.L. Bearne).
https://doi.org/10.1016/j.bbagen.2018.08.008
Received 11 June 2018; Received in revised form 20 July 2018; Accepted 6 August 2018
Availableonline10August2018
0304-4165/©2018ElsevierB.V.Allrightsreserved.

G.D. McCluskey, S.L. Bearne
BBA-GeneralSubjects1862(2018)2714–2727
(Fig. 1A). All these events must be properly synchronized to ensure that
CTP is produced efficiently. As an added layer of complexity, CTPS is
highly regulated by its nucleotide ligands. The substrates ATP and UTP
promote oligomerization of the enzyme to active tetramers [22–24].
The product CTP can also effect this change in oligomerization state
[25], as well as act as a feedback inhibitor [22, 26]. Because CTPS is the
only known enzyme that catalyzes the de novo formation of CTP from
UTP, and because CTP plays an important role in both membrane
phospholipid [27–29] and nucleic acid biosynthesis [30], CTPS has
been studied as a potential antiviral [31], antiprotozoal [32–38], an-
tineoplastic, [30, 39] and immunosuppressive [40] drug target. Eu-
karyotic CTPS homologues have been shown to be regulated by phos-
phorylation [41–44] and, more recently, the enzyme has also been
shown to form filaments both in vitro [45, 46] and in vivo [47], which
may constitute an additional level of regulation [48–52]. Finally, gua-
nosine-5′-triphosphate (GTP) is an allosteric activator of the glutami-
nase reaction [53] that induces a conformational change [54–57] sta-
bilizing the tetrahedral intermediates formed during Gln hydrolysis
[58] and may act as an activator in concert with the 4-phospho-UTP
intermediate [21].
The X-ray crystal structures of CTPSs from Escherichia coli (EcCTPS)
[26, 59, 60] and Mycobacterium tuberculosis [61] revealed the presence
of a 25-Å tunnel connecting the glutaminase and synthase domains that
NH3 must pass through during catalysis (Fig. 1). Although no structures
of CTPS have been solved with bound GTP, or guanosine analogues,
modeling studies suggest that GTP binds in the glutaminase domain at
an opening leading to a solvent-filled “vestibule” that constitutes part of
the NH3 tunnel (Fig. 1C) [59]. CTPS can also utilize free (i.e., exogenous)
NH3 as a substrate, though the entry site for exogenous NH3 has not
been conclusively identified. That NH3-dependent CTP generation is
inhibited by GTP, however, suggests they may share a common binding
site [62]. The NH3 tunnel, starting in the glutaminase domain, is
partially formed by the residues surrounding the catalytic triad (Glu
517–His 515–Cys 379) that catalyzes the hydrolysis reaction [58, 59,
63]. The tunnel continues to a constriction formed by 3 residues – Pro
54, His 57, and Val 60 – at the opening to the synthase domain that may
comprise an NH3 gate. These putative NH3 gate residues are highly
conserved among CTPSs (Fig. 2), and form the boundary between the
glutaminase and synthase domains, suggesting that the gate plays a
vital role in synchronizing the delivery of NH3 to the preformed 4-
phospho-UTP intermediate. His 57 is believed to function as a “door”
that swings open or shut over the constriction, depending on whether or
not UTP is bound [59]. According to high-resolution crystal structures
of wild-type EcCTPS and molecular models of the tunnel, the constric-
tion is only ~2.4 Å in diameter [26, 59], despite the molecular diameter
of NH3 being closer to 4 Å [64]. Taking this into account, the tunnel
captured in X-ray diffraction studies must undergo a conformational
change to allow for the effective transfer of NH3 to the synthase do-
main, or the constriction is an artefact of the crystallization conditions.
The proximity of the gate to the putative GTP binding site suggests a
possible role for GTP in regulating opening of the NH3 gate, thus cou-
pling spatially distant reactions by modulating the interdomain flow of
NH3.
Using site-directed mutagenesis, we replaced Val 60 of EcCTPS by
five amino acids (Ala, Cys, Asp, Trp, and Phe) at the most constricted
part of the tunnel. We then employed a combination of kinetics and
biophysical analyses to investigate the role of Val 60 in interdomain
NH3 transport. While the substitutions revealed the exquisite sensitivity
of the enzyme to changes at the interface between its glutaminase and
synthase domains, the Phe substitution unveiled the coordinated role of
GTP binding and the glutaminase activity in facilitating passage of NH3
through the tunnel gate, thereby suggesting an additional role for GTP
as an allosteric effector.
Fig. 1. Catalytic mechanism and mutation strategy for ex-
ploring inter-domain NH3 transport in EcCTPS. (A) The re-
action catalyzed by CTPS involves the transfer of the nascent
NH3, generated through GTP-activated hydrolysis of L-gluta-
mine (Gln) in the glutaminase domain, to the synthase do-
main where it reacts with UTP that has been activated
through ATP-dependent phosphorylation at the 4-position to
form CTP. The structural mimic of the glutamyl-enzyme in-
termediate formed following treatment of EcCTPS with 6-
diazo-5-oxo-L-norleucine (DON) is shown above the inter-
mediate. Amino acid residues of the wild-type (red) and V60F
(cyan) EcCTPS variants comprising the putative NH3 gate are
shown as sticks alongside a tunnel model (surface) to illus-
trate the constriction. (B) The NH3 tunnel (dark mesh) of an
EcCTPS monomer connects the glutaminase (green) and syn-
thase (blue) domains. Amino acids making up the surface of
EcCTPS are depicted as surface representations with the
tunnel modeled in mesh. The tunnel was modeled into the
wild-type EcCTPS structure (PDB 2AD5, [26]) using CAVER
Analyst 1.0 [75] for PyMOL with a probe radius of 1.2 Å. (C)
The NH3 tunnel passes through a molecular gate comprised of
Pro 54, His 57, and Val 60. The catalytic cysteine and amino
acids making up the NH3 gate (space-filling representations)
are shown. The putative GTP-binding site is indicated by the
dashed red circle and resides adjacent to the gate.
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G.D. McCluskey, S.L. Bearne
BBA-GeneralSubjects1862(2018)2714–2727
Fig. 2. Amino acid sequence neighboring the NH3 gate re-
sidues. (A) Partial HMM logo for 5324 predicted CTP synthase
protein sequences. Val 60 (E. coli numbering) is highlighted in
green. Skylign [91] was employed to construct this hidden
Markov model (HMM) logo from an alignment of 5324 pre-
dicted CTP synthase protein sequences obtained from the
Pfam database [92]. The relative amino acid probabilities for
positions 57 and 60 (E. coli numbering) are provided in Sup-
plementary Tables S3 and S4, respectively. (B) Amino acid
sequence alignment of a portion of 9 representative CTPSs
containing a putative NH3 tunnel. Invariant residues (*) and
residues showing conservation between groups of strongly (:)
or weakly (.) similar properties are indicated. Val 60 (green,
E. coli numbering), Pro 54 (blue), and His 57 (blue) are
highlighted. In descending order the proteins included in the
alignment are as follows: Escherichia coli (taxid:562), Trypa-
nosoma brucei (taxid:5691), Lactococcus lactis (taxid:1358),
Thermus thermophilus (taxid:274), Homo sapiens (taxid:9606),
Sulfolobus solfataricus (taxid:2287), Saccharomyces cerevisiae
(taxid:4932), Schizosaccharomyces pombe (taxid:4896), and
Mycobacterium tuberculosis (taxid:83332). Alignment ren-
dering was conducted using Clustal Omega [93].
2. Materials and methods
enzyme [58]. The concentration of each His6-tagged EcCTPS variant
was determined from its absorbance at 280 nm using a molar extinction
coefficient (ε) of 40,340 M−1 cm−1 for all EcCTPSs except for V60W
EcCTPS for which ε = 45,840 M−1 cm−1. Molar extinction coefficients
were calculated using the ProtParam tool available on the ExPASy
server (http://web.expasy.org/protparam) [65].
2.1. General
All chemicals, unless stated otherwise, were purchased from Sigma-
Aldrich Canada Ltd. (Oakville, ON, Canada). The pET-15b expression
system (Novagen) and HisBind resin (Novagen) were purchased from
EMD Millipore (Etobicoke, ON, Canada). Synthetic DNA oligonucleo-
tides for site-directed mutagenesis were purchased from Integrated
DNA Technologies (Coralville, IA). Plasmid preparations for site-di-
rected mutagenesis and bacterial transformations were conducted using
QIAprep Spin Mini-prep Kits (Qiagen, Toronto, ON, Canada). For HPLC
experiments, a Waters 510 pump and 680 controller were used for
solvent delivery, and injections were carried out with a Rheodyne 7725i
sample injector fitted with a 20-μL injection loop. Analytes were de-
tected with a Waters 474 scanning fluorescence detector or a Waters
486 absorbance detector, as indicated. Circular dichroism studies were
carried out using a JASCO J-810 spectropolarimeter (Jasco Inc., Easton,
MD).
2.3. Assay of CTP formation
The rate of EcCTPS-catalyzed conversion of UTP to CTP was de-
termined at 37 °C by following the change in absorbance at 291 nm
(Δε = 1338 M−1 cm−1) [22]. The reaction mixture contained EcCTPS
in assay buffer (70 mM Na+-HEPES, pH 8.0, containing 10 mM MgCl2
and 0.5 mM EGTA) and what was determined to be the saturating
concentrations of ligands (Supplementary Table S2), unless stated
otherwise. EcCTPS (10–30 μg/mL for wild-type, V60A, V60C, V60D,
V60W, and DON-V60F; 120–400 μg/mL for V60F) and nucleotides
(Supplementary Table S2) were pre-incubated at 37 °C followed by the
addition of the NH3 source (NH4Cl, NH4OAc, or Gln) to initiate the
reaction. For reactions using NH4Cl or NH4OAc (5.0–150 mM), KCl or
2.2. Site-directed mutagenesis and enzyme purification
NaOAc were used, respectively, to maintain the ionic strength at
+
0.15 M. The [NH3] present at pH 8.0 was calculated using a pKa (NH4
)
The pET-15b-CTPS1 plasmid [58], containing the CTPS open
reading frame from Escherichia coli, was used as the template for site-
directed mutagenesis. Site-directed mutagenesis was conducted using
the QuickChange Site-Directed Mutagenesis Kit (Stratagene Inc., La
Jolla, CA) with KAPA HiFi DNA polymerase (Kapa Biosystems, Wil-
mington, MA). The synthetic oligodeoxynucleotide primers are given in
Supplementary Table S1. The entire plasmid open reading frame was
commercially sequenced (Robarts Research, London, ON, Canada) to
verify that no other mutations in the nucleotide sequence were in-
troduced.
value of 9.24 (i.e., [NH3] = 0.0575·[NH4Cl]total) [54]. For reactions
using Gln (0.05–6.0 mM for wild-type, V60A, V60C, and V60D;
2–50 mM for V60F), GTP was present at saturating concentrations,
unless stated otherwise (Supplementary Table S2). The concentrations
of the EcCTPS variants utilized in the assays were chosen so that reli-
able initial velocities could be measured. The values of Km, kcat, and
kcat/Km were determined by fitting of Eq. (1) to the initial velocity data.
The dependence of CTP formation on GTP (0–2.0 mM) was measured
with either Gln (6.0 mM for wild-type, V60A, V60C, and V60D;
50.0 mM for V60F) or NH4OAc (150 mM for DON-V60F). The values of
kact, KA, and ko were determined by fitting of Eq. (2) to the initial ve-
locity data. The dependence of CTP formation on UTP (0–2.0 mM for
wild-type; 0–4.0 mM for V60A, V60C, V60D, V60F, and DON-V60F;
0–5.0 mM for V60W) and ATP (0–4.0 mM) was examined with either
Gln (6.0 mM for wild-type, V60A, V60C, and V60D; 50 mM for V60F) or
NH4OAc (150 mM for V60W and DON-V60F). All kinetic experiments
with DON-V60F were conducted in the presence of a saturating con-
centration of GTP (1.0 mM), unless stated otherwise. Eq. (3) was fit to
the initial velocity data using non-linear regression analysis to estimate
the values of Vmax/[E]T, [S]0.5, and n when ATP and UTP were ex-
amined as the variable substrates. Inhibition of Gln-dependent CTP
formation by salts was determined by incubating the indicated EcCTPS
Wild-type and mutant forms of recombinant EcCTPS were purified
from E. coli BL21(DE3) cells transformed with either the pET-15b-
CTPS1, pET-15b-CTPSH57A, pET-15b-CTPSV60A, pET-15b-CTPSV60C,
pET-15b-CTPSV60D, pET-15b-CTPSV60W, or pET-15b-CTPSV60F
plasmids as described previously [58]. Soluble EcCTPS variants bearing
an N-terminal His6-tag were purified by metal ion affinity chromato-
graphy using established protocols [58] and dialyzed against Na+
-
HEPES buffer (70 mM, pH 8.0) containing MgCl2 (10 mM) and EGTA
(0.5 mM) (i.e., assay buffer). Recombinant enzyme preparations
were > 98% pure as determined by analysis using SDS-PAGE (8%) (see
Supplementary Fig. S1). The His6-tag was not removed from the re-
combinant enzymes since the tag does not affect the activity of the
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G.D. McCluskey, S.L. Bearne
BBA-GeneralSubjects1862(2018)2714–2727
variants with increasing concentrations of NaCl, KCl, or NaOAc
(0–150 mM). Wild-type, V60A, V60C, V60D, and V60F EcCTPSs were
pre-incubated with saturating concentrations of GTP, ATP, and UTP
(Supplementary Table S2) and the indicated concentration of salt. Re-
actions were initiated by addition of a saturating concentration of Gln
(Supplementary Table S2) and Eq. (4) was fit to the relative velocity (vi/
vo) data to estimate the IC50 and n values for the inhibition of the en-
zyme by salt.
N4-OH-CTP formation was calculated using ΔA300/Δt, while the initial
rate of CTP formation was calculated using Eq. (6) [69], where l is the
pathlength.
Δ[CTP]
ΔA291 − 1.73ΔA300
vi =
=
Δt
ΔtΔε291l
(6)
To determine whether Gln and/or GTP could enhance utilization of
exogenous NH2OH, wild-type, V60F, and DON-V60F EcCTPSs were
assayed as described above with saturating concentrations of the in-
dicated nucleotides (Supplementary Table S2). For wild-type EcCTPS
(40 μg/mL), the reaction was initiated by the addition of NH2OH•HOAc
(100 mM) alone, or simultaneously with Gln (6.0 mM). Similarly, re-
actions catalyzed by V60F and DON-V60F EcCTPSs (110–125 μg/mL)
were initiated by the addition of NH2OH•HOAc (25 mM) alone, or si-
multaneously with Gln (50 mM). The ionic strength was maintained at
0.10 M using NaOAc for all variants. For wild-type EcCTPS, Eqs. (7) and
(8) were fit to the initial velocity data for CTP and N4-OH-CTP forma-
tion, respectively, which were based on the kinetic mechanism shown
in Supplementary Scheme S1. Due to substrate inhibition of V60F cat-
alysis, Eq. (9) was fit to the initial velocity data for N4-OH-CTP for-
mation. Data are the average of three independent experiments SD.
kcat [E]T [S]
vi =
Km + [S]
(1)
kact [E]T [GTP]
vi = ko
+
KA + [GTP]
(2)
(3)
(4)
Vmax [S]n
vi =
[S]n0.5 + [S]n
IC5n0
vi
vo
=
IC5n0 + [I]n
2.4. Coupling ratio determinations
Gln
(k KSNH2OH
)
d [CTP]/dt
cat
Glutaminase activities of the wild-type and Val 60 EcCTPS variants
were assayed by measuring the levels of Gln and glutamate derivatized
by o-phthaldialdehyde in the presence of β-mercaptoethanol using RP-
HPLC as described previously [57, 66]. Assays were conducted in assay
buffer containing saturating concentrations of the appropriate ligands
(Gln, GTP, ATP, and UTP) (Supplementary Table S2), and enzyme
(3.0–5.0 μg/mL for wild-type, V60A, V60C, V60D, and V60W;
20–30 μg/mL for V60F) in a total volume of 1.0 mL. To investigate the
integrity of the NH3 tunnel, the coupling efficiency between the glu-
taminase and synthase active sites was calculated using Eq. (5) [57]. To
investigate the “bottleneck” effect (vide infra) of V60F, Gln hydrolysis
and CTP formation were determined using RP-HPLC and UV/vis spec-
troscopy, respectively, as described above except with saturating, near
Km, and sub-saturating concentrations of Gln. The coupling ratio was
also determined at saturating concentrations of Gln, but with the con-
centration of GTP reduced to 0.006 mM for wild-type EcCTPS and
0.08 mM for V60F (i.e., [GTP] ≈ KA/5). For these glutaminase assays,
the concentrations of wild-type EcCTPS were 3.5 μg/mL (at saturating
[Gln]), 6.9 μg/mL (at near Km of Gln), 4.0 μg/mL (at sub-saturating
[Gln]), and 3.9 μg/mL (at sub-saturating [GTP]); and the concentra-
tions of V60F EcCTPS were 10.0 μg/mL (at saturating [Gln]), 26.0 μg/
mL (at near Km of Gln), 23.7 μg/mL (at sub-saturating [Gln]), and
19.5 μg/mL (at sub-saturating [GTP]).
=
KSNH2OH + [NH2OH]
[E]T
(7)
(8)
NH2OH
d [N4-OH-CTP]/dt
k
[NH2OH]
cat
=
=
KSNH2OH + [NH2OH]
[E]T
NH2OH
d [N4-OH-CTP]/dt
k
[NH2OH]
cat
2
[NH OH]
KSNH2OH + [NH2OH] +
2
[E]T
K
′
(9)
NH2OH
2.6. Derivatization of EcCTPS with 6-diazo-5-oxo-L-norleucine (DON)
To investigate whether acyl-enzyme formation could enhance the
utilization of exogenous NH3, the wild-type, V60A, and V60F EcCTPSs
(20 μM) were derivatized with DON (2.0 mM) in assay buffer containing
ATP, UTP, and GTP (all NTPs at 1.0 mM) following the protocol of
Koshland and co-workers [71, 72]. The reaction was incubated for 1 h
at 37 °C prior to dialysis for 12 h against fresh assay buffer lacking
NTPs. The resulting DON-modified variants were used immediately in
kinetic assays. The effects of GTP on the utilization of exogenous NH3
were investigated using unmodified wild-type EcCTPS, DON-modified
wild-type EcCTPS (DON-EcCTPS), DON-V60A, and DON-V60F
(10–30 μg/mL) in assay buffer containing saturating concentrations of
ligands (Supplementary Table S2) and NH4OAc (150 mM) as the ni-
trogen donor. Eq. (4) was used to fit the initial velocity data for the
inhibition of wild-type, DON-EcCTPS, and DON-V60A EcCTPSs. Acti-
vation of DON-V60F by GTP was analyzed using non-linear regression
analysis by fitting of Eq. (2) to the initial velocity data.
(vi/[E]T )CTP formation
coupling ratio =
(vi/[E]T )Glu formation
(5)
2.5. Assay of N4-OH-CTP formation
Competition between nascent and exogenous nitrogen sources was
investigated using Gln as the source of nascent NH3, and NH2OH•HOAc
as the exogenous nitrogen donor. NH2OH•HCl was converted to the
acetate salt [67] to avoid inhibition by chloride ions. Utilization of Gln
or NH2OH results in the conversion of UTP to either CTP or N4-hydroxy-
CTP (N4-OH-CTP), which can be measured individually by monitoring
the change in absorbance at 291 nm where, Δε291 = 1338 M−1 cm−1
[22] or Δε291 = 4023 M−1 cm−1 [68], respectively, or measured si-
multaneously by monitoring the change in absorbance at 291 nm and
300 nm where Δε300 for conversion of UTP to CTP is negligible and
Δε300 = 3936 M−1 cm−1 for the conversion of UTP to N4-OH-CTP [68,
69]. Unlike ammonium salts, no calculation is needed to account for the
total concentration of the nucleophilic form of NH2OH because it has a
pKa value well below the assay pH of 8.0 (i.e., pKa of +NH3OH = 6.03
[70]). In the presence of both Gln and NH2OH•HOAc, the initial rate of
2.7. Dynamic light scattering (DLS)
The oligomerization states of all the EcCTPS variants were analyzed
by DLS as previously described [52] using a BI-200SM goniometer and
laser scattering system fitted with a Brookhaven Mini-L30 diode laser
(637 nm, 30 mW; Brookhaven Instruments, Holtsville, NY). In brief,
enzyme (30 μg/mL) was mixed with saturating concentrations of UTP
and ATP (Supplementary Table S2) and filtered into a quartz cuvette.
The filtered sample was equilibrated at 37 °C for 10 min prior to re-
cording measurements at an angle of 60° for a total duration of 2 min at
37 °C. Intensity-weighted distributions of the hydrodynamic diameter
(dH) were acquired by fitting of a non-negative least squares (NNLS)
algorithm to the autocorrelation functions using Brookhaven Instru-
ments DLS software v. 5.89.
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G.D. McCluskey, S.L. Bearne
BBA-GeneralSubjects1862(2018)2714–2727
2.8. Tunnel modeling and molecular dynamics (MD) simulations
were excised, processed, and analyzed using LC-ESI-MS/MS as de-
scribed previously [76].
MD simulations were conducted on wild-type and V60F EcCTPSs in
order to gain structural insight into how the mutation might impact the
overall protein structure, and how the tunnel might be constricted.
Homology models for wild-type and V60F EcCTPSs were built using
SWISS-MODEL [73] to remove all ligands and restore missing residues
that were not observed in the X-ray diffraction analysis of the original
structure using a wild-type EcCTPS structure as a template (PDB: 2AD5)
[26]. The resulting 3D models were energy minimized prior to simu-
lation using the method of steepest descent (50,000 steps, 100 ps) and a
force constant of 1000 kJ mol−1 nm−2. Each model was solvated using
an explicit 3-point water model (SPC/E) in a cubic solute-box (143 ×
143 × 143 Å). Simulations were conducted using GROMACS Version
5.0.4 with the GROMOS96 54a7 force field [74]. Temperature and
pressure were maintained at 300 K and 1 atm, respectively. Bond
lengths were constrained using the LINCS algorithm and all atoms of
the system were subject to the dynamics simulation.
Initially, the V60F EcCTPS model was simulated once for 10 ns with
trajectory coordinates saved at 20-ps intervals. Wild-type and V60F
EcCTPS models were subsequently subjected to 100-ns simulations with
no alterations to the other simulation parameters. Three-dimensional
models of the NH3 tunnel and statistical data were generated for the
initial structures derived from the simulations, since the constriction
present in V60F EcCTPS became too great to model later in the simu-
lation. CAVER Analyst 1.0 [75] was used to generate a 3D tunnel that
passes through the NH3 gate using the starting coordinates 107 × 95 ×
52 Å, and the initial search area was set to 2 Å with a probe size of
0.6 Å. All other CAVER settings were kept as the defaults. For the wild-
type EcCTPS structure (PDB: 2AD5), the same coordinates were used,
but with a probe size of 1.2 Å.
3. Results
To explore the role of the putative NH3 gate in coordinating the
reactions required for CTPS catalysis, we conducted site-directed mu-
tagenesis to alter His 57 and Val 60. Substitution of the conserved His
57 “door” residue with alanine (H57A) yielded an EcCTPS variant that
retained the ability to hydrolyze Gln only 3-fold slower than wild-type
EcCTPS, but was unable to catalyze CTP formation (Supplementary Fig.
S2). The fact that His is fully conserved at this position in CTPS
homologues (Supplementary Table S3) suggests that this residue plays a
critical role in catalysis. Because substitution of His 57 by Ala had such
a detrimental effect on EcCTPS-catalyzed CTP formation, we focused
our attention on the gate residue Val 60, located at the most constricted
part of the NH3 tunnel. Examination of the sequence logo (Fig. 2) re-
vealed that residues bearing short alkyl (Pro, Ile, and Ala) or neutral
polar (Thr and Cys) side chains constitute the majority of (predicted)
natural variants at position 60 (E. coli numbering), while larger, aro-
matic, and acidic or basic substitutions are not as abundant in the
alignment (Supplementary Table S4). Consequently, we substituted Val
60 with a Phe residue with the anticipation that the increased steric
bulk would block or impede the passage of NH3 (Fig. 1A). Additionally,
we substituted Val 60 with Ala, Cys, Asp, and Trp to examine the effects
of some other side chains on catalysis. Though more “natural” sub-
stitutions (V60A and V60C) were well-tolerated, the V60D, V60W, and
V60F substitutions were more disruptive to catalysis. With the excep-
tion of V60D, the Val 60-substituted EcCTPS variants shared similar
secondary structures as determined by circular dichroism spectroscopy
(Supplementary Fig. S3).
2.9. Circular dichroism (CD)
3.1. NH3-dependent CTP formation
CD spectra for the EcCTPS variants (0.2 mg/mL) in Tris-SO4 buffer
(2 mM, pH 8.0) containing MgSO4 (10 mM) were obtained in triplicate
over a wavelength range of 190–260 nm using a 0.1-cm light path at
37 °C. For each EcCTPS variant, the average ellipticity of a buffer blank
was subtracted from the observed average of ellipticity values obtained
for the proteins.
Current high-resolution EcCTPS structures reveal that the NH3
tunnel contains a ~2.4-Å constriction that is primarily formed by the
Pro-His-Val gate [26, 59]. These structures, therefore, do not depict
active forms of the enzyme and conformational changes are required to
open the NH3 tunnel to permit passage of NH3 (diameter ≈ 4 Å) [64].
To determine whether the ability of the EcCTPS variants to utilize
exogenous NH3 as a substrate was affected by the amino acid sub-
stitutions, the kinetic parameters for NH3-dependent CTP formation
were determined (Table 1, Supplementary Figs. S4-S10). Conservative
mutations (V60A and V60C) had minimal effect on the utilization of
NH3 derived from either NH4Cl or NH4OAc. The efficiency (kcat/Km) of
2.10. Mass spectrometry (MS)
Both the V60F and DON-V60F EcCTPS variants were analyzed using
mass spectrometry. Gel bands from SDS-PAGE containing each variant
Table 1
Kinetic parameters for exogenous and nascent NH3-dependent catalysis of wild-type and Val 60-substituted CTPS variantsa,b
.
Nitrogen source
Kinetic parameterc
CTPS variant
Wild-type
V60A
V60C
V60D
V60W
V60F
DON-V60F
e
NH4Cl-dependent CTP formation
NH4OAc-dependent CTP formation
Gln-dependent CTP formation
Km (mM)
2.15
9.50
4.43
1.4
7.7
5.3
0.24
6.0
24.7
0.14d
0.53d
0.12d
0.7
3.3
5.1
0.61
3.12
5.1
0.37
5.2
0.1
0.2
1.2
0.04
0.08
0.2
0.01
0.2
0.9
3.3
5.7
1.8
4.2
7.5
1.8
0.24
5.14
21.6
0.7
0.8
0.1
0.2
0.2
0.1
0.04
0.03
4.1
3.6
0.99
0.27
1.6
1.1
0.7
0.5
1.6
2.9
0.4
0.07
0.01
0.5
0.1
0.2
0.1
0.2
0.5
4.1
2.2
0.31
0.05
0.1
0.1
0.1
–
–
–
1.6
1.2
0.73
3.1
2.2
0.3f
0.1f
e
kcat (s−1
)
0.95
0.25
1.5
e
kcat/Km (mM−1 s−1
Km (mM)
)
)
)
0.08f
0.1
0.94
0.16
0.17
5.9
0.82
0.14
0.08f
0.01f
0.01f
0.2
0.06
0.01
0.1f
0.2f
kcat (s−1
)
0.5
0.1
1.6
kcat/Km (mM−1 s−1
Km (mM)
1.1
0.72
0.05f
0.02d
–
–
e
e
kcat (s−1
)
0.4d
0.7d
–
–
e
e
kcat/Km (mM−1 s−1
14.2
–
–
e
e
a
See Supplementary Figs. S4–S10 for corresponding initial rate plots.
b
c
d
e
f
See Supplementary Table S2 for the saturating concentrations of unvaried ligands.
Values are the averages of three independent experiments SD.
Data from reference [90].
Activity too low to be determined reliably.
Contained 1.0 mM GTP.
2718

G.D. McCluskey, S.L. Bearne
BBA-GeneralSubjects1862(2018)2714–2727
Table 2
Kinetic parameters for NTP-dependent catalysis of wild-type and Val 60-substituted EcCTPS variantsa,b
.
Activator/substrate
Kinetic parameterc
EcCTPS variant
Wild-type
V60A
V60C
V60D
V60Wg
V60F
DON-V60Fg
f
GTP
UTP
ATP
KA (mM)
0.03
7.1
0.5
0.11
4.2
1.8
0.18
5.6
1.5
0.01d
0.3d
0.1
0.15
5.9
0.03
0.2
0.13
4.9
0.3
0.15
5.10
1.4
0.19
5.1
0.01
0.2
0.09
0.01
0.03
0.1
0.03
0.1
0.1
0.78
1.31
0.15
0.32
1.7
1.42
0.29
1.5
0.09
0.07
0.02
0.05
0.2
0.06
0.02
–
0.42
1.0
–
0.74
1.05
1.65
0.31
0.99
2.0
0.04
0.1
0.31
1.76
0.25
1.00
1.86
1.75
0.47
1.68
2.28
0.02
f
kact (s−1
)
–
0.03
0.05
0.06
0.09
0.06
0.01
0.06
0.14
f
f
ko (s−1
)
0.17
0.25
5.69
1.37
0.22
5.6
0.03
0.02
0.11
0.08
0.01
–
[S]0.5
Vmax/[E]T
n
[S]0.5
Vmax/[E]T
n
0.01d
1.2
1.4
2.16
0.96
1.46
2.27
0.1
0.1
0.15
0.12
0.07
0.08
0.08
0.12
0.08
0.06
0.06
0.2d
0.1d
0.04e
e
0.2
0.3
0.11
0.1
0.20
0.2e
1.29
1.3
1.54
0.2
a
See Supplementary Figs. S4–S10 for corresponding initial rate plots.
b
c
d
e
f
See Supplementary Table S2 for the saturating concentrations of unvaried ligands.
Values are the averages of three independent experiments SD.
Data from reference [90].
Data from reference [52].
Activity too low to be determined reliably.
g
NTP-dependent kinetics for V60W and DON-V60F were measured with 150 mM NH4OAc, while Gln was used as the NH3 source for wild-type and all other
EcCTPS variants.
V60A (kcat/Km = 5.1 mM−1 s−1) was slightly greater than that of wild-
type EcCTPS (kcat/Km = 4.43 mM−1 s−1), owing to a higher affinity
(decreased Km) for exogenous NH3, while V60C was slightly less effi-
low to be measured reliably (Table 1). Despite its inability to utilize
exogenous NH3 effectively as a substrate, V60F EcCTPS could utilize
Gln as a substrate, albeit its efficiency was reduced ~176-fold (kcat
/
cient (kcat/Km = 1.8 mM−1 s−1
)
due to a lower turnover number
Km = 0.14 mM−1 s−1) relative to wild-type EcCTPS. This marked re-
duction in catalytic efficiency arose from a 7-fold decrease in the
(kcat = 5.7 s−1). These observations suggest that small hydrophobic
residues are required for optimal tunnel and/or gate function. Con-
sistent with this hypothesis, V60D exhibited a much lower catalytic
efficiency with exogenous NH3 (kcat/Km = 0.27 mM−1 s−1 (NH4Cl) and
0.7 mM−1 s−1 (NH4OAc)) than wild-type EcCTPS and the EcCTPS var-
iants bearing the conservative substitutions, though the Km for NH4Cl-
derived NH3 (3.6 mM) was 2-fold higher than that for NH4OAc-derived
NH3 (1.6 mM), suggesting that the presence of Cl− impaired NH3
binding. Indeed, NaCl and KCl were more potent inhibitors of the Val
60-substituted EcCTPSs, but acetate had much less impact than the
chloride salts (Supplemental Fig. S11). V60W EcCTPS had a Km value
with NH4OAc-derived NH3 (1.5 mM) similar to that of wild-type
EcCTPS, but the turnover number was approximately 5-fold lower
(kcat = 1.6 s−1) (Table 1). Most interestingly, we found that V60F
EcCTPS was unable to use exogenous NH3 as a substrate from either
NH4Cl or NH4OAc. Only if GTP, which is normally an inhibitor of NH3-
dependent CTP formation [62], was added to the reaction mixture
could CTP formation be detected. Even in the presence of GTP, the
activity of V60F was only detectable with NH4OAc due to the lack of
concomitant inhibition by Cl−, and the turnover (kcat) and catalytic
turnover number (kcat = 0.82 s−1
) and a 25-fold increase in Km
(5.9 mM), relative to wild-type EcCTPS (Table 1).
Because Gln-dependent CTP formation requires the presence of the
allosteric effector GTP for full activity, the GTP-dependent activation
parameters were measured for each EcCTPS variant (Table 2). Every
substitution for Val 60 caused a reduction in the GTP-binding affinity
(1/KA). V60A (KA = 0.15 mM) and V60C (KA = 0.13 mM) EcCTPSs had
5- and 4-fold reduced affinities for GTP, respectively, relative to wild-
type EcCTPS (KA = 0.03 mM), though the activation rate constant (kact
)
was similar to that of wild-type EcCTPS for both variants (Table 2).
Notably, the GTP-binding affinity for V60D (KA = 0.78 mM) and V60F
(KA = 0.42 mM) EcCTPSs were 24- and 13-fold weaker, respectively,
than the GTP-binding affinity of wild-type EcCTPS. Moreover, the ac-
tivation rate constants were reduced 5- and 7-fold for V60D and V60F,
respectively, relative to wild-type EcCTPS, indicating that the acidic
and bulky aromatic substitutions were detrimental to GTP-dependent
activation of Gln-dependent CTP formation. The GTP-dependent ki-
netics could not be measured reliably for V60W EcCTPS because of the
inability of this variant to utilize Gln as a substrate. Clearly, amino acid
substitutions at position 60 all cause local structural perturbations that
have a detrimental effect on the ability of GTP to bind and activate the
enzyme. This effect is in accord with notion that the putative GTP
binding site is located near Val 60 [54, 57, 59, 77].
efficiency were about 50- and 30-fold lower (kcat = 0.16 s−1; kcat
/
Km = 0.17 mM−1 s−1), respectively, than the values obtained for wild-
type EcCTPS (kcat = 7.7 s−1; kcat/Km = 5.3 mM−1 s−1) (Table 1).
3.2. Gln-dependent CTP formation
3.3. UTP- and ATP-dependent kinetics
As was observed for the effect of the substitutions on the kinetic
parameters for NH3-dependent CTP formation, the conservative sub-
stitutions (V60A and V60C) had little effect on catalysis when Gln was
the substrate, unlike V60D, V60W, and V60F. The V60A and V60C
variants had kcat/Km values of 14.2 and 21.6 mM−1 s−1, respectively,
similar to the wild-type efficiency of 24.7 mM−1 s−1 (Table 1, Supple-
mentary Figs. S4-S10). Neither the affinity of V60A and V60C EcCTPSs
for glutamine (Km = 0.37 mM and 0.24 mM, respectively) nor their
turnover values (kcat = 5.2 s−1 and 5.14 s−1, respectively) differed
significantly from those of wild-type EcCTPS (Km = 0.24 mM;
kcat = 6.0 s−1). On the other hand, V60D EcCTPS exhibited an 8-fold
decrease in catalytic efficiency (kcat/Km = 2.9 mM−1 s−1), and the rates
of Gln-dependent CTP formation catalyzed by V60W EcCTPS were too
UTP and ATP are not only required as substrates, but also promote
oligomerization of the enzyme to form the active tetramers [24]. Val 60
is located near the dimer-dimer interface, and may also be close-enough
to the synthase domain such that the Val 60-substituted variants might
have impaired affinity for these NTP substrates. The UTP- and ATP-
dependent kinetics were measured using Gln as the NH3 source to ex-
plore this possibility, except for V60W EcCTPS where its inability to
utilize Gln as a substrate necessitated the use of NH4OAc instead. All
variants had very similar [S]0.5 values for ATP binding
([S]0.5 ≈ 0.2–0.3 mM), with the exception of V60W EcCTPS, which had
a 5-fold lower affinity relative to wild-type EcCTPS (Table 2, Supple-
mentary Figs. S4-S10). On the other hand, the UTP-binding site
2719

G.D. McCluskey, S.L. Bearne
BBA-GeneralSubjects1862(2018)2714–2727
Table 3
Coupling efficiencies for wild-type and Val 60-substituted EcCTPSs at saturating ligand concentrationsa,b
.
EcCTPS variant
Wild-type
V60A
V60C
V60D
V60W
V60F
c
e
CTP formation (vi/[E]T, s−1
)
6.0
5.99
1.00
0.4
0.23
0.08
4.74
3.8
1.15
0.24
0.3
0.17
5.14
5.2
0.99
0.03
0.7
0.13
1.39
1.27
1.09
0.16
0.14
0.15
–
0.85
1.14
0.75
0.12
0.09
0.12
c
Gln hydrolysis (vi/[E]T, s−1
coupling efficiencyd
)
0.08
N/A
0.02
a
See Supplementary Table S2 for saturating ligand concentrations.
See Supplementary Fig. S12 for representative kinetic plots.
Values are the averages of three independent experiments SD.
Coupling efficiencies were calculated using Eq. (5).
Activity too low to be detected.
b
c
d
e
appeared to be more sensitive to changes at residue position 60. Al-
though the variants with the conservative substitutions (V60A and
V60C) exhibited similar UTP-dependent turnover numbers compared to
wild-type EcCTPS (Vmax/[E]T ≈ 5–6 s−1), substitution of Val 60 with a
an acidic residue (V60D) or a large aromatic residue (V60W and V60F)
caused an approximately 4-fold reduction in the turnover numbers
(Table 2). Interestingly, only the substitutions with amino acids with
bulky side chains cause a marked reduction in the UTP-binding affinity
of 11- and 7-fold for V60W ([S]0.5 = 1.2 mM) and V60F
([S]0.5 = 0.74 mM) EcCTPSs, respectively, relative to wild-type EcCTPS
([S]0.5 = 0.11 mM). Overall, these data indicated that the less con-
servative substitutions for Val 60 had a greater impact on the synthase
active site(s) than the more “natural” V60A and V60C mutations.
the glutaminase activity for the V60W variant was very low (kcat ≈ vi/
[E]T = 0.08 s−1, Table 3). The relatively high rate of activity with
exogenous NH3, but poor catalysis of Gln hydrolysis, suggested that the
glutaminase reaction was somehow compromised by the substitution by
Trp.
V60F EcCTPS was the only Val 60 variant that exhibited a defect in
NH3 transport. At saturating concentrations of Gln, V60F EcCTPS pro-
duced nascent NH3 at a rate 1.3-fold faster than the rate at which CTP
was formed, resulting in a coupling efficiency of 75% (Table 3). This
reduced coupling efficiency could arise from either NH3 leaking away
from the tunnel or a “bottleneck” effect in which a rapid build-up of
NH3 occurs within the tunnel because of congestion at the now-con-
stricted gate. We slowed the glutaminase reaction down by reducing the
concentrations of either Gln or GTP to explore this possibility (Table 4,
Supplementary Fig. S12). At concentrations of Gln near its Km value,
wild-type and V60F EcCTPSs had coupling efficiencies of 98% and
105%, respectively, indicating that the overall rate of CTP generation
was not substantially different from the rate of NH3 formation in the
glutaminase domain. At sub-saturating concentrations of Gln, the V60F
substitution failed to block inter-domain coupling as well, since 100%
of the nascent NH3 was converted into CTP (Table 4). These data sug-
gested that the V60F substitution did not introduce a leak, nor did it
hinder the passage of nascent NH3, except slightly when the rate of NH3
formation was high. Moreover, this “bottleneck” effect was also re-
lieved by slowing the rate of formation of the nascent NH3 by reduction
of the concentration of GTP at saturating concentrations of Gln. These
observations indicate that the inability of the enzyme to efficiently
transfer the NH3, produced at high rates of Gln hydrolysis, from the
glutaminase domain to the synthase domain was indeed the cause of the
reduced NH3 transfer in the V60F variant (Table 4). Still, the rate of Gln
hydrolysis catalyzed by V60F EcCTPS at saturating concentrations of
Gln (vi/[E]T = 1.14 s−1) was lower than that of wild-type EcCTPS (vi/
[E]T = 5.99 s−1) by ~5-fold, indicating that the glutaminase reaction
was also impaired by the V60F substitution (Table 3). The possibility
that the V60F mutation introduces a pronounced alteration in the
3.4. Glutaminase activity and coupling efficiencies
While the passage of exogenous NH3 appeared to be blocked within
the V60F EcCTPS variant, nascent NH3 was able to traverse the NH3
tunnel to yield CTP, albeit at a much lower rate than wild-type EcCTPS.
The low rate of Gln-dependent CTP formation could have arisen from
the added steric bulk of the Phe residue at position 60 of V60F causing a
deformation in the tunnel leading to loss of nascent NH3 directly from
the tunnel (i.e., a leaky tunnel), or a more extensive effect on the glu-
taminase and/or synthase machinery. We determined the efficiency of
NH3 transport from the glutaminase domain to the synthase domain for
the wild-type and Val 60-substituted EcCTPSs by measuring the number
of CTP molecules formed per molecule of NH3 arising from Gln (i.e.,
coupling ratio, Eq. (5)). Like wild-type EcCTPS, the V60A, V60C, and
V60D variants all exhibited ~100% coupling efficiency (Table 3, Sup-
plementary Fig. S12), indicating that the NH3 tunnel was intact.
However, V60D EcCTPS exhibited reduced overall glutaminase activity
relative to wild-type EcCTPS, which accounted for its lower rate of CTP
formation when using Gln as a substrate (Tables 1 and 3). Similarly,
though we were unable to determine a coupling efficiency for V60W
EcCTPS since the Gln-dependent CTP formation was not measurable,
Table 4
Coupling efficiencies for wild-type and V60F EcCTPS at non-saturating concentrations of Gln or GTPa,b
.
Sub-saturating [Gln]c
Sub-saturating [GTP]d
Wild-type
c
EcCTPS variant
[Gln] ≈ Km
Wild-type
V60F
Wild-type
V60F
V60F
e
CTP formation (vi/[E]T, s−1
)
3.32
3.38
0.98
0.3
0.05
0.2
0.06
0.44
0.42
1.05
8.0
0.03
0.09
0.23
0.33
0.31
1.06
0.03
0.01
0.01
0.05
0.24
0.24
1.00
1.2
0.01
0.02
0.09
1.44
1.42
1.01
6.0
0.11
0.02
0.08
0.25
0.27
0.93
50.0
0.02
0.02
0.08
e
Gln hydrolysis (vi/[E]T, s−1
coupling efficiencyf
[Gln] (mM)
)
a
Cf. the saturating concentrations of Gln were 6.0 mM and 50.0 mM for wild-type and V60F, respectively (Supplementary Table S2).
See Supplementary Fig. S12 for representative kinetic plots.
[GTP] = 1.0 mM
[GTP] = 0.006 mM and 0.080 mM (i.e., [GTP] ≈ KA/5) for wild-type and V60F EcCTPSs, respectively.
Values are the averages of three independent experiments SD.
Coupling efficiencies were calculated using Eq. (5).
b
c
d
e
f
2720

G.D. McCluskey, S.L. Bearne
BBA-GeneralSubjects1862(2018)2714–2727
structure of the glutaminase domain could not be ruled out; though,
circular dichroism spectroscopy of V60F EcCTPS showed no appreciable
difference in its secondary structure relative to wild-type EcCTPS
(Supplementary Fig. S3).
NH3 was strongly inhibited by increasing concentrations of NH2OH, like
wild-type EcCTPS (Fig. 3B).
Having concluded that both nascent and exogenous amines likely
take the same route to the synthase domain, we incubated V60F EcCTPS
with NH2OH in the presence and absence of GTP and/or Gln to de-
termine whether GTP binding and/or the glutaminase activity enhances
the passage of exogenous NH2OH to the synthase domain. In the pre-
sence of a saturating concentration of Gln, the rate of N4-OH-CTP for-
mation was enhanced ~3-fold more than with NH2OH alone (Fig. 3C).
However, unlike when NH4OAc was the exogenous amine (Table 1),
GTP alone was not able to enhance the utilization of the bulkier
NH2OH. Wild-type EcCTPS did not exhibit any Gln- or GTP-dependent
increase in NH2OH utilization (Fig. 3C), consistent with the observation
that the enzyme can efficiently utilize either exogenous NH3 or NH2OH
as substrates and their passage to the synthase site is, therefore, nor-
mally unimpeded [57].
3.5. Utilization of exogenous NH2OH
Evidently, exogenous NH3 failed to be utilized as a substrate by V60F
EcCTPS in the absence of GTP, but nascent NH3 was, somehow, able to
overcome the effect of the V60F substitution. Because the passage of
nascent NH3 through the tunnel occurs in the presence of bound GTP
and Gln hydrolysis within the glutaminase domain, we assessed whe-
ther Gln and/or GTP could enhance exogenous NH3-dependent catalysis
using an approach similar to that described by Willemöes for studies on
the activation of CTPS from Lactococcus lactis [69]. In brief, NH2OH
may be used as an exogenous nitrogen source resulting in the formation
of N4-OH-CTP, which absorbs light at a higher wavelength than CTP
allowing for the simultaneous determination of CTP or N4-OH-CTP
produced from nascent or exogenous nitrogen sources, respectively.
However, we first had to establish that nascent (NH3) and exogenous
(NH2OH) amines follow the same route in the E. coli homologue in
order to draw a comparison between how the two sources are affected
by Gln. For wild-type EcCTPS, the rate of CTP formation arising from
nascent NH3 declined with a corresponding rise in N4-OH-CTP as the
concentration of NH2OH was increased (Fig. 3A), suggesting that the
two nucleophilic amines (NH3 and NH2OH) compete for reaction with
the 4-phospho-UTP intermediate. When the same experiment was
conducted using V60F EcCTPS, N4-OH-CTP formation was much slower,
as might be expected due to the increased bulk of NH2OH and the
presumably obstructed tunnel; however, CTP formation from nascent
3.6. Effects of acyl-enzyme formation
Although the utilization of exogenous NH2OH by V60F EcCTPS in-
creased upon addition of Gln, whether the enhanced rate of N4-OH-CTP
formation arose because of concomitant Gln binding or hydrolysis was
ambiguous. EcCTPS catalyzes the hydrolysis of Gln at a basal rate in the
absence of GTP, thus either binding or hydrolysis of Gln may have
enhanced NH2OH utilization. During Gln hydrolysis, the side chain
thiolate of Cys 379 acts as the nucleophile forming a glutamyl-enzyme
intermediate and releasing NH3. The thioester intermediate is subse-
quently hydrolyzed yielding Glu [58, 63]. To ascertain whether for-
mation of the glutamyl-enzyme intermediate (Fig. 1A) formed during
the hydrolysis of Gln enhanced the utilization of an exogenous nitrogen
Fig. 3. Competition between nascent NH3 and exogenous
NH2OH and the activating effects of Gln and GTP on the rates
of N4-OH-CTP formation catalyzed by wild-type, V60F, and
DON-V60F EcCTPSs. The rates of CTP (◯) and N4-OH-CTP
(△) formation for the wild-type (A) and V60F (B) EcCTPS
variants were measured in the presence of saturating con-
centrations of Gln (Supplementary Table S2) and varying
concentrations of NH2OH•HOAc (0–100 mM). The ionic
strength was maintained at 0.10 M with NaOAc. Curves for
CTP formation are fits of Eq. (7) to the initial velocity data,
and curves for the formation of N4-OH-CTP are fits of Eq. (8)
(wild-type) or Eq. (9) (V60F) to the initial velocity data (see
Supplementary Scheme S1). For CTP formation by wild-type
NH2OH
EcCTPS, the apparent values of (kcatGlnKSNH2OH) and KS
were 252
18 mM s−1 and 44
4 mM, respectively. For
N4-OH-CTP formation by V60F EcCTPS, (kcatGlnKSNH2OH) and
NH2OH
KS
were 4
1 mM s−1 and 8
3 mM, respectively.
For N4-OH-CTP formation by wild-type EcCTPS, the apparent
NH2OH
NH2OH
values of kcat
and KS
were 13.4
0.9 s−1 and
74
9 mM, respectively. For N4-OH-CTP formation by V60F
NH2OH
NH2OH
EcCTPS the apparent values of kcat
K′NH2OH were 5
,
KS
,
and
1 s−1, 54
19 mM, and 38
15 mM,
respectively. The activating effects of Gln and GTP on the
rates of NH2OH-dependent N4-OH-CTP formation by wild-
type (C, cyan), V60F (C, orange), or DON-V60F (D) EcCTPSs
were investigated by pre-incubating the enzymes with satur-
ating concentrations of ATP and UTP (Supplementary Table
S2) before initiation of the reaction with NH2OH (100 mM for
wild-type EcCTPS; 25 mM for V60F and DON-V60F) and,
where indicated, a saturating amount of Gln and/or GTP
(Supplementary Table S2). Data are expressed as the relative
change in vi/[E]T for N4-OH-CTP formation upon treatment
with the activating ligand with respect to the control sample
(i.e., Gln and GTP = 0 mM or ‘–’).
2721

G.D. McCluskey, S.L. Bearne
BBA-GeneralSubjects1862(2018)2714–2727
source by V60F EcCTPS, we covalently modified the active site Cys 379
of V60F with DON to mimic the structure of the glutamyl-enzyme in-
termediate (see Supplementary Figs. S13 and S14). Despite having an
inactivated glutaminase domain, the DON-modified wild-type enzyme
retains the ability to utilize exogenous NH3 as a substrate [71, 72]. The
rate constant for NH4OAc-dependent CTP formation by DON-V60F
EcCTPS in the absence of GTP (ko = 0.25 s−1, Table 2) was greater than
the corresponding rate constant for the NH4OAc-dependent CTP for-
mation (kcat = 0.16 s−1, Table 1) catalyzed by the unmodified V60F
variant in the presence of GTP. This observation indicated that the basal
activity was increased by the DON modification. DON-V60F EcCTPS,
however, was still activated by GTP and once a saturating concentra-
tion of GTP was obtained, DON-V60F EcCTPS was 14-fold more active
(KA = 0.42 mM) (Table 2). Together, these data indicated that the
greater rate enhancement observed for DON-V60F EcCTPS in the pre-
sence of GTP, relative to the unmodified V60F variant, was likely due to
a conformational change arising from alkylation of the enzyme rather
than an increase in affinity for GTP (although we cannot rule out the
possibility that the allosteric effects of GTP may differ between the
modified and unmodified V60F variants). Interestingly, the DON-
EcCTPS and DON-V60F variants exhibited similar activity with NH4OAc
at saturating concentrations of GTP (vi/[E]T = 2.66 s−1 and vi/
[E]T = 1.45 s−1, respectively) suggesting that they both have the same
levels of activity under these conditions. Assuming this was coin-
cidence, the GTP-dependent inhibition of the DON-V60A variant was
also examined in the presence of NH4OAc. While V60A EcCTPS nor-
mally has roughly 2-fold less activity than wild-type EcCTPS when
utilizing NH4OAc as an NH3 source, the DON-V60A variant still ex-
hibited similar activity to the other modified variants at a saturating
concentration of GTP (Fig. 4). These observations suggest that there is a
basal rate of NH3-dependent CTP formation by the DON-modified
variants where GTP-dependent inhibition is compensated for by the
GTP-dependent activation effect. That DON does not restore the affi-
nities of V60F EcCTPS for GTP, UTP, and ATP to wild-type levels, nor
ameliorate the inhibition by Cl−, suggests that the effect of DON
leading to enhanced rates of NH3-dependent CTP formation arises pri-
marily from its effect on the NH3 gate.
with exogenous NH3 (derived from NH4OAc) as
a substrate
(kcat = 2.2 s−1
,
Table 1) than the unmodified V60F variant
(kcat = 0.16 s−1, Table 1) under identical conditions (Table 1). Indeed,
NH2OH utilization by DON-V60F EcCTPS was also enhanced, but unlike
the unmodified V60F variant, only in the presence of GTP (Fig. 3D),
thereby suggesting that DON-V60F EcCTPS was not a perfect mimic of
the glutamyl-enzyme. Alkylation of V60F EcCTPS by DON was also
unable to completely “rescue” the ability of the variant to utilize NH3 as
a substrate since the values of kcat were also 8- and 3.5-fold lower than
wild-type EcCTPS using either NH4Cl or NH4OAc as substrates, re-
spectively. The difference in kcat values with either NH4Cl or NH4OAc as
substrates also suggests that modification by DON did not alleviate
inhibition by Cl− (Table 1).
3.7. Quaternary structure of the EcCTPS variants
The activation of NH3-dependent CTP formation catalyzed by V60F
and DON-V60F EcCTPSs was unexpectedly dependent on GTP, which is
normally an inhibitor of this activity [62]. We examined the GTP-de-
pendent inhibition of NH3-dependent CTP formation catalyzed by un-
modified and modified (DON-EcCTPS) wild-type EcCTPSs as a contrast
to the activating effects of GTP on the DON-V60F variant. As reported
previously [53], we found that GTP inhibited DON-EcCTPS
(IC50 = 0.08 mM) more effectively than the unmodified enzyme
(IC50 ≈ 3.6 mM), but failed to ablate CTP formation entirely (Fig. 4).
While DON-EcCTPS had a higher apparent affinity for GTP than wild-
type EcCTPS based on the IC50 values, DON-V60F (KA = 0.31 mM) had
only slightly stronger affinity for GTP than unmodified V60F
EcCTPS is most active as a tetramer [24, 78], and changing the
chemical nature of Val 60 at the dimer-dimer interface could possibly
alter the ability of the V60-substituted variants to oligomerize, resulting
in the diminished catalytic activity observed. For example, increasing
the steric bulk (i.e., V60F or V60W) or increasing acidity (i.e., V60D) of
the amino acid side chain at position 60 could have impaired assembly
of the active tetramer – especially considering that some variants ex-
hibited diminished ability to bind ATP and/or UTP that drive the oli-
gomerization process. Moreover, the sensitivity of these variants to Cl−
was similar to that of CTPS from L. lactis, which has been attributed to
salt-induced tetramer dissociation [68]. Hence, the apparent sensitivity
of Val 60 variants to Cl− could have caused a reduction in the pool of
the active tetrameric species, and the “rescue” of V60F by Gln and DON
could conceivably have arisen from an increase in the tetrameric pool of
enzyme. Consequently, the oligomerization state of each EcCTPS var-
iant was analyzed by DLS using assay buffer containing saturating
concentrations of UTP and ATP to induce tetramerization. For the DLS
analyses, wild-type EcCTPS was equilibrated without NTPs to measure
the mean hydrodynamic diameter of
a
dimer in solution
(dH = 8 3 nm) as a control for non-tetramerized protein (Fig. 5). The
mean hydrodynamic diameters of EcCTPS tetramers were also mea-
sured in the presence of saturating concentrations of UTP and ATP with
(dH = 15
3 nm) or without GTP (dH = 15
4 nm) to determine the
hydrodynamic diameter of EcCTPS tetramers under these conditions. In
the presence of saturating concentrations of UTP and ATP, the V60A,
V60C, V60D, and V60W variants had mean hydrodynamic diameters of
Fig. 4. Effects of GTP on NH3-dependent CTP formation catalyzed by EcCTPS
variants. Unmodified wild-type (◯, red), modified wild-type (DON-EcCTPS,
△, green), DON-V60A (◇, violet), and DON-V60F (☐, blue) EcCTPSs were
incubated with saturating concentrations of ATP, UTP, and NH4OAc
(Supplementary Table S2) and increasing concentrations of GTP (0–2.0 mM).
The data represent the average initial velocities from three independent
experiments SD. Curves for the wild-type, DON-EcCTPS, and DON-V60A
variants are fits of Eq. (4) to the initial velocity data. The IC50 values were
12
2, 12
2, 12
3, and 16
4 nm, respectively, indicating
little or no change in oligomerization relative to wild-type EcCTPS
under the same conditions (Fig. 5). V60F had only a slightly lower dH
value of 13
was observed upon addition of Gln (50 mM). While GTP induced a
slight increase in the dH value to 14 3 nm, the enzyme also had a dH
value of 14 3 nm in the presence of both Gln and GTP suggesting
3 nm compared to wild-type EcCTPS, and no increase
that these ligands did not greatly impact oligomerization state of V60F
EcCTPS (Fig. 5). Furthermore, DON-V60F EcCTPS, which is activated by
GTP, did not exhibit any increase in mean hydrodynamic diameter
following addition of GTP. Thus, the activation of the NH3-dependent
CTP formation of V60F EcCTPS by Gln and GTP, and of DON-V60F
EcCTPS by GTP, was not due to a change in the oligomerization
3.6
0.8 mM (extrapolated) for wild-type EcCTPS, 0.08
0.02 mM for DON-
EcCTPS, and 2.2
0.6 mM (extrapolated) for DON-V60A. Hill (n) values for
wild-type EcCTPS, DON-EcCTPS, and DON-V60A variants were 0.87
0.56 0.08, and 0.50 0.08, respectively. In the case of DON-EcCTPS, the
IC50 was calculated using 0 ≤ [GTP] ≤ 0.25 mM. The curve for DON-V60F is a
fit of Eq. (2) and the kinetic parameters can be found in Table 2.
0.09,
2722

G.D. McCluskey, S.L. Bearne
BBA-GeneralSubjects1862(2018)2714–2727
Fig. 5. DLS Analysis of the quaternary structure of wild-type and V60-substituted EcCTPS variants. DLS was used to determine the mean hydrodynamic diameter (dH)
for the wild-type (WT) and V60-substituted EcCTPS variants equilibrated with saturating concentrations of UTP and ATP (Supplementary Table S2) at 37 °C. Gln and/
or GTP were also added, where indicated, as controls to ensure that the glutaminase-mediated activation of V60F was not caused by a shift in the oligomerization
equilibrium towards the tetrameric species. Wild-type EcCTPS was also equilibrated in the absence of NTPs (WT dimer) as a control for non-oligomerized (i.e.,
dimeric) enzyme. Data are represented as Gaussian fits to aggregate relative intensities from three experiments with the average dH
SD indicated for each
condition. Parametric, unpaired t-tests conducted on the means derived from Gaussian-fits to the dH distributions from three independent DLS experiments reveal that
the dH values for the tetramers are not statistically different; however, the dH values for the tetramers (V60F EcCTPS (ATP + UTP) are statistically different from that
of the dimer (i.e., for V60F EcCTPS (ATP + UTP) vs. wild-type EcCTPS, p = 0.02).
equilibrium to favor formation of the active tetramer. Since DLS and the
kinetic assays were performed under identical conditions, this further
supports the conclusion that V60F and DON-V60F EcCTPSs are tetra-
mers with localized structural perturbations consistent with constricted
NH3 tunnels.
kinetic interrogation of this mechanism. Remarkably, the V60F variant
had limited ability to catalyze CTP formation when exogenous NH3 was
employed as the substrate, but Gln-derived NH3 was utilized much
more effectively. Furthermore, GTP was required for the utilization of
exogenous NH3 by the V60F variant despite the fact that GTP normally
inhibits NH3-dependent CTP formation [62, 79]. The requirement for
GTP, and the putative proximity of its binding site to Val 60, suggested
that GTP induced a conformational change in the vicinity of the gate to
effect NH3 transport. Though we were unable to obtain an X-ray
structure for the V60F variant, a 100-ns molecular dynamics (MD) si-
mulation was performed to determine how the mutation may have af-
fected the gate. Direct comparison between the energy-minimized
homology model of V60F EcCTPS and the X-ray crystal structure of
wild-type EcCTPS [26, 59, 60] indicated that the NH3 gate of V60F
EcCTPS was constricted by ~0.7 Å in radius relative to the tunnel of the
wild-type enzyme (Supplementary Fig. S15). We were unable to model
a tunnel within the enzyme at later times during the MD simulation due
to further constriction of the gate. Additionally, the V60F EcCTPS
model showed that the aromatic side chain of the Phe residue inter-
acted with the imidazole group of His 57 via an edge-on interaction for
much of the simulation which may have further blocked the tunnel exit
(Supplementary Fig. S16). Unfortunately, later time points also showed
some localized conformational disruption (primarily domain motion
about the inter-domain linker [59]) of the V60F model relative to wild-
type EcCTPS. However, kinetic data clearly demonstrated that nascent,
but not exogenous, NH3 could bypass this seemingly obstructed gate,
suggesting that the glutaminase activity plays a role in opening the
blocked gate. This mechanism of controlling the opening of the gate is
reminiscent of a similar mechanism utilized by glucosamine-6-phos-
phate synthase (GlmS), which cannot utilize exogenous NH3 [80]. In-
stead, GlmS has evolved an intrinsically blocked NH3 tunnel that opens
only during Gln hydrolysis [81]. A model summarizing the effects of the
glutaminase activity, GTP binding, and modification by DON (vide
infra) on the utilization of the exogenous nitrogen sources NH3 and
NH2OH is presented in Fig. 6.
4. Discussion
Substitutions of conserved residues often have significant detri-
mental consequences for catalysis by the enzyme being modified.
Despite having substituted Val 60 with two naturally-occurring amino
acid residues found in EcCTPS homologues, no Val 60-substituted var-
iant was completely unaffected. Even though conversion of Val 60 to
the naturally-occurring Ala or Cys substitutions was relatively in-
nocuous, the affinities of these variant enzymes for GTP were reduced
(Tables 1 and 2). This effect was even more pronounced in the V60D
and V60W variants, which supports previous hypotheses that the GTP-
binding site is located nearby the NH3 gate (barring any long-range
effects) [26, 57, 59]. However, none of these EcCTPS variants exhibited
impaired NH3 transport, and much of the reduced catalytic efficiencies
resulted from a reduction in the glutaminase activity (Table 3). V60W
was the most markedly affected EcCTPS variant since it was unable to
hydrolyze Gln, but retained the ability to utilize exogenous NH3. Since
the side chain methyl groups of Val 60 reside ~10 Å from the amide
carbon of Gln in the structure if EcCTPS with bound substrate (PDB ID
5TKV, [60]), it is plausible that mutation of Val 60 at the NH3 gate may
directly affect catalysis at the glutaminase site, as well as alter the
conformation of the NH3 gate to affect inter-domain NH3 transport.
4.1. “Pinching” the NH3 tunnel
Substitutions of Val 60 were originally prepared to investigate how
NH3 is transported between domains because the constriction shown in
the crystal structures of EcCTPS appears too narrow to allow the pas-
sage of NH3 [26, 59, 60]. Assuming there is a conformational change in
the tunnel that governs NH3 transport, we attempted to perturb the
function of the gate through site-directed mutagenesis to enable a
Because the efficiency (kcat/Km) of Gln-dependent CTP formation
catalyzed by V60F EcCTPS was reduced 176-fold, relative to wild-type
2723

G.D. McCluskey, S.L. Bearne
BBA-GeneralSubjects1862(2018)2714–2727
Fig. 6. Model describing the utilization of exogenous NH3 and
NH2OH by V60F EcCTPS variants. For simplicity, a single
monomer is shown with the positions of the glutaminase site,
GTP-binding site, NH3 tunnel, and synthase site schematically
illustrated. In the absence of GTP and Gln, the V60F variant is
unable to effectively utilize exogenous NH3 or NH2OH as
substrates (A). Upon binding of GTP, a conformational change
occurs that is consistent with partial opening of the tunnel
allowing for the use of exogenous NH3, but not NH2OH (B).
Alternatively, in the presence of Gln, a conformational change
occurs that permits the variant to catalyze the formation of
CTP from either exogenous NH3 or NH2OH, or from the nas-
cent NH3 derived from the hydrolysis of Gln (C), the latter
being promoted by the binding of GTP (thick arrow in D).
Finally, mimicking the glutamyl-enzyme intermediate
through modification by DON with activation by GTP (E)
induces a local structural change consistent with further
opening of the tunnel for more effective use of exogenous NH3
and NH2OH as substrates (cf. B and E). (For graphic re-
presentation, the surface representation of the monomer from
wild-type EcCTPS (PDB 2AD5, [26]) is shown. Cys 379 in the
glutaminase active site and Val 60 (rather than Phe) are
shown in sphere representation and colored yellow and red,
respectively.)
EcCTPS (Table 1), coupling assays were conducted with the V60F var-
iant to determine whether the lower rates of CTP formation resulted
from a leak, a bona fide blockage, or reduced rates of Gln hydrolysis.
Even though there was a reduction in the intrinsic glutmainase activity
of V60F EcCTPS relative to wild-type EcCTPS (Table 3), there existed
the possibility that the tunnel was “clogged” by the rapid build-up of
NH3 at saturating concentrations of Gln such that the constricted NH3
gate caused a “bottleneck” effect. When the concentrations of Gln were
lowered to a level similar to its Km value, or to a sub-saturating amount,
the observed coupling efficiency increased such that 100% of the NH3
produced in the glutaminase domain was utilized to generate CTP
(Table 4), thereby ruling out the possibility of NH3 leaking from the
tunnel. This “bottleneck” effect was also relieved following a reduction
in the concentration of GTP, even at saturating concentrations of Gln,
signifying that the impaired coupling observed when both Gln and GTP
are present at their saturating concentrations, is consistent with the
notion that the more constricted gate of V60F EcCTPS simply cannot
cope with the rapid build-up of NH3.
4.2. Opening of the V60F tunnel constriction
Evidently there was a disruption in the NH3 tunnel of V60F EcCTPS
that was relieved by Gln hydrolysis (Fig. 6C) and/or GTP binding
(Fig. 6B) because Gln-dependent CTP formation (i.e., transfer of nascent
NH3 through the gate) occurred much more efficiently than exogenous
NH3-dependent CTP formation. We therefore tested whether Gln and/
or GTP could increase the rate of CTP formation from an exogenous
nitrogen source. Since the Gln- and NH3-derived CTP products are in-
distinguishable, we employed NH2OH as an exogenous amine to mimic
exogenous NH3. We first established that both NH2OH and nascent NH3
compete for the same 4-phospho-UTP intermediate since EcCTPS is a
tetramer that exhibits half-of-the-sites reactivity with Gln [72] and
subunits lacking bound Gln might still react with exogenous amines
[69]. Levitski and Koshland [71] concluded that exogenous NH3 com-
petes with nascent NH3 derived from Gln based on the observation that
the rates of CTP formation at pH 9.25 were equal (but not additive).
Willemoës [69] demonstrated directly that exogenous and nascent
amines compete for the same 4-phospho-UTP in CTPS from L. lactis. We
confirmed that nascent NH3 and NH2OH also compete in EcCTPS by
2724

G.D. McCluskey, S.L. Bearne
BBA-GeneralSubjects1862(2018)2714–2727
conducting the same experiment with wild-type and V60F EcCTPSs
(Fig. 3A and B, respectively). The rate of N4-OH-CTP formation was
much slower with the V60F variant, possibly due to the increased bulk
of NH2OH and the presumably constricted tunnel. Consistent with the
indications that the glutaminase activity was required for the passage of
NH3 in V60F EcCTPS, the formation of N4-OH-CTP was markedly en-
hanced in the presence of Gln (Figs. 3C and 6C). Though no GTP-de-
pendent effect was observed, GTP may not be able to effect a great
enough conformational change to enable passage of the bulkier NH2OH
through the gate as it did with exogenous NH3 (Fig. 6B).
mutagenesis and subsequent kinetic analyses. Carbamoyl phosphate
synthase (CPS) [84–86], glutamine-dependent NAD+ synthetase
(NadE) [14], GlmS [80, 81], phosphoribosylformylglycinamidine syn-
thase [15], Gln phosphoribosylpyrophsphate amidotransferase [87],
and imidazole glycerol phosphate synthase [88, 89] are just a few ex-
amples of amidotransferases that have been extensively studied and
structurally characterized with respect to their ability to synchronize
multiple reactions. The tunnel of NadE contains several aperture-like
constrictions that open/close depending on what combination of li-
gands are bound, and substitution of the conserved Leu 489 by Phe
Gln was more effective at promoting passage of exogenous amine
through the constricted tunnel of V60F EcCTPS than GTP, but whether
it was Gln binding or hydrolysis remained unclear. Gln is hydrolyzed in
the glutaminase domain at a basal rate when GTP is absent, and this
slower process may be sufficient for enhancing inter-domain NH3
transport in V60F EcCTPS. We mimicked the formation of the glutamyl-
enzyme intermediate using covalent modification by DON [72] to de-
termine whether such an acyl-enzyme intermediate mimic could induce
a conformational change in the NH3 gate to allow passage of exogenous
NH3. Indeed, it is not unreasonable to expect that the transient gluta-
mylation of Cys 379 at the glutaminase active site could induce a
conformational change (e.g., NH3 gate opening) to facilitate the trans-
port of each newly formed NH3 to the synthase domain since con-
formational changes coordinated with ligand binding events have been
demonstrated for other amidotransferases [5, 8, 12, 82, 83]. The re-
sulting DON-V60F variant was able to catalyze CTP formation, using
either NH4Cl or NH4OAc, at an efficiency (kcat/Km) that was ~4-fold
greater than that of V60F EcCTPS (Table 1), but still required GTP for
activation. Unlike the unmodified V60F variant, DON-V60F EcCTPS
also required GTP for enhanced utilization of NH2OH (Figs. 3D and 6D),
which may be due to the static nature of the alkylated enzyme (as
opposed to the hydrolyically labile acyl-enzyme intermediate) or due to
an inability of GTP to facilitate opening of the NH3 gate wide enough
for the bulkier NH2OH to pass in the absence of Gln or its mimic. The
basal glutaminase activity may be sufficient for opening the tunnel of
V60F EcCTPS, but only when the full catalytic cycle is in progress.
Nevertheless, alkylation of V60F EcCTPS at Cys 379 of the glutaminase
active site greatly enhanced NH3-dependent CTP formation, supporting
the notion that the glutaminase activity plays a role in opening the NH3
gate.
reduced
inter-domain
coupling
efficiency
[14].
Mutation
(αP360A/αH361A/βR265A) of CPS resulted in a perforation of its NH3
tunnel leading to nascent NH3 leaking from the tunnel; however, full
catalytic activity with exogenous NH3 was retained [85]. In the present
study, we obtained an unusual result in that a single-amino acid sub-
stitution blocked the use of exogenous NH3, but retained Gln-dependent
activity, suggesting that opening of the NH3 gate is effected, in part, by
the “upstream” glutaminase activity.
X-Ray crystal structures of EcCTPS are challenging to obtain as
evidenced by the dearth of reported structures [26, 59, 60]; and no
high-resolution structures of EcCTPS have yet been solved with GTP
bound. Although this leaves us without direct observation of how the
Val 60 side chain substitutions affect the overall structure and/or
conformation of EcCTPS, our extensive mutational and kinetic analyses
indicate that Val 60 plays a central role that impacts glutaminase ac-
tivity, NTP binding, inhibition by Cl−, and coupling of reactions be-
tween domains. Clearly, substitutions at position 60 produce a local
disruption of structure that affects a variety of functions. Most sig-
nificantly, however, the local disruption of structure resulting from the
slight increase in steric bulk due to the substitution of Val 60 with Phe
was sufficient to induce an effect that was consistent with “pinching” of
the NH3 tunnel, permitting us to uncover the relationship between
processes that synchronize the transfer of NH3 from the glutaminase
domain to the synthase domain. Though wild-type EcCTPS does not
require Gln hydrolysis to efficiently utilize exogenous NH3, presumably
because its NH3 gate is more open, the V60F variant has just enough of
a constriction in the NH3 gate to furnish us with an enzyme that reveals
how the glutaminase activity and GTP-dependent activation can act in
concert to mediate inter-domain NH3 transport.
That GTP activates NH3-dependent CTP formation by V60F and
DON-V60F EcCTPSs at all is striking because GTP normally inhibits the
utilization of exogenous NH3 [62, 79]. Consequently, we also tested the
effects of GTP on the rates of NH3-dependent CTP formation for wild-
type EcCTPS, DON-EcCTPS, and DON-V60A EcCTPSs (Fig. 4). The DON
modifications enhanced GTP binding as determined from the inhibition
of wild-type EcCTPS when exogenous NH3 was employed as the ni-
trogen source, but never fully obviated CTP formation. The DON-
EcCTPS, DON-V60A, and DON-V60F variants had similar rates of CTP
formation at saturating concentrations of GTP indicating that mod-
ification of V60F EcCTPS by DON, by and large, “rescued” the enzyme
by restoring near-wild-type levels of activity under these conditions
(Fig. 4). The slightly lower rates of CTP formation catalyzed by the
DON-V60A and DON-V60F variants with respect to DON-EcCTPS are
likely due to the slight inhibitory effects of acetate. Although it seems
counter-intuitive, it may be that DON-V60F EcCTPS (and V60F EcCTPS)
is inhibited by GTP, i.e., GTP may have dual, separable effects on DON-
V60F EcCTPS compared to DON-EcCTPS, with both enzymes inhibited
similarly but with a V60F-specific effect that relieves the constriction in
the NH3 tunnel.
Acknowledgements
We thank Dr. Alejandro Cohen of the Dalhousie Faculty of Medicine
Proteomics Facility for his assistance with mass spectrometry experi-
ments.
Funding
This work was supported by an operating grant from the Canadian
Institutes of Health Research (no. 114895, S.L.B.) and a Scotia Support
Grant from the Nova Scotia Heath Research Foundation (no. 140;
S.L.B.). G.D.M. is a trainee in the Cancer Research Training Program of
the Beatrice Hunter Cancer Research Institute, with funds provided by
Craig's Cause Pancreatic Cancer Society, a CIBC Graduate Scholarship
in Medical Research, and The Terry Fox Research Institute.
Notes
The authors declare no competing financial interest.
4.3. Concluding remarks
Appendix A. Supplementary data
Several studies have been conducted to understand how amido-
transferases coordinate NH3 transport with “downstream” amidoliga-
tion reactions in a synthase domain, generally through site-directed
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.bbagen.2018.08.008.
2725

G.D. McCluskey, S.L. Bearne
BBA-GeneralSubjects1862(2018)2714–2727
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