Limited trypsin-catalysed proteolysis of both wild-type and mutant CTP synthases was analysed by monitoring CTPS activity vide infra at specific time points and by SDS/PAGE.. N-Terminal s
Trang 1Limited proteolysis of Escherichia coli cytidine 5¢-triphosphate
synthase Identification of residues required for CTP formation
and GTP-dependent activation of glutamine hydrolysis
Dave Simard, Kerry A Hewitt, Faylene Lunn, Akshai Iyengar and Stephen L Bearne
Department of Biochemistry and Molecular Biology, Dalhousie University, Halifax, Nova Scotia, Canada
Cytidine 5¢-triphosphate synthase catalyses the
ATP-dependent formation of CTP from UTP using either
ammonia orL-glutamine as the source of nitrogen When
glutamine is the substrate, GTP is required as an allosteric
effector to promote catalysis Limited trypsin-catalysed
proteolysis, Edman degradation, and site-directed
muta-genesis were used to identify peptide bonds C-terminal to
three basic residues (Lys187, Arg429, and Lys432) of
Escherichia coliCTP synthase that were highly susceptible to
proteolysis Lys187 is located at the CTP/UTP-binding site
within the synthase domain, and cleavage at this site
destroyed all synthase activity Nucleotides protected the
enzyme against proteolysis at Lys187 (CTP > ATP >
UTP > GTP) The K187A mutant was resistant to
pro-teolysis at this site, could not catalyse CTP formation, and
exhibited low glutaminase activity that was enhanced slightly
by GTP K187A was able to form tetramers in the presence
of UTP and ATP Arg429 and Lys432 appear to reside in an
exposed loop in the glutamine amide transfer (GAT) domain Trypsin-catalyzed proteolysis occurred at Arg429 and Lys432 with a ratio of 2.6 : 1, and nucleotides did not protect these sites from cleavage The R429A and R429A/ K432A mutants exhibited reduced rates of trypsin-catalyzed proteolysis in the GAT domain and wild-type ability to catalyse NH3-dependent CTP formation For these mutants, the values of kcat/Kmand kcatfor glutamine-dependent CTP formation were reduced 20-fold and 10-fold, respect-ively, relative to wild-type enzyme; however, the value of Km
for glutamine was not significantly altered Activation of the glutaminase activity of R429A by GTP was reduced 6-fold at saturating concentrations of GTP and the GTP binding affinity was reduced 10-fold This suggests that Arg429 plays
a role in both GTP-dependent activation and GTP binding Keywords: activation; amidotransferase; CTP synthase; glutaminase; proteolysis; site-directed mutagenesis
CTP synthase [CTPS; EC 6.3.4.2; UTP:ammonia ligase
(ADP-forming)] catalyses the ATP-dependent formation of
CTP from UTP using eitherL-glutamine or NH3 as the
nitrogen source (Scheme 1) [1,2] This glutamine
amido-transferase is a single polypeptide chain containing 545
amino acids and consisting of two domains The C-terminal
glutamine amide transfer (GAT) domain catalyses the
hydrolysis of glutamine, and the nascent NH3derived from
glutamine hydrolysis is transferred to the N-terminal
synthase domain where the amination of UTP is catalysed
[3,4] CTPS belongs to the Triad family of glutamine
amidotransferases [5,6] which utilizes a Cys-His-Glu triad to
catalyse glutamine hydrolysis and also includes anthranilate
synthase, carbamoyl phosphate synthase,
formylglycin-amidine synthase, GMP synthase, imidazole glycerol
phos-phate synthase, and aminodeoxychorismate synthase
CTPS catalyses the final step in the de novo synthesis of cytosine nucleotides Because CTP has a central role in the biosynthesis of nucleic acids [7] and membrane phospho-lipids [8], CTPS is a recognized target for the development
of antineoplastic agents [7,9], antiviral agents [9,10], and antiprotozoal agents [11–13] Recently, CTP synthase inhibition has been shown to potentiate the cytotoxic effects
of the anticancer drug 1-b-D-arabinofuranosylcytosine [14] and anti-HIV therapies [15]
CTPS from E coli is the most thoroughly characterized CTPS with respect to its physical and kinetic properties, and
is regulated in a complex fashion [1] GTP is required as a positive allosteric effector to increase the efficiency (kcat/Km)
of glutamine-dependent CTP synthesis 45-fold but has a negligible effect on the reaction when NH3is the substrate [16,17] In addition, the enzyme is inhibited by the product CTP [18], exhibits negative cooperativity for glutamine [19], and displays positive cooperativity for ATP and UTP
Scheme 1 CTP-forming reactions catalysed by CTPS.
Correspondence to S L Bearne, Department of Biochemistry and
Molecular Biology, Dalhousie University, Halifax, Nova Scotia,
Canada B3H 1X5 Tel.: +1 902 494 1974, Fax: + 1 902 494 1355,
E-mail: sbearne@is.dal.ca
Abbreviations: CTPS, CTP synthase; GAT, glutamine amide transfer;
GF-HPLC, gel-filtration-HPLC; PVDF, poly(vinylidene difluoride).
Enzymes: CTP synthase (EC 6.4.3.2).
(Received 28 February 2003, revised 17 March 2003,
accepted 21 March 2003)
Trang 2[18–20] ATP and UTP act synergistically to promote
tetramerization of the enzyme to its active form [20]
The structure of CTPS has not yet been determined and
hence little is known about the enzyme’s tertiary structure
However, analysis of crystal structures of the Triad
amidotransferases GMP synthase and carbamoyl
phos-phate synthase reveal that the structures of the GAT
domains are probably closely related among all Triad
enzymes [21,22] Site-directed mutagenesis studies and
sequence comparisons have revealed structural and
cata-lytic roles of several amino acid residues within the GAT
domain of CTPS, including residues of the catalytic triad
(Cys379, His515, and Glu517) [3], residues comprising the
oxyanion hole (Gly351, Gly377, Gly381, and possibly
adjacent hydrophobic residues) [23], and residues between
Ala346 and Tyr355 that appear to play an important
structural role [4] Recently, Willemoe¨s reported that
Thr431 and Arg433 in the GAT domain of Lactococcus
lactis CTPS play a role in GTP-dependent activation of
glutamine hydrolysis [24]
Our knowledge about the synthase domain is much more
limited Analyses of mutant CTP synthases from Chlamydia
trachomatis[25], hamster [26], and yeast [27] have revealed
that mutations which render cells resistant to both the
cytotoxic effects of cyclopentenylcytosine and feedback
inhibition by CTP occur between residues 116 and 229
(E coli numbering), with many of the mutations clustering
between residues 146 through 158 Hence, this region of the
synthase domain is believed to form part of the
CTP-binding site Competitive inhibition experiments have
suggested that for E coli CTPS, this site may also be the
UTP-binding site [18] The locations of the ATP- and
GTP-binding sites have not yet been identified Recent studies
from our laboratory have revealed that residues Asp107 and
Leu109 in the synthase domain of E coli CTPS facilitate
efficient coupling of glutamine hydrolysis to CTP synthesis
[28]
To learn more about the structure of CTPS, we
inves-tigated controlled proteolysis of the enzyme Using limited
trypsin-catalysed proteolysis and site-directed mutagenesis,
we have identified peptide bonds C-terminal to three basic
residues of E coli CTPS that are highly susceptible to
proteolysis One residue, Lys187, is located at the CTP/
UTP-binding site within the synthase domain and is
essential for catalysis but not for enzyme tetramerization
The other two residues, Arg429 and Lys432 appear to reside
in an exposed loop that is important for both GTP binding
and GTP-dependent allosteric activation of glutamine
hydrolysis
Materials and methods
Materials
HisÆBind resin and thrombin cleavage capture kits were
from Novagen; broad range protein markers were from
New England Biolabs; Pfu Turbo DNA polymerase was
from Stratagene Inc.; nucleotides, a-chymotrypsin from
bovine pancreas (54 UÆmg)1), Pronase from Streptomyces
griseus (4.7 UÆmg)1), protease V8 from Staphylococcus
aureus (1000 UÆmg)1), thermolysin from Bacillus
thermo-proteolyticus rokko(55 UÆmg)1), and TPCK-treated trypsin
from bovine pancreas (10 900 UÆmg)1), and all other chemicals were from Sigma-Aldrich Canada Ltd Oligo-nucleotide primers for DNA sequencing and site-directed mutagenesis were commercially synthesized by ID Labor-atories (London, ON, Canada) QIAprep spin plasmid miniprep kit (Qiagen Inc.) was used for the preparation of plasmids for mutagenesis and transformation DNA sequencing was conducted at the Dalhousie University– NRC Institute for Marine Biosciences Joint Laboratory (Halifax, NS, Canada) and the Robarts Research Institute (London, ON, Canada), while the N-terminal amino acid sequencing was carried out at the Eastern Que´bec Proteo-mics Core Facility (Ste-Foy, QC, Canada) Predictions of secondary structure were conducted using the programs 3-D PSSM[29],GOR4 [30],HNN[31],J-PRED[32],PREDATOR[33],
PSIPRED [34], and SSPRO [35] Sequence alignments were conducted usingCLUSTALW[36]
Enzyme expression and purification Wild-type and mutant forms of recombinant E coli CTPS were expressed in and purified from E coli strain BL21(DE3) cells transformed with either mutant or wild-type plasmid pET15b-CTPS1 as described previously [16] This construct encodes the CTPS gene product with an N-terminal hexahistidine tag Thrombin-catalysed cleavage
of the histidine tag from soluble enzymes (new N-terminus, GSHMLEM1…) was carried out as described previously [16] The resulting enzyme was dialysed into Hepes buffer (70 mM, pH 8.0) containing EDTA (0.5 mM) and MgCl2 (10 mM) The results of purification and cleavage pro-cedures were routinely monitored using SDS/PAGE The amino acid residues in the recombinant wild-type and mutant enzymes are numbered according to the sequence of the wild-type E coli enzyme starting with M1as position one
Mutagenesis The plasmid pET15b-CTPS1 [16] was used as the template for site-directed mutagenesis Site-directed mutagenesis was conducted using the Quikchange Site-Directed Mutagenesis Kit (Stratagene Inc.) and following the manufacturer’s protocol The synthetic deoxyoligonucleotide forward (F) and reverse (R) primers used to construct the mutants were: 5¢-GCGTCTGGTGAAGTCGCAACCAAACCGACT CAG-3¢ (F, K187A), 5¢-GCTGAGTCGGTTTGGTT GCGACTTCACCAGACGC-3¢ (R, K187A), 5¢-CGG CAACGTTGAAGTTGCTAGCGAGAAGAGCG-3¢ (F, R429A), 5¢-CGCTCTTCTCGCTAGCAACTTCAACGT TGCCG-3¢ (R, R429A), 5¢-GCAACGTTGAAGTT GCTAGCGAGGCGAGCGATCTCG-3¢ (F, R429A/ K432A), 5¢-CGAGATCGCTCGCCTCGCTAGCAACT TCAACGTTGC-3¢ (R, R429A/K432A), where the posi-tions of the mismatches are underlined Potential mutant plasmids were isolated and used to transform competent DH5a cells These cells were used for plasmid maintenance and for all sequencing reactions The entire mutant genes were sequenced to verify that no other alterations of the nucleotide sequence had been introduced Competent
E coli strain BL21(DE3) cells were used as the host for target gene expression
Trang 3Enzyme assay and protein determinations
CTPS activity was determined at 37C using a continuous
spectrophotometric assay by following the rate of increase
in absorbance at 291 nm resulting from the conversion of
UTP to CTP (De¼ 1338ÆM )1Æcm)1) [18] The standard
assay mixture consisted of Hepes buffer (70 mM, pH 8.0)
containing EDTA (0.5 mM) and MgCl2 (10 mM), CTPS,
and saturating concentrations of UTP (1 mM) and ATP
(1 mM) in a total volume of 1 mL Enzyme and nucleotides
were preincubated together for 2 min at 37C followed by
addition of substrate (NH4Cl or glutamine) to initiate the
reaction Total NH4Cl concentrations used in the assays
were 5, 10, 20, 30, 50, 60, 80, and 100 mM, and CTPS
concentrations were 3.0 lgÆmL)1 (wild-type),
3.0 lgÆmL)1(R429A), and 4.0 lgÆmL)1 (R429A/K432A)
For glutamine assays, concentrations of glutamine
were 0.1, 0.2, 0.3, 0.5, 1.0, 2.0, 3.0, and 6.0 mM and
CTPS concentrations were 4.0 lgÆmL)1 (wild-type),
1.4 mgÆmL)1 (R429A), and 0.9 mgÆmL)1 (R429A/
K432A) The concentration of GTP was maintained at
0.25 mM for all assays when glutamine was used as the
substrate In addition, the ionic strength was maintained at
0.25Min all spectrophotometric assays by the addition of
KCl The apparent activation constant (KA) for R429A
CTPS (0.4 mgÆmL)1) with respect to GTP was determined
for glutamine-dependent CTP formation as described
previously [28]
All kinetic parameters were determined in triplicate and
average values are reported Initial rate kinetic data was fit
to Eqn (1) by nonlinear regression analysis using the
program ENZYMEKINETICS v1.5 (1996) from Trinity
Soft-ware (Plymouth, NH) In Eqn (1), viis the initial velocity,
Vmax (¼ kcat[E]T) is the maximal velocity at saturating
substrate concentrations, [S] is the substrate concentration
(glutamine or NH3), and Kmis the Michaelis constant for
the substrate Values of Kmfor NH3were calculated using
the concentration of NH3 present at pH 8.0 {pKa
(NH4+)¼ 9.24 [37]} Values of kcat were calculated for
CTPS variants with the hexahistidine tag removed using
the molecular masses (Da) of 61 029 (wild-type), 60 944
(R429A), and 60 887 (R429A/K432A) The reported
errors are standard deviations Except where noted
otherwise, protein concentrations were determined using
the Bio-Rad Protein Assay (Bio-Rad Laboratories Ltd.)
with BSA standards
mi¼ Vmax½S
Glutaminase activity
Values of kcat for the hydrolysis of glutamine, at fixed
saturating concentrations of glutamine (6 mM), UTP
(1 mM), and ATP (1 mM) were determined as described
previously [38] Data describing the dependence of the
apparent kcat values on the concentration of GTP were
fitted to Eqn (2) for hyperbolic nonessential activation
kinetics where KAis the apparent activation constant, kois
the turnover number in the absence of GTP, and kactis
the turnover number at saturating concentrations of GTP
[39]
kapparentcat ¼
koþ kact
GTP
KA
1þ ½GTPK
A
Limited proteolysis Initially, wild-type CTPS was subjected to limited proteo-lysis by several endopeptidases including trypsin, chymo-trypsin, pronase, thermolysin and V8 protease Proteolysis was conducted in Hepes buffer (70 mM, pH 8.0) containing EDTA (0.5 mM) and MgCl2 (10 mM) at 37C using a CTPS/protease ratio (lg protein) of 60 : 1 in a total volume
of 1 mL Limited proteolysis using pronase and thermolysin was also conducted in potassium phosphate buffer (50 mM,
pH 7.2) containing EDTA (1 mM) and MgCl2 (10 mM) EDTA was omitted from the buffers for all thermolysin-catalysed reactions Proteolysis experiments were conducted
in the absence and presence of ATP (10 mM) and UTP (10 mM) During the proteolysis reactions, aliquots (25 lL) were removed from the reaction mixture over the course of
1 h, and transferred to gel loading buffer [25 lL; Tris/HCl (170 mM, pH 6.8) containing dithiothreitol (120 mM), SDS (5.4%, w/v), Bromophenol blue (0.03%, w/v) and glycerol (27.2%, w/v)] to terminate the reaction The samples were then boiled for 5 min and the proteolytic fragments were separated using SDS/PAGE (12% gels) Fragments were visualized by staining with Coomassie blue R-250 and subsequent de-staining in a solution of methanol/H2O/ acetic acid (45 : 45 : 10) Detailed studies were subsequently conducted using trypsin as trypsin-catalysed proteolysis gave different fragments depending on whether the nucleo-tides were absent or present
Limited trypsin-catalysed proteolysis of both wild-type and mutant CTP synthases was analysed by monitoring CTPS activity (vide infra) at specific time points and by SDS/PAGE Trypsin proteolysis reactions (1 mL total volume) contained either wild-type or mutant CTPS (0.20 mgÆmL)1), and were conducted for 1 h in Hepes buffer (70 mM, pH 8.0) containing EDTA (0.5 mM) and MgCl2 (10 mM) at 37C Reactions were initiated by addition of trypsin (0.1 lgÆmL)1) and aliquots (100 lL) were removed every 10 min over 1 h and assayed for activity using NH3as the substrate The cleavage fragments produced from trypsin-catalysed proteolysis were analysed using SDS/PAGE (12% and 20% gels) Reactions were conducted as described above, with the exception that at 10,
20, 30, and 60 min, aliquots (20 lL) were removed and transferred to gel loading buffer (15 lL) to terminate the reaction A zero time point was obtained using 3 lL of the enzyme stock solution ( 1.5 mgÆmL)1) used for the reaction The ability of various ligands to protect both wild-type and mutant CTP synthases from proteolysis was examined using ATP (10 mM), UTP (10 mM), ATP and UTP together (10 mM each), CTP (0.1, 0.5, and 1.0 mM), GTP (2.5 mM) andL-glutamine (10 mM)
Inactivation assays Aliquots from two separate proteolysis reactions were assayed using both NH4Cl (100 mM) and glutamine
Trang 4(10 mM) as substrates, as described above Inactivation of
CTPS activity followed first-order kinetics, and the apparent
first-order rate constants for inactivation were calculated
from plots of the percent activity remaining as a logarithmic
function of the time of incubation The ability of various
ligands to protect both wild-type and mutant CTPS from
proteolysis was examined using ATP (0.5, 1.0, 2.0, and
10 mM), UTP (0.5, 1.0, 2.0, and 10 mM), ATP and UTP
together (10 mM each), GTP (0.25 mM), andL-glutamine
(10 mM) at the concentrations indicated
N-Terminal sequence analysis
Approximately 190 lg total protein containing CTPS and
the various trypsin-catalysed cleavage fragments, produced
after a 90-min proteolysis reaction, were separated using
SDS/PAGE (12% or 20% gels) and subsequently
trans-ferred to a poly(vinylidene difluoride) (PVDF) membrane
[Immun-Blot 0.2 lm (Bio-Rad Laboratories Ltd) for
fragments with molecular masses of 25, 28 and 53 kDa;
Immobilon-Psq0.2 lm PVDF (Millipore Ltd) for the
fragment with a molecular mass of 10 kDa] as described
by Wilson and Yuan [40] Electroblotting was conducted
in CAPS buffer (0.01M, pH 11.0) containing methanol
(10%) Whole enzyme and cleavage fragments were located
on the PVDF membrane by staining with Coomassie blue
followed by destaining in 50% methanol Sections (50 mm2)
of the PVDF membrane with adsorbed protein were
submitted for N-terminal amino acid sequence analysis
CD spectra
CD spectra were obtained using a JASCO J-810
spectro-polarimeter and were recorded for both the wild-type and
mutant enzymes (K187A, R429A, R429A/K432A) over the
range 190–260 nm in the absence of nucleotides A marked
decrease in buffer transparency was observed below
190 nm and therefore all spectra were truncated at this
wavelength The resulting CD spectra obtained from
enzyme solutions (0.2 mgÆmL)1) in Bis-Tris propane buffer
(10 mM, pH 8.0) containing MgSO4(10 mM) were analysed
for percent a-helix and b-sheet structure using CDNN CD
Spectra Deconvolution v 2.1 developed by G Bo¨hm [41]
Protein concentrations were determined
spectrophotomet-rically at 280 nm using an extinction coefficient equal to
38 030ÆM )1Æcm)1 for the wild-type, K187A, R429A, and
R429A/K432A CTP synthases
Tetramerization of CTPS
The ability of the K187A CTPS to form tetramers was
evaluated using gel-filtration-HPLC (GF-HPLC) with
native tryptophan fluorescence detection Wild-type and
mutant CTP synthases, and standard proteins were eluted
under isocratic conditions using Hepes buffer (pH 8.0,
0.07M) containing MgCl2(10 mM) and EDTA (0.5 mM) at
a flow rate of 1.0 mLÆmin)1 on a BioSep–SEC-S 3000
column (7.80· 300 mm; Phenomenex, Torrance, CA) A
Waters 510 pump and 680 controller were used for solvent
delivery Injections were made using a Rheodyne 7725i
sample injector fitted with a 20-lL injection loop The eluted
proteins were detected by native protein fluorescence
(excitation and emission wavelengths of 285 nm and
335 nm, respectively) using a Waters 474 scanning fluores-cence detector GF-HPLC of both wild-type and mutant enzymes was conducted in the absence and presence of ATP (1 mM) and UTP (1 mM), and the retention times were compared with those observed for the wild-type enzyme The column was standardized using the following proteins (0.5 mgÆmL)1): bovine thyroglobulin (669 kDa), b-amylase (200 kDa), BSA (66 kDa), and carbonic anhydrase (29 kDa) Chromatograms were analysed using PEAKSIM-PLEsoftware from Mandel Scientific (Guelph, ON, Canada) The retention time of bovine thyroglobulin was used to estimate the column void volume (Vo)
Results
Limited proteolysis of CTPS CTPS from E coli was subjected to controlled proteolysis
by five endopeptidases (pronase, a-chymotrypsin, V8 pro-tease, thermolysin, and trypsin) in the absence or presence of the nucleotides ATP and/or UTP (data not shown) These preliminary experiments revealed that only treatment of CTPS with trypsin produced a limited number of cleavage fragments over the course of 1 h, of which the formation of some fragments was suppressed in the presence of ATP and/
or UTP Thus, trypsin was used in the present study to investigate the accessibility of regions in CTPS to proteolytic cleavage in the presence and absence of various ligands Limited trypsin-catalysed cleavage of wild-type CTPS Limited trypsin-catalysed proteolysis of wild-type CTPS produced different cleavage fragments depending on whe-ther ATP and/or UTP were absent or present in the reaction mixture (Fig 1) In the absence of nucleotides, trypsin-catalysed cleavage of CTPS (63 kDa) produced four fragments with molecular masses corresponding to 10, 25,
28, and 53 kDa However, in the presence of either ATP or UTP (data not shown), ATP and UTP together, or CTP, only fragments with molecular masses of 10 and 53 kDa were produced indicating that these nucleotides protected CTPS from cleavage at the site which produced the 25- and 28-kDa fragments Neither glutamine nor GTP protected CTPS from limited trypsin-catalysed digestion
The sites of trypsin-catalysed cleavage yielding each of the fragments were identified using Edman degradation to obtain the N-terminal amino acid sequence of each fragment (Table 1), and the known nucleotide sequence encoding the enzyme [3] The N-terminal sequence of the 25- and 53-kDa fragments were the same as that of the whole recombinant wild-type protein indicating that the cleavage site was located at the C-terminus of each of these two fragments The N-terminal sequence of the 28-kDa fragment indicated that one of the cleavage sites was Lys187 N-terminal analysis of the 10-kDa fragment produced two sequences indicating that cleavage occurred
at Arg429 and Lys432 with a ratio of 2.6 : 1, respectively The cleavage pattern observed in the absence of any protecting ligands, and the sites identified using N-terminal analysis are summarized in Fig 2 The molecular mass of each polypeptide fragment, calculated using the known
Trang 5amino acid sequence [3], is in excellent agreement with the values deduced from SDS/PAGE calibration (Table 1)
Limited trypsin-catalysed cleavage of mutant CTP synthases
To confirm that the cleavage sites identified using N-terminal analysis were indeed correct, site-directed mut-agenesis was used to construct two single mutants (K187A and R429A) and one double mutant (R429A/K432A) Limited trypsin-catalysed proteolysis of K187A CTPS in the absence of nucleotides produced only the 10- and 53-kDa fragments (Fig 3A), a cleavage pattern which was identical to that observed for wild-type CTPS in the presence of ATP and UTP (Fig 1B) Proteolysis of R429A in the absence of nucleotides gave fragments with molecular masses of approximately 10, 25, 38, and 53 kDa (Fig 3B) The 38-kDa fragment accumulated because rapid cleavage at Arg429 no longer occurred while cleavage at
Fig 1 SDS/PAGE analysis of trypsin-catalysed cleavage of
recom-binant wild-type CTPS in the absence and presence of ligands For each
gel: lane 1 contains molecular mass standards and lanes 2–6 contain
wild-type CTPS treated with trypsin for 0, 10, 20, 30, and 60 min,
respectively In the absence of any ligands (A), the wild-type protein is
rapidly cleaved to yield a 10-kDa (not shown except in E) and a
53-kDa fragment, the latter which is subsequently cleaved to yield two
fragments with molecular masses of 25 and 28 kDa In the
pres-ence of ATP and UTP (10 m M each) (B), and CTP (2.5 m M ) (C), only
production of the 10- and 53-kDa cleavage fragments is observed In
the presence of GTP (2.5 m M ) (D) and L -glutamine (10 m M ) (E),
fragments with molecular masses of 10, 25, 28, and 53 kDa are
pro-duced All gels are 12% except for (E) which is 20%.
Table 1 N-terminal amino acid sequences of trypsin-catalysed cleavage fragments.
Molecular massa (kDa) N-terminal sequence
Cleavage site identifiedb
10 (13.1) S430EKSDLGGTM (major) c Arg429 (12.8) S433DLGGTMRL (minor)c Lys432
a Apparent molecular mass for full-length recombinant wild-type
E coli CTPS and fragments determined from SDS/PAGE calib-ration are given The corresponding molecular masses calculated using the known amino acid sequence are given in parentheses b The amino acid listed is that which provides the carbonyl function to the scissile peptide bond The numbers correspond to the numbering for wild-type E coli CTPS c The ratio of the major peptide to minor peptide was 2.6 : 1 and was determined by integration of the HPLC chromatogram peaks corresponding to the phenylthiohydantoin derivatives of the N-terminal serines.
Fig 2 Fragments generated by limited
trypsin-catalysed proteolysis of wild-type CTPS.
Peptide bond cleavage occurs C-terminal to
Lys187 in the synthase domain, and Arg429
and Lys432 in the GAT domain The CTP/
UTP-binding site and residues comprising the
catalytic triad (Cys379, His515, and Glu517)
are also shown.
Trang 6Lys187 divided the protein into the 25- and 38-kDa
fragments The 10- and 53-kDa fragments were formed in
much lower amounts than observed with wild-type CTPS
because of slow cleavage at Lys432 In the presence of ATP
and UTP, cleavage of R429A at Lys187 was suppressed and
only the 10- and 53 kDa fragments were formed because of
slow cleavage at Lys432 (Fig 3C) Limited proteolysis of the
double mutant (R429A/K432A) in the absence of
nucleo-tides produced only two fragments with molecular masses
corresponding to 25 and 38 kDa consistent with cleavage
occurring only at Lys187 (Fig 3D) In the presence of ATP
and UTP, proteolysis of R429A/K432A CTPS was com-pletely suppressed (Fig 3E)
Inactivation and protection studies Treatment of wild-type and mutant CTP synthases with trypsin produced time-dependent loss of both NH3 -depend-ent activity and glutamine-depend-depend-ent activity (Fig 4), which followed first-order kinetics up to at least 90% of the reaction The observed first-order inactivation rate constants for CTPS activity assayed using either NH3or glutamine as the substrate are given in Table 2 In the absence of ligands, the observed first-order rate constant for trypsin-catalysed proteolysis of CTPS was slightly greater when glutamine-dependent CTP formation was measured than when NH3-dependent CTP formation was measured
A reduction in the observed first-order rate constants for the inactivation of the NH3-dependent activity was observed for increasing concentrations of ATP, UTP, and CTP consis-tent with each of these nucleotides providing protection from trypsin-catalysed cleavage Interestingly, in the
Fig 3 SDS/PAGE analysis of trypsin-catalysed cleavage of mutant
CTP synthases in the absence and presence of ligands (A) Lane 1,
molecular mass standards; lane 2, trypsin (at 7000 times the
concen-tration used in the proteolysis reactions); lanes 3–7 contain K187A
CTPS treated with trypsin for 0, 10, 20, 30, and 60 min, respectively.
The wild-type protein is rapidly cleaved to yield a 10- (not shown) and
a 53-kDa fragment, the latter which is not cleaved to yield the 25- and
28-kDa fragments For each gel shown in B through E, lane 1 contains
molecular mass standards and lanes 2–6 contain mutant CTPS treated
with trypsin for 0, 10, 20, 30, and 60 min, respectively Limited
pro-teolysis of R429A CTPS (B) in the absence of nucleotides produced
fragments with molecular masses of 10, 25, 38, and 53 kDa However,
in the presence of ATP and UTP (10 m M each) (C), only the 10- and
53-kDa fragments are produced Limited proteolysis of R429A/
K432A CTPS (D) in the absence of nucleotides produces fragments
with molecular masses of 25 and 38 kDa However, in the presence of
ATP and UTP (10 m M each) (E), no cleavage fragments were
pro-duced over the course of 1 h indicating that proteolysis was greatly
suppressed All gels are 12%.
Fig 4 Time-dependent inactivation of wild-type CTPS by trypsin (A) Inactivation of CTPS-catalysed NH 3 -dependent CTP formation in the absence of ligands (s) and in the presence of ATP (10 m M , n), UTP (10 m M , h), and ATP and UTP combined (10 m M each, ,) Panel B shows the inactivation of CTPS-catalysed glutamine-dependent CTP formation in the absence of nucleotides (s) and in the presence of UTP (10 m M , n), ATP (10 m M , h), and ATP and UTP combined (10 m M
each, ,) In both panels, the activity of the enzyme in the absence of trypsin is also shown (d).
Trang 7presence of ATP and UTP, the rate constant for
inactiva-tion of glutamine-dependent activity was greater than that
observed for inactivation of the NH3-dependent activity
This observation is consistent with the cleavage sites in the
GAT domain not being protected by these nucleotides and
the resulting 53-kDa fragment still possessing NH3
-depend-ent activity Although all CTPS ligands tested (ATP, UTP,
CTP, GTP, and glutamine) protected CTPS from
inactiva-tion to some degree, the most effective protecinactiva-tion was
afforded by CTP
The observed first-order inactivation rate constants for
the NH3-dependent activity of the R429A and R429A/
K432A CTP synthases were less than that observed for
wild-type CTPS Apparently, reduced cleavage within the
GAT domain results in less rapid cleavage within in the
synthase domain (i.e at Lys187) and hence a lower value for
the rate constant for the loss of NH3-dependent activity
Inactivation of the K187A enzyme could not be studied
because this enzyme was inactive (vide infra)
CD
The secondary structural content of wild-type CTPS and
the three mutant enzymes was analysed using CD
spectroscopy Fig 5 shows that the secondary structure
content of all the mutant proteins is similar to that of the
wild-type enzyme, except that the a-helix content of the
K187A and R429A/K432A mutants is slightly reduced
while the content of antiparallel b-sheet structure is slightly
increased, relative to wild-type CTPS Although no
signi-ficant gross perturbations in secondary structure are evident in the mutant proteins, the possibility that the mutations cause a localized perturbation of secondary structure or conformational change cannot be ruled out Mutant enzyme kinetics
The kinetic parameters kcatand Kmfor CTP formation were determined with respect to NH3and glutamine for each of the mutant enzymes except for K187A CTPS which was inactive (Table 3) Direct examination of the conversion of glutamine to glutamate (glutaminase activity) revealed that K187A CTPS was able to catalyse the hydrolysis of glutamine, but had a value of kcat that was half of that observed for wild-type CTPS in the absence of GTP When the concentration of GTP was increased to 1 mM, the value
of kactwas increased fivefold, compared to 50-fold for wild-type CTPS (Fig 6)
R429A and R429A/K432A CTP synthases displayed similar kinetic properties Each mutant had close to wild-type NH3-dependent activity; however, glutamine-depend-ent CTP formation was impaired Interestingly, Km for glutamine only increased 1.3- to 1.7-fold indicating that the mutations had little effect on glutamine binding However,
kcat was reduced 15-fold for each mutant so that the catalytic efficiency (kcat/Km) of glutamine-dependent CTP formation was decreased 25- and 19-fold for the R429A and R429A/K432A CTP synthases, respectively
The kinetics of R429A CTPS were investigated in detail
to determine if the impaired glutamine-dependent CTP formation was caused by an inability of GTP to activate glutamine hydrolysis In the presence of ATP and UTP (1 mM each) and saturating glutamine (6 mM), GTP (0.25 mM) caused a 2.5-fold increase in kcatfor glutamine-dependent CTP formation catalysed by R429A CTPS compared to a 30-fold increase for wild-type CTPS (data not shown) Concentrations of GTP above 0.25 mM(up to
1 mM) did not enhance the observed rate of CTP formation Direct examination of the glutaminase activity revealed that kcatwas reduced approximately 10-fold for the R429A and R429A/K432A enzymes relative to wild-type CTPS with the concentration of GTP equal to 0.25 mM(Table 3) More detailed analysis of the glutaminase activity of R429A CTPS (Fig 6) revealed that GTP binding and kact were reduced approximately 10-fold and sixfold, relative to wild-type CTPS Thus mutation of Arg429 to alanine impairs both GTP binding and allosteric activation of glutamine hydrolysis
Comparison of the kcatvalues for the glutaminase activity and glutamine-dependent CTP formation catalysed by R429A CTPS reveals that ammonia is produced from glutamine hydrolysis at a rate that is slightly higher than the rate at which CTP is formed This observation suggests that there may be a partial uncoupling of the glutaminase and synthase reactions, however, the kcatvalues for the corres-ponding reactions catalysed by the R429A/K432A mutant are experimentally equal
Oligomerization of CTPS
To determine if the K187A mutant was inactive because
it was unable to form tetramers, we investigated the
Table 2 Observed rate constants for inactivation of recombinant
wild-type and mutant CTP synthasesa ND, Not determined.
Protecting
ligand
Concentration (m M )
k obs (· 10)2min)1)
NH 3 as substrate
L -glutamine
as substrate
None
(R429A/K432A)
10.0 0.4 (± 0.1) 1.9 (± 0.2)
10.0 0.7 (± 0.1) 3.9 (± 0.9) ATP and UTP 10 + 10 0.15 (± 0.07) 0.8 (± 0.2)
Controlb 0 0.075 (± 0.009) 0.07 (± 0.04)
a All k obs values are for inactivation of wild-type CTPS except
where indicated otherwise.bControl refers to the inactivation of
CTPS that is observed during incubation of the enzyme at 37 C
for 1 h in the absence of added trypsin.
Trang 8Fig 5 CD analysis of wild-type and mutant CTP synthases (A) Spectra for wild-type, K187A, R429A, and R429A/K432A CTP synthases are shown Each spectrum is the average of three scans for each CTPS variant (B) The relative amount of each type of sec-ondary structure is indicated for each CTPS variant Error bars represent the standard deviation of the mean for three independent trials.
Table 3 Kinetic parameters for wild-type and mutant CTP synthases ND, not determined.
Reaction (substrate) Kinetic parameter a
CTPS variants
L -glutamate
formation (fixed
L -glutamine with
varying [GTP])
k cat (s)1) [GTP] ¼ 0.25 m M 5.01 ± 0.18 0.13 ± 0.03 0.48 ± 0.03 0.49 ± 0.07
a
Assay conditions are as described in Materials and methods [ATP] ¼ [UTP] ¼ 1 m M bNo activity was observed (i.e < 0.5% wild-type CTPS activity).cValues could not be determined accurately because of the low activity.
Trang 9ability of this mutant to form tetramers in the presence of
nucleotides using GF-HPLC The observed molecular
masses for wild-type CTPS in the absence of nucleotides
and in the presence of ATP and UTP were 123 and
251 kDa, respectively These values are similar to the
predicted values of 122 and 245 kDa, based on the amino
acid sequence of the recombinant mutant protein, and are
consistent with wild-type CTPS existing primarily as
dimers in the absence of ATP and UTP, and with a
shifting of the equilibrium to favour the tetrameric species
in the presence of ATP and UTP [42] The K187A
mutant had an apparent molecular mass of 178 kDa in
the absence of nucleotides This value is slightly higher
than that observed for the wild-type enzyme and
corres-ponds to the enzyme existing as 30% tetramer as
calculated using Eqn (3) [20], where X is the fraction of
the enzyme in the tetramer form and the molecular
masses of the dimer (121 944 Da) and the tetramer
(243 888 Da) are those predicted based on the monomer
molecular mass of 60 972 Da for recombinant K187A
lacking the histidine tag In the presence of ATP and
UTP, the observed molecular weight for K187A was
259 kDa Thus it appeared that K187A CTPS was
capable of forming tetramers in the presence of saturating
concentrations of UTP and ATP, similar to the wild-type
enzyme
molecular mass¼ð243 888Þ
2
Xþ ð121 944Þ2ð1 XÞ ð243 888ÞX þ ð121 944Þð1 XÞ ð3Þ
Discussion
Limited proteolysis has been used to delineate the structural
organization of several amidotransferases including
aspa-ragine synthase [43], carbamoyl phosphate synthase [44–50], anthranilate synthase [51], and glucosamine-6-phosphate synthase [52] This methodology has been particularly useful for identifying both ligand-binding sites and, in the case with glucosamine-6-phosphate synthase, an exposed hinge region that, when cleaved by a-chymotryp-sin, led to separation of the enzyme into its GAT and synthase domains Our interest in delineating structural aspects of E coli CTPS led us to examine the susceptibility
of CTPS to controlled proteolysis In preliminary experi-ments with endopeptidases of different specificity, we identified trypsin as the enzyme of choice Trypsin-catalysed cleavage of wild-type CTPS generated four fragments in the absence of ATP and UTP, but only two fragments in the presence of these nucleotides Determination of the N-terminal sequence of these fragments, in conjunction with the known nucleotide sequence of the E coli pyrG gene [3], permitted us to identify three principal cleavage sites: Lys187 in the synthase domain, and Arg429 and Lys432 in the GAT domain A summary of the fragmen-tation pattern arising from trypsin-catalysed cleavage at these sites is presented in Fig 2
Lys187 resides in a region of the synthase domain that is highly conserved among CTP synthases from different organisms This region, between residues 116 and 229, has been suggested to comprise the CTP/UTP-binding site [18,25–27] Our observation that both CTP and UTP afford effective protection to CTPS from trypsin-catalysed clea-vage at Lys187 also supports the notion that this residue is located in the CTP/UTP-binding site Interestingly, ATP also provides protection against cleavage and does so better than UTP Such protection could arise because: (a) ATP binds at an adjacent site and sterically blocks access of trypsin to Lys187; (b) ATP-induced tetramerization yields a quaternary structure in which the Lys187 site is not accessible to trypsin; or (c) ATP induces a conformational change in CTPS to yield a conformation in which Lys187
is no longer exposed to bulk solvent
Replacement of Lys187 by an alanine residue yielded a protein that was resistant to limited trypsin-catalysed proteolysis in the synthase domain, supporting our conclu-sion that cleavage occurred C-terminal to this residue The K187A mutant could not catalyse the formation of CTP, however, it retained the ability to form tetramers in the presence of nucleotides, and exhibited a very low level of GTP-dependent glutaminase activity which was enhanced slightly by GTP Interestingly, in the absence of nucleotides, K187A existed as 30% tetramer suggesting that neutrali-zation of positive charge at residue 187 might play a role in promoting enzyme tetramerization Indeed, hydrophobic interactions between dimers of E coli CTPS have been suggested to play a role in the formation of tetramers [53] Predictions of the secondary structure of the highly conserved region of amino acid sequence between residues
185 and 192 suggest that Lys187 constitutes part of a conserved loop It is not clear whether nucleotides protect this putative loop from proteolytic cleavage because nucleotide binding directly blocks access of trypsin to the cleavage site or, because nucleotide binding causes a change
in the enzyme’s conformation or quaternary structure (i.e tetramerization) that subsequently conceals the cleavage site from trypsin
Fig 6 Glutaminase activity for mutant CTP synthases The values of
kapparentcat for the hydrolysis of glutamine by K187A (d) and R429A (s)
CTP synthases are shown Inset: values of kcatapparentfor the hydrolysis of
glutamine by wild-type CTPS The curves shown are from a fit of the
data to Eqn (2) and the values of k o , k act , and K A are given in Table 3.
Trang 10Finally, we note that studies on the chemical modification
of E coli CTPS with thiourea dioxide led Roberston et al
[54] to conclude that lysine residues were important for
catalysis To our knowledge, the present study represents
the first identification of a catalytically essential lysine
residue in E coli CTPS involved in either amido-/NH3
transfer or UTP phosphorylation
Arg429 and Lys432 reside in a region of amino acid
sequence within the GAT domain that is partially conserved
only among CTP synthases from some sources (Fig 7) In
accord with our expectations, nucleotides offered no
protection against cleavage at these sites but replacement
of these residues by alanine (i.e R429A and R429A/K432A)
yielded mutant enzymes that were more resistant to
proteolytic cleavage in the GAT domain These mutant
enzymes displayed wild-type activity with respect to
NH3-dependent CTP formation and wild-type affinity for
glutamine, but glutamine-dependent CTP formation was
markedly impaired Interestingly, although these mutations
in the GAT domain did not impair the enzyme’s ability to
utilize NH3as a substrate (i.e the activity associated with
the synthase domain [16]), they did cause the rate of loss of
NH3-dependent activity during limited trypsin-catalysed
proteolysis to be less than would have been predicted based
on the inactivation rate constant observed for wild-type
CTPS (Table 2) This is consistent with previous reports that suggested interactions between the GAT and synthase domains within the tertiary structure of the enzyme [17,28,55] The existence of such interactions is also supported by our observation that mutation of Lys187 to alanine in the synthase domain severely impairs the glutaminase activity in the GAT domain
Our observations that R429A CTPS binds GTP with reduced affinity ( 10-fold) and, at saturating concentra-tions of GTP, the apparent kcat value for glutamine-dependent CTP formation is reduced sixfold suggest that Arg429 plays a role in both binding GTP and the mechanism for allosteric activation of glutamine hydrolysis Secondary structure predictions suggest that Arg429 and Lys432 are located within a region where a b-strand undergoes a transition into a loop structure The ability of trypsin to catalyse cleavage adjacent to these residues suggests that this loop is exposed to bulk solvent Despite the fact that this region is not highly conserved between organisms, it does appear to be required for E coli CTPS to catalyse glutamine turnover Arg429 and Lys432 lie close to
a conserved sequence motif [GG(TS)(ML)RLG] within the GAT domain (shaded residues 436–442 in Fig 7) that was recently identified by Willemoe¨s [24] Using site-directed mutagenesis experiments on CTPS from L lactis,
Fig 7 Sequence comparison of a portion of the C-terminus (GAT domain) of CTP synthases For the protein sequences shown, invariant residues (*), conservative substitutions (:), and semiconservative substitutions (.) are indicated The two residues (Arg429 and Lys432) identified as cleavage sites during limited trypsin proteolysis and mutated in the present study are indicated (›) These residues reside in a region of the primary structure that is not conserved among different organisms The conserved sequence motif (GG[TS][ML]RLG) identified by Willemoe¨s [24] is shaded The proteins included in the alignment are as follows (accession numbers in parentheses): Girardia intestinalis (AAB41453.1), Synechococcus (Q54775), Spiro-plasma citri (P52200), Synechocystis (P74208), Bacillus subtilis (P13242), Mycobacterium leprae (S72961), Mycobacterium bovis (AAB48045.1), Methanococcus jannaschii (Q58574), Chlamydia trachomatis (Q59321), Haemophilus influenzae (P44341), Neisseria meningitidis (CAB84970.1), Nitrosomonas europaea (AAC33441.1), Azospirillum brasilense (P28595), Campylobacter jejuni (CAB72520.1), Heliobacter pylori (O25116), Borrelia burgdorferi (O51522), Cricetulus griseus (P50547), Mus musculus (P70698), Homo sapiens (NP_001896.1), Arabidopsis thaliana (AAC78703.1), Saccharomyces cerevisiae H (URA-8, P38627), Saccharomyces cerevisiae G (URA-7, P28274), Plasmodium falciparum (AAC36385.1), Lactococcus lactis (CAA09021.2), and Escherichia coli (AAA69290.1) Numbering shown is for the E coli sequence.