Mechanistic investigations reveal that Ap3A formation requires: a that the second step of Ap4A formation is slightly reversible, thereby leading to a modest reappearance of adenylate int
Trang 1Michael Wright, Nonlawat Boonyalai, Julian A Tanner, Alison D Hindley and Andrew D Miller Imperial College Genetic Therapies Centre, Department of Chemistry, Imperial College London, London, UK
Aminoacyl-tRNA synthetases (aaRSs) are a
heteroge-neous family of around 20 distinct enzymes involved in
maintaining the fidelity of protein synthesis through
the specific esterification of an amino acid to the 2¢- or
3¢-hydroxyl group of the 3¢-terminal adenosine of the
cognate tRNA(s) during the translation process [1]
Most prokaryotic aaRSs are usually coded for by
sin-gle, unique genes (unlike eukaryotes), but Escherichia
coli lysyl-tRNA synthetase (LysRS) is an exception in
that it exists as two distinct synthetase isoforms, LysS
and LysU These two isoforms share a high degree of sequence identity (88%) but appear to have evolved for different purposes LysS is constitutively expressed under normal growth conditions and is responsible for the normal tRNA charging activity, whereas LysU
is the product of a normally silent gene, but can be induced to a high-level expression under selected physiological conditions, including heat shock, oxi-dative stress, and anaerobiosis [2,3] LysU is a highly efficient diadenosine 5¢,5¢¢¢-P1,P4-tetraphosphate
Keywords
Ap4A; Ap3A; dinucleoside polyphosphates;
heat shock response; LysU
Correspondence
A D Miller, Imperial College Genetic
Therapies Centre, Department of Chemistry,
Flowers Building, Armstrong Road, Imperial
College London, London, SW7 2AZ, UK
Fax: +44 20 75945803
Tel: +44 20 75945773
E-mail: a.miller@imperial.ac.uk
(Received 21 March 2006, revised 31 May,
accepted 6 June 2006)
doi:10.1111/j.1742-4658.2006.05361.x
Heat shock inducible lysyl-tRNA synthetase of Escherichia coli (LysU) is known to be a highly efficient diadenosine 5¢,5¢¢¢-P1,P4-tetraphosphate (Ap4A) synthase However, we use an ion-exchange HPLC technique to demonstrate that active LysU mixtures actually have a dual catalytic activ-ity, initially producing Ap4A from ATP, before converting that tetraphos-phate to a triphostetraphos-phate LysU appears to be an effective diadenosine 5¢,5¢¢¢-P1,P3-triphosphate (Ap3A) synthase Mechanistic investigations reveal that Ap3A formation requires: (a) that the second step of Ap4A formation is slightly reversible, thereby leading to a modest reappearance
of adenylate intermediate; and (b) that phosphate is present to trap the intermediate (either as inorganic phosphate, as added ADP, or as ADP generated in situ from inorganic phosphate) Ap3A forms readily from
Ap4A in the presence of such phosphate-based adenylate traps (via a
‘reverse-trap’ mechanism) LysU is also clearly demonstrated to exist in a phosphorylated state that is more physically robust as a catalyst of Ap4A formation than the nonphosphorylated state However, phosphorylated LysU shows only marginally improved catalytic efficiency We note that
Ap3A effects have barely been studied in prokaryotic organisms By con-trast, there is a body of literature that describes Ap3A and Ap4A having substantially different functions in eukaryotic cells Our data suggest that
Ap3A and Ap4A biosynthesis could be linked together through a single prokaryotic dual ‘synthase’ enzyme Therefore, in our view there is a need for new research into the effects and impact of Ap3A alone and the intra-cellular [Ap3A]⁄ [Ap4A] ratio on prokaryotic organisms
Abbreviations
aaRS, aminoacyl-tRNA synthetase; ACPR, 2-amino-6-chloropurine ribonucleoside; AMMPR, 2-amino-6-mercapto-7-methylpurine
ribonucleoside; AMPCP, adenosine 5¢-(a,b-methylene) diphosphate; AMPPCP, adenosine 5¢-(b,c-methylene) triphosphate; Ap 3 A, diadenosine 5¢,5¢¢¢-P 1 ,P 3 -triphosphate; Ap4A, diadenosine 5¢,5¢¢¢-P 1 ,P 4 -tetraphosphate; AppCH2ppA, methylene-substituted Ap4A analogue;
HA, hydroxyapatite; MMMP, 2-methyl-6-mercapto-7-methylpurine.
Trang 2(Ap4A) synthase, and the production of Ap4A under
conditions of cellular stress appears to be a primary
function [4–7]
The synthesis of Ap4A catalysed by LysU occurs in
two steps (Scheme 1) The first (step 1) involves the
formation of a lysyl-adenylate intermediate in which
the amino acid is activated through combination with
the a-phosphate of the first nucleotide substrate ATP,
in a process involving the simultaneous displacement
of pyrophosphate In step 2, the c-phosphate of a
sec-ond nucleotide substrate ATP combines with
enzyme-bound lysyl-adenylate, thereby generating Ap4A and
liberating free l-lysine [8] Step 1 is highly specific and
conservative; ATP can only be replaced by deoxy-ATP
nucleotide substrates Step 2 is much more catholic,
and the second nucleotide substrate ATP can be
replaced by a variety of diphosphate, triphosphate or
tetraphosphate nucleotide substrates This flexibility in
step 2 means that LysU can be an efficient platform
catalyst for the synthesis of a wide variety of natural
and artificial polyphosphates [9–12]
Most previous studies of LysU catalysis have focused
on Ap4A synthase activity, but there have also been
several reports concerning the unexpected coisolation of diadenosine 5¢,5¢¢¢-P1,P3-triphosphate (Ap3A) from LysU catalysis mixtures [8,9,13,14] Since ADP is not a first nucleotide substrate for LysU (that is, LysU cannot catalyse the formation of lysyl-adenylate directly from ADP and l-lysine), this Ap3A has been assumed to be a minor side product emanating from the combination of the lysyl-adenylate intermediate with residual ADP pre-sent in ATP Therefore, we decided to determine whe-ther Ap3A was indeed a minor side product of LysU catalysis or in fact a genuine second main product alongside Ap4A At the same time, we intended to investigate the behaviour of LysU with respect to phos-phorylation, looking for potential linkages between LysU-mediated catalysis and phosphorylation state The results of our investigations are reported here
Results and Discussion
Ap4A/Ap3A synthase duality Typically, when a standard LysU catalysis mixture
is prepared (comprising LysU, excess l-lysine and
N N
N N
NH 2
O
OH HO O P O
NH2
H 2 N N
N
N
N
NH 2
O
OH
HO
O
POPOPO
O O O
H 2 N
OH
NH 2
O +
PPi, H 2 O
2.Pi
inorganic pyrophosphatase
N N
N N
NH 2
O
OH HO O P O P O P O P O
O O O O O
O
OH OH N N N
N
NH 2
Step 2
+ L-lysine
Step 1
3.Mg 2+
N N
N N
NH 2
O
OH HO O P O P O P O
O O O O
O
OH OH N N N
N
NH 2
ADP
Zn 2+
Step 3
ATP
Zn 2+
N N
N N
NH 2
O
OH HO O P O P
O O O + L -lysine
Step 4
Pi
(a)
(e)
(d)
Zn 2+
Scheme 1 LysU-catalysed Ap4A and Ap3A synthesis Both synthetic mechanisms are catalysed in the presence of Mg 2+ , Zn 2+ and inorganic pyrophosphatase The pathways share a common step 1 in the formation of a lysyl-adenylate intermediate (a) from ATP (b) and L -lysine with release of pyrophosphate (cleaved to phosphate by inorganic pyrophosphatase) Steps 2 and 3 are the combination of this intermediate and either ATP to form Ap4A (c), or ADP (if present) to form Ap3A (d), with the concurrent release of L -lysine Once the extraneous ATP (b) has been exhausted, partial reversal of step 2 results in the cleavage of Ap4A (c) back to lysyl-adenylate (a) and ATP (which in turn forms further intermediate) In this situation, step 4 allows the produced lysyl-adenylate (a) to slowly react with inorganic phosphate to give ADP (e), which then allows the formation of Ap3A (d) The overall reaction is thus: 2ATP fi Ap 4 A + 2Pi fi Ap 3 A + 3Pi.
Trang 3inorganic pyrophosphatase in a buffer containing
Mg2+ and Zn2+), added ATP (> 99%, purified by
ion exchange chromatography) will be converted into
Ap4A after 20–30 min at 37C Thereafter, significant
quantities of Ap3A will be formed at the expense of
Ap4A, such that triphosphate may easily become the
major product after 1 h (depending upon the
concen-trations of LysU and substrate involved) This
phe-nomenon was clearly observed using an assay based
on a SOURCE 15Q ion exchange column attached to
an HPLC system set up to take repeated aliquots from
enzyme incubation mixtures every 9 min, thereby
allowing for the quantitative measurement of
individ-ual nucleotide substrate and diadenosine
polyphos-phate concentrations as a function of time The results
obtained from the incubation of ATP (5 mm) with
LysU in a typical catalysis mixture are shown in
Fig 1 From multiple data sets of this kind, we
observed that the fast initial Ap4A synthesis
(170 ± 40 min)1) converted the majority (perhaps all)
of the available ATP to tetraphosphate This was
fol-lowed by a slower process of Ap4A-to-Ap3A
conver-sion (12 ± 3 min)1) with concurrent rise of ADP
levels to about 0.1 mm (initially negligible) over the
next 1.5 h This dual catalytic phenomenon runs
coun-ter to the general expectation of LysU as a primary
Ap4A synthase and deserves an explanation In the
absence of initially available ADP, it is apparent that
ADP is somehow generated in situ and then acts as an
alternative second nucleotide substrate in step 2,
com-bining with lysyl-adenylate to form Ap3A (Scheme 1,
step 3) However, what is the origin of this in situ
ADP?
Initially, we considered two possibilities: option 1,
that LysU may act as an ATP-to-ADP hydrolase; or
option 2 that LysU may be a symmetrical Ap4A hydrolase The rapidity of ATP consumption in the first stage of polyphosphate formation with no appar-ent formation of ADP appeared to rule out option 1
In fact, significant ADP was not seen in reaction mix-tures until > 30 min from the start, by which time no ATP remained (Fig 1) Instead, the kinetics of ADP appearance and Ap4A disappearance suggested that
Ap4A may be the source of ADP according to option
2 Consistent with option 2, when a mixture of ATP and adenosine 5¢-(b,c-methylene) triphosphate (AMPPCP) 1 : 1 (m⁄ m) is combined with a LysU cata-lysis mixture, AppCH2ppA (a methylene-substituted
Ap4A analogue) forms without apparent formation of
Ap4A, and subsequently Ap3A is not isolated even after 3 h at 37 C (beyond which time, LysU begins to denature) [10] Even a single bisphosphonate methylene linkage is known to confer resistance to hydrolysis [15,16], and AMPPCP is unable to function as a first nucleotide substrate for LysU (Scheme 1, step 1) It is, however, an apparently more potent second nucleotide substrate than ATP, able to trap the lysyl-adenylate intermediate in a nonreversible equivalent to step 2 (Scheme 1), and leading to the exclusive formation of AppCH2ppA [9] The subsequent failure to generate
Ap3A could then be accounted for by the inability
of AppCH2ppA to undergo symmetrical hydrolysis to form ADP, also owing to the presence of the bisphos-phonate methylene linkage
In order to obtain further evidence for a putative
‘symmetrical Ap4A hydrolase’ activity, Ap4A was incubated with LysU in a standard catalysis mixture (comprising Tris⁄ HCl buffer) minus inorganic phos-phate or any other nucleotide Against expectations,
no ADP was generated over 3 h (Fig 2A) However,
A
0 1 2 3 4 5
B
t / min
90 min
63 min
36 min
9 min
t / min
Fig 1 (A) Ion exchange HPLC measurement of mononucleotide and dinucleotide levels observed in a LysU catalysis mixture containing
5 m M ATP over 2 h Traces show A260during each separation (B) Integration and normalization of the traces to give relative concentrations for each component Compounds are identified as: n, ATP; s, Ap 4 A; d, ADP; , Ap 3 A; h, AMP Errors are ± 0.05 m M
Trang 4when a mixture of ADP and Ap4A 1 : 1 (m⁄ m) was
incubated with LysU in an identical catalysis mixture
(minus inorganic phosphate or any other nucleotide),
then Ap3A was generated rapidly with an estimated
turnover number for the conversion of Ap4A to
Ap3A of 90 ± 5 min)1 (10-fold higher than observed
previously) (Fig 2B) The turnover rate was observed
to increase further as the ADP-to-Ap4A ratio was
increased This latter result is consistent with a partial
reversibility of Ap4A formation (Scheme 1, step 2),
giving rise to an adenylate intermediate in situ that
can then be trapped by ADP to give Ap3A at the
expense of Ap4A (Scheme 1, step 3) The absence of
any observed accumulation of ATP formed from
reverse step 2 suggests that this is rapidly recombined with lysine to form an adenylate intermediate (Scheme 1, step 1) that would then be rapidly trapped
by ADP once again to give Ap3A Evidence in sup-port of this reverse-trap process was obtained by sub-stituting for AMPPCP or adenosine 5¢-(a,b-methylene) diphosphate (AMPCP) for ADP Trap products AppCH2ppA and ApCH2ppA, respectively, were generated exclusively, completely consistent with our proposed reverse-trap mechanism Hence, faced with such evidence that LysU is clearly not an Ap4A hydrolase (Fig 2A), we needed to come up with
an alternative explanation for the source of ADP generated in situ
0 1 2 3 4 5
t/min
0
1
2
3
4
5
t/min
0
1
2
3
4
5
t/min
0 1 2 3 4 5
t/min
Fig 2 Evidence for LysU catalytic duality (A) A LysU catalysis mixture containing 5 m M Ap4A shows no significant conversion to Ap3A nor hydrolysis to ADP over 2 h A constant AMP background is seen due to trace contaminants in the Ap4A stock, but no ADP or Ap3A is pro-duced (B) A catalysis mixture containing 5 m M Ap 4 A and 5 m M ADP shows rapid loss of ADP and Ap 4 A (fitting exponential decay curves), and concurrent synthesis of Ap3A The turnover of ADP is about twice that of Ap4A, which matches the Ap4A + 2.ADP fi 2.Ap 3 A + 2.Pi stoichiometry (C) A LysU catalysis mixture containing 5 m M Ap4A and made up in 50 m M potassium phosphate buffer, pH 7.8 Apparent phosphate attack on lysyl-adenylate results in greatly increased turnover of Ap 4 A to ADP and Ap 3 A Longer incubation times (> 2 h) show that the conversion of Ap4A to Ap3A continues, while the concentration of ADP stabilizes at approximately 1 m M (D) A LysU catalysis mix-ture containing 5 m M ATP and made up in 50 m M potassium phosphate buffer The presence of inorganic phosphate disrupts the formation
of Ap 4 A, and the major product under these conditions is ADP Compounds are identified as: n, ATP; s, Ap 4 A; d, ADP; , Ap 3 A; h, AMP Errors are ± 0.05 m M
Trang 5Returning to the original Ap4A synthesis scenario
(Fig 1), we came to the realization that there could be
an alternative option (option 3) During the initial
synthesis of Ap4A, the main byproduct of step 1 is
inorganic phosphate formed through cleavage of
pyro-phosphate by the action of an inorganic
pyrophospha-tase enzyme (contained in the LysU catalysis mixture)
Therefore, we considered that this inorganic phosphate
may also be capable of trapping residual adenylate
intermediate arising from the partial reversibility of
Ap4A formation (Scheme 1, step 4), thereby generating
ADP in situ An equivalent process to this (albeit much
slower) has been remarked upon in studies on E coli
glycyl-tRNA synthetase [17] Thereafter, ADP thus
formed in situ would be in a position either to trap
further adenylate deriving from the partial reversibility
of Ap4A formation or to combine with adenylate
gen-erated in the normal way from ATP, thereby forming
Ap3A in either case All the proposed mechanistic
steps are summarized in Scheme 1 Given this, when
ATP is incubated in a standard LysU catalysis mixture
(Fig 1), then the observed variations in concentrations
of mononucleotide and dinucleotide species with time
can be accounted for in the following way Early ATP
consumption and Ap4A formation are rapid, consistent
with an initial process in which step 1 is committed
and step 2 is kinetically very favourable Thereafter,
the partial reversibility of step 2 allows the reverse-trap
mechanism to give ADP (from inorganic phosphate)
and then Ap3A (from ADP; Scheme 1) The
appear-ance of the lysyl-adenylate intermediate would appear
to be rate-limiting (at least in part) for the formation
of both ADP and Ap3A, although the former
clearly must convert readily into the latter (Scheme 1,
step 3) at the expense of Ap4A, keeping the overall
solution concentration of ADP at a minimum while
the concentration of end-product Ap3A begins to rise
steadily
Evidence in support of our proposed mechanism
and the adenylate-trapping function of inorganic
phos-phate was obtained in the following way Ap4A was
incubated with LysU in a catalysis mixture containing
50 mm potassium phosphate but minus any
nucleo-tides In this instance, > 20% of Ap4A was observed
to convert to Ap3A over 2 h This contrasts with the
previous situation where Ap4A alone incubated in a
LysU catalysis mixture (Tris⁄ HCl buffer, no inorganic
phosphate) failed to convert into Ap3A over a 3-h
per-iod (compare Fig 2C with Fig 2A) Furthermore,
when ATP alone was incubated with LysU in a
cata-lysis mixture comprising 50 mm phosphate buffer,
then Ap3A⁄ Ap4A synthase activities as a whole were
adversely affected, with fully 30% of the initial ATP
being converted to ADP via attack of inorganic phos-phate on the adenylate intermediate (compare Fig 2D with Fig 1B) Curiously, unlike ADP and inorganic phosphate, AMP did not appear to be able to trap adenylate The reasons for this are unclear but are likely to be due to LysU active site topographies and unfavourable steric contacts [18] Finally, the summar-ized mechanistic steps (Scheme 1) can be used to pro-vide an alternative explanation for our original observation that AppCH2ppA is the exclusive product when ATP and AMPPCP 1 : 1 (m⁄ m) are combined with a LysU catalysis mixture As stated above, ATP
is the only possible first nucleotide substrate of LysU, whereas both ATP and AMPPCP could be second nucleotide substrates [9,10] Given the situation in which the formation of AppCH2ppA is essentially irreversible but the formation of Ap4A is partially reversible (as described above), the final outcome is consistent with the formation of only transient Ap4A, since adenylate intermediate regeneration at its expense would then be trapped by excess remaining AMPPCP Therefore, our data describing the exclusive formation
of AppCH2ppA can be seen as a simple variation of our newly proposed reverse-trap mechanism for the
Ap3A synthase activity of LysU
Phosphorylation state duality Previous purification protocols for LysS and LysU have described the use of hydroxyapatite (HA) med-ium columns to subfractionate samples of LysU and LysS into different charged ‘isoforms’ [4,5,19] HA makes this possible, as it is a crystalline form of cal-cium phosphate that interacts differently with globular proteins depending upon their charge [20] In our hands, LysU (previously purified by S300 and Q-Seph-arose chromatography [14]) could be easily resolved into two main subfractions by elution through an HA column, the first subfraction eluting at 20% and the second at 30% from a linear gradient of potassium phosphate (10–300 mm) (Fig 3) Both subfractions were confirmed to be greater than 95% pure LysU by SDS⁄ PAGE However, since HA resolves proteins by charge, we concluded that these two different subfrac-tions should contain LysU isoforms of different overall charge at neutral pH
Differences in protein phosphorylation state were considered the most likely explanation for the emer-gence of these two different LysU isoforms Therefore, separate aliquots of each subfraction were analysed for phosphate by means of the malachite green assay [21] calibrated against a standard curve generated with known concentrations of inorganic phosphate The
Trang 6results clearly suggest that the 20% subfraction was
unphosphorylated whereas the 30% subfraction
contained LysU phosphorylated at the level of a single
phosphate per monomer (Fig 4), in line with our
explanation Furthermore, western blot analyses of the
two subfractions using a phosphothreonine antibody
confirmed that not only was the 30% subfraction
phosphorylated, but also was the position of
phos-phorylation on the hydroxyl group of a threonine
residue (Fig 4) LysU has a number of
surface-access-ible threonine residues according to the X-ray crystal
structure [2] Therefore, enzyme digestion and
frag-ment MS is likely to be the most effective way to
deter-mine precisely which threonine residue is involved
Phosphorylated forms of aminoacyl-tRNA
synthe-tase enzymes are known For example, phosphorylated
forms of eukaryotic lysyl-tRNA from rat liver have been characterized [22,23], as have phosphorylated forms of other aminoacyl-tRNA synthases (multiply phosphorylated on the serine amino acid residue) from rabbit reticulocytes [24] The role of these phosphory-lation events has not been explored to any great extent, although nonphosphorylated yeast LysRS is known to be more specific for lysine than its threon-ine-phosphorylated equivalent [22] Furthermore, with regards to Ap4A synthesis, phosphorylation of rabbit reticulocyte SerRS and ThrRS has been shown to enhance the catalytic rates of Ap4A synthesis rates by two-fold and six-fold, respectively [25] Consequently,
we elected to characterize the 20% and 30% subfrac-tions of LysU in order to determine if there were any major differences In particular, in view of the dual catalytic behaviour of LysU described here, we were curious to characterize the effects or otherwise of phosphorylation upon the catalytic rates for Ap4A and
Ap3A synthesis A variety of catalysis characterization techniques were employed to identify differences A radioactive assay using [14C]ATP was used to accurately measure total polyphosphate synthesis over a fixed per-iod, allowing for the calculation of average turnover under kcatconditions (moles Ap3 ⁄ 4A per min per mole LysU at 37C) Typical results were 134 ± 10 min)1 (20% subfraction) and 150 ± 12 min)1 (30% subfrac-tion), suggesting little difference between
phosphorylat-ed LysU and the nonphosphorylatphosphorylat-ed isoform within experimental error On the other hand, phosphorylated LysU was able to retain activity (70 ± 5% activity
20%
30%
20%
21.5 kDa
45.0 kDa
66.2 kDa
97.4 kDa
30%
0.0 0.5 1.0 1.5
30%
20%
A 81
C
Fig 4 LysU phosphorylation (A) SDS ⁄ PAGE of 20% and 30% subfractions (see Fig 3), both showing clean bands matching the expected mass for LysU monomer (B) Calibration of Malachite green assay (C) Anti-phosphothreonine western blot of 20% and 30% subfractions, with only the 30% subfraction showing positive results.
0 50 100 150 200 250 300 350
0
10
20
30
40
50
A280
Elution volume / ml
0.0 0.2 0.4 0.6 0.8
1.0 20%
30%
Fig 3 Hydroxyapatite (HA) chromatography The HA column was
loaded with LysU and then eluted with a stepwise gradient of
potassium phosphate buffer, pH 6.5 (10–300 m M ); fractions were
analysed for absorbance at 280 nm Illustration of the elution profile
monitored by absorbance showing two main subfractions of LysU
eluting at 20% and 30% gradient.
Trang 7after 7 days) for significantly longer after storage at
4C than unphosphorylated LysU (30 ± 5% activity
after 7 days), suggesting that phosphorylated LysU is
significantly more stable than nonphosphorylated
LysU
A 1H-NMR spectroscopy assay was then used to
characterize the conversion of ATP to Ap4A by the 20%
and 30% subfractions of LysU, respectively For each
individual NMR assay, conversion of ATP into Ap4A
can be observed with time in an NMR tube by
monitor-ing the disappearance as a function of time of the H2
and H8 proton signals of adenine (of ATP) matched by
the appearance of the H2 and H8 proton signals of
adenine (of Ap4A) Ap4A adenine signals are shifted
slightly upfield due to the p-stacking of adenine rings in
Ap4A (not observed with ATP) [26] Assuming
steady-state conditions, initial rates of catalysis of Ap4A
forma-tion may be calculated from H2 and H8 proton peak
integrations, and used to determine the main kinetic
steady-state parameters for ATP and l-lysine [14,27]
The effects of [ATP] and [l-lysine] on Ap4A formation
rates were determined by taking one substrate in excess
and varying the concentration of the other (excess
con-centrations estimated from previous results as 10 mm
ATP and 2 mm lysine), keeping the concentrations of
20% or 30% subfractions of LysU constant (400 nm,
dimer concentration) (Table 1) Unsurprisingly the kcat
constants of the two LysU isoforms were found to be
essentially identical within experimental error, although
the specificity constant (kcat⁄ KM) of phosphorylated
LysU for l-lysine appears to be 2–3-fold higher than
that of nonphosphorylated LysU The reason appears
to be that phosphorylated LysU has both a
margin-ally higher kcat for l-lysine and a lower value of KM,
indicative of a situation wherein LysU phosphorylation
has enabled l-lysine to be an improved substrate under
these given assay conditions
Next, a colorimetric-coupled assay was used to
char-acterize the formation of the lysyl-adenylate
intermedi-ate by the 20% and 30% subfractions of LysU,
respectively In each individual assay, the formation
of inorganic phosphate (produced by the action of inorganic pyrophosphatase following lysyl-adenylate formation) was coupled with the release of a UV-active dye 2-methyl-6-mercapto-7-methylpurine (MMMP) [28,29] from a chromogenic substrate 2-amino-6-mercapto-7-methylpurine ribonucleoside (AMMPR) The coupling enzyme purine nucleoside phosphorylase catalyzes the combination of inorganic phosphate with AMMPR, leading to MMMP release This coupled reaction must occur faster than the formation of lysyl-adenylate Accordingly, only a low fixed LysU concen-tration (20 nm dimer concenconcen-tration) was used per assay, together with a correspondingly high excess of ATP or l-lysine and a low incubation temperature of
20C (which significantly slows the catalysis of Ap4A synthesis) The concentrations of AMMPR, purine nucleoside phosphorylase and inorganic pyrophospha-tase required were determined by experiment, and the assay was calibrated against known concentrations of potassium phosphate The kinetic parameters deter-mined are shown (Table 2) Since this coupled assay monitors specifically the formation of lysyl-adenylate alone and not Ap4A synthesis, these parameters can be attributed solely to the binding and chemical equilib-rium of step 1 (Scheme 1) alone Once again, values of
kcat were found to be essentially identical within experimental error, but the value of the specificity con-stant (kcat⁄ KM) for ATP of phosphorylated LysU was between one- and two-fold higher than that of non-phosphorylated LysU The reason for this is that phos-phorylated LysU has a lower value of KM for ATP, indicative of a situation wherein phosphorylation of LysU has enabled ATP to be a mildly improved substrate for LysU-mediated catalysis under these assay conditions Finally, a complete repetition of the SOURCE 15Q ion exchange chromatography assays (as described earlier) suggested that the rates of forma-tion of Ap3A from Ap4A were unaffected by the state
of LysU phosphorylation (results not shown)
Table 1 Kinetic constants derived from 1H-NMR Ap 4 A synthesis
assays of 20% and 30% subfractions.
KM
kcat (s)1)
kcat⁄ K M
(s)1Æm M )1)
LysU 20% subfraction
For lysine 23 ± 12 l M 1.8 ± 0.1 78 ± 40
LysU 30% subfraction
For lysine 10 ± 5 l M 2.9 ± 0.2 290 ± 120
Table 2 Kinetic constants derived from lysyl-adenylate, enzyme-coupled assays of 20% and 30% subfractions.
K M k cat (s)1) k cat ⁄ K M
LysU 20% subfraction For ATP 9.6 ± 3 m M 16.0 ± 2.5 1.7 ± 0.8 s)1Æm M )1
For lysine 0.27 ± 0.14 l M 8.6 ± 1.1 32 ± 20 s)1Æl M )1
LysU 30% subfraction For ATP 4.0 ± 1.2 m M 12.2 ± 1.5 3.0 ± 1.3 s)1Æm M )1
For lysine 0.34 ± 0.11 l M 10.1 ± 0.7 30 ± 11 s)1Æl M )1
Trang 8Roles of dualities
We have demonstrated that dimeric LysU has dual
Ap4A and consecutive Ap3A synthase activities, and
that it exists in either a phosphorylated or
nonphos-phorylated state The phosnonphos-phorylated LysU appears to
be a more robust enzyme than nonphosphorylated
LysU, although in catalytic terms they do not appear
to have substantially different activities This suggests
that modifying LysU-catalysed Ap4A or Ap3A
forma-tion is probably not the main funcforma-tion of
phosphoryla-tion Nevertheless, in our view, the Ap4A⁄ Ap3A
synthase and phosphorylation state dualities of LysU
do appear to be linked, albeit not strongly The Ap3A
synthase activities of LysU appear to be the product
of a number of possible contributory mechanisms
Both ADP and inorganic phosphate are present in
cells, so Ap3A could be formed directly from ATP and
ADP (where ADP replaces ATP as the second
nucleo-tide substrate in Scheme 1, step 2), or from Ap4A by
the illustrated reverse-trap mechanism (Scheme 1,
reverse step 2, then steps 3 and 4) However, there is
insufficient information in the current literature to
make sense of this catalytic behaviour In eukaryotic
cells, there seems to be a general tendency for Ap4A
and Ap3A to have antagonistic effects in vivo In
par-ticular, the relative concentrations of these
polyphos-phates appear to be indicative of cellular status, with
human cultured cells showing high Ap4A⁄ Ap3A ratios
when undergoing induced apoptosis and the reverse
during differentiation [30,31] Ap4A 10 lm was also
shown to be sufficient to trigger apoptosis in a variety
of human and mouse cell lines, a concentration not
significantly higher than that seen in human adrenal
vein blood serum [32]
Similarly, Ap3A has been reported to act as a
coin-ducer of cell differentiation when combined with protein
kinase C activators This antagonistic behaviour has
also been seen in a number of other studies For
exam-ple, submicromolar concentrations of Ap3A have been
reported to induce platelet aggregation, in contrast to
Ap4A which causes disaggregation [33], and these
com-pounds have been shown to have opposing effects on
rabbit interocular pressure as well [34] Regrettably,
there has been no evidence produced to date to suggest
that Ap4A and Ap3A should have antagonistic effects
in vivoin prokaryotes as well as in eukaryotes
There-fore, although Ap4A may appear to act as an immediate
modulator of stress responses in prokaryotes [30,35,36],
analogous links between Ap3A and longer-term
prok-aryotic stress accommodation, or high concentrations of
Ap4A and failure of the stress response, must remains
hypothetical Our data presented here suggest that
Ap3A and Ap4A biosynthesis are linked together through a single prokaryotic dual ‘synthase’ enzyme Therefore, in our view, there is now an urgent need for new research into the effects and impact of Ap3A alone and the intracellular [Ap3A]⁄ [Ap4A] ratio on prokaryo-tes in order to make sense of the dual enzymology of LysU in a fuller, more complete biological context
Conclusion
LysU has dual Ap3A⁄ Ap4A synthase activities and dual phosphorylation state behaviour These dualities are linked, but only weakly There is insufficient know-ledge about the role of Ap3A in biological prokaryotic systems to understand the implications of these duali-ties More research into the function of Ap3A in prok-aryotes is necessary
Experimental procedures
General
LysU enzyme was overexpressed and purified according to previously published protocols [18] All LysU concentra-tions are of the dimer LysU concentration was determined from A280 measurement using an extinction coefficient of
30 580 m)1Æcm)1 [37] All compounds used were obtained from Sigma Aldrich (St Louis, MO) unless otherwise stated
Ion exchange HPLC assays
A custom 2 mL SOURCE 15Q packed ion exchange col-umn (Amersham Biosciences HR 5⁄ 10; Piscataway, NJ) was attached to an Agilent 1100 series HPLC column (Agi-lent, Palo Alto, CA), equipped with an autosampler, a ther-mostatted sample chamber (37C) and a UV absorbance detector set at 260 nm The column was loaded (injection aliquots 10 lL) in 5 mm Tris⁄ HCl buffer, pH 8.0, and
elut-ed with a gradient of NaCl (0–45%) over 5 min The stand-ard LysU catalysis mixture (500 lL) comprised 50 mm Tris⁄ HCl, pH 7.8, 10 mm MgCl2, 160 lm ZnCl2, 5 mm nu-cleotides, 2 mm l-lysine, 5 units of inorganic pyrophospha-tase (Roche, Laval, Canada) and 1 lm LysU The contents
of fractions postelution were identified by their retention time compared with standards (AMP 3.85 min, AMPPCP 4.39 min, ADP 4.43 min, ATP 4.72 min, Ap4A 5.13 min) Mononucleotide and dinucleotide concentrations were calculated from peak area, assuming 260 nm extinction coefficients to be roughly equivalent for AMP through ATP and double for Ap3A and Ap4A Ap3A was synthes-ized by incubating 5 mm ATP in a standard LysU mixture overnight, and then extracted from the mixture using a SOURCE 15Q ion exchange column (50 mL) (Amersham Biosciences), loaded in water and eluted with a gradient of
Trang 92 m triethylammonium hydrogencarbonate buffer (0–70%)
[17] Appropriate fractions were combined and freeze dried
Contents were characterized by ESI-MS (Bruker Esquire
3000, negative ionization; Billerica, MA), giving a PMI of
754.9 m⁄ z, and ion exchange HPLC, giving a single peak
at 4.71 min retention time
HA column
An HA biogel column (3· 20 cm) was mounted on an
FPLC (Amersham Biosciences, Piscataway, NJ) system and
equilibrated with 10 mm potassium phosphate, pH 6.5,
containing 2 mm b-mercaptoethanol, at a flow rate of
1.5 mLÆmin)1 LysU (3 mg) was dialysed to phosphate
buf-fer and loaded onto the column The column was eluted at
1.5 mLÆmin)1 with a gradient (10–300 mm) of potassium
dihydrogen phosphate, holding at 10%, 20% and 30% to
collect fractions (detected by A280 absorbance) The first
10% peak was discarded and the remainder were
concen-trated separately using a stirred cell with a 30 kDa
molecu-lar weight cutoff filter, before being stored at 4C in 20%
glycerol
Malachite green assay
The reagent was made up from one part 0.2% Malachite
green in 1 m HCl to five parts 1 m HCl, and two parts
10% ammonium molybdate in 3 m HCl The reagent was
centrifuged to clear precipitate, and an aliquot (100 lL)
was added to an aliquot of LysU (300 lL in water) A810
was measured after 5 min Equal amounts of protein
solu-tion and 10% Mg(NO3)2 in 95% ethanol were combined
and then vigorously dehydrated in a glass flask over a
strong flame The ‘ash’ was redissolved in 0.5 m HCl prior
to assay [38]
Anti-threonine western blot
SDS⁄ PAGE gels of the two LysU fractions (20% and
30%) were electroblotted onto nitrocellulose membranes at
25 V, 0.2 mA for 2 h [15], in 20 mm Tris⁄ HCl, pH 8.0,
con-taining 152 mm glycine and 20% methanol Nonspecific
binding was blocked with 10% free BSA in TBS (20 mm
Tris⁄ HCl, 137 mm NaCl), pH 8.2, 0.05% Tween-20 for 1 h
at room temperature Primary antibodies (mouse
monoclo-nal IgG2b anti-phosphothreonine, Sigma-Aldrich) were
added at 1 : 1000 dilution, and the whole mixture was
incu-bated for 12 h at 4C with gentle rocking The membrane
was then washed with TBS (20 mL), pH 8.2, Tween-20
0.05%; 5· 10 min washes were performed at room
tem-perature Thereafter, the secondary antibody (goat
anti-mouse IgG conjugated to horse radish peroxidase; Santa
Cruz Biotechnology, Santa Cruz, CA) was added at
1 : 5000 dilution and the whole mixture was incubated for
1 h Finally, the membrane was washed again and then incubated with 2· 5 mL chemiluminescence reagents (Santa Cruz) for 1 min before wrapping in clear film and exposing to photographic film
Radioactive [14C]ATP turnover assay
Initially, a LysU reaction mixture of 20 mm Hepes, pH 7.8, was prepared, comprising 150 mm KCl, 25 mm ATP,
25 mm MgCl2, 0.5 mm l-lysine, and 150 lm ZnCl2 Reaction buffer (50 lL) was added to an Eppendorf tube (1.5 mL), followed by [14C]ATP (2.5 lL; 0.125 lCi), 0.25 lg of inorganic pyrophosphatase and a concentrated aliquot of LysU (5 lL) This LysU catalysis mixture was then incubated at 37C for 40 min and then briefly boiled
to denature the enzymes After this, 10 units of alkaline phosphatase was added to degrade remaining nucleotides and the mixture was incubated at 37C for a further 2 h
An aliquot (40 lL) of the mixture was then washed through three-ply DE-81 filters (ion exchange paper) with 5· 1 mL
of fresh 25 mm ammonium bicarbonate The filters were then transferred to 5 mL of scintillation fluid [Brady’s fluid: 10% (v⁄ v) methanol, 6% (w ⁄ v) naphthalene, 2% (v ⁄ v) ethylene glycol, 0.4% (w⁄ v) diphenyloxazole in 1,4-dioxane] and shaken vigorously for 20 min The average LysU turn-over was calculated from the scintillation count (minus background) as moles Ap4A per min per mole of LysU at
37C
1H-NMR assays
Individual NMR assays were preformed at 37C in a 5 mm NMR tube using a 400 MHz Bruker NMR spectrometer set
up with spectra taken every 2 min over a period of 20 min Each spectrum was a compilation of 64 scans acquired over
a period of 1 min Initially, LysU reaction mixtures of
20 mm Hepes, pH 7.8, 150 mm KCl, 5 mm MgCl2, 150 lm ZnCl2, 1–50 mm ATP, 0.05–5 mm l-lysine, 0.03 mg inor-ganic pyrophosphatase and 20% D2O were prepared For each NMR assay, an aliquot (600 lL) of an appropriate LysU reaction mixture was equilibrated at 37C in the NMR tube, after which a LysU aliquot (400 nm) was intro-duced by needle and spectral acquisition was begun ATP
1
H-NMR signals were seen initially at: 8.55 p.p.m (H2) and 8.29 p.p.m (H8), and this was followed by the emergence of
Ap4A signals with time at: 8.40 p.p.m (H2) and 8.18 p.p.m (H8) The H8 signals were seen to be the most reliable indi-cator of relative concentrations; therefore, the initial rate of conversion of ATP into Ap4A observed in each given NMR assay was determined from H8 signal integrations Initial rate data were then processed by standard Michaelis–Men-ten data-fitting to give values of KMand kcat for ATP con-version into Ap4A
Trang 10AMMPR-coupled assay
This assay was performed on a thermostatted Ultraspec III
(Amersham Biosciences) set to detect A360 at 20C from
a 0.7 mL quartz cuvette This assay uses AMMPR, which
was prepared in a three-step, one-pot reaction from the
reagent 2-amino-6-chloropurine ribonucleoside (ACPR,
1 g) ACPR was dissolved in distilled dimethyl formamide
(4 mL) and the solution was then placed in a stirred flask
(50 mL) under nitrogen Methyl iodide (2 mL) was added,
and the mixture was stirred for 20 h at room temperature
before thiourea (1 g) was added The mixture was stirred
for a further 30 min, after which 2 m ammonia in
meth-anol was added dropwise until the mixture reached
neut-ral pH Finally, the mixture was poured into stirred
acetone (200 mL), causing AMMPR to precipitate out of
solution The pale yellow solid was filtered and dried
under nitrogen before storage at ) 20 C The yield was
64%, and spectral characterization matched the literature
[16]
Purine nucleotide phosphorylase enzyme (Sigma-Aldrich)
was repurified before use by means of a Mono-Q column
(1· 10 cm) packed in 50 mm Tris ⁄ HCl, pH 7.6, and eluted
with a gradient of NaCl (0–1.5 m) at a flow rate of 3 mLÆ
min)1 Individual reactions were tested for activity by
incu-bation with 5 mm potassium phosphate and 150 lm
AMMPR Active fractions were pooled, concentrated using
3 kDa cutoff centricon concentrators and dialysed into
20 mm Hepes, pH 7.8, before storage as a precipitate in
3.2 m ammonium sulphate at 4C
Initially for the AMMPR assay, aliquots (500 lL each)
of a LysU reaction mixture comprising 20 mm Hepes,
pH 7.8, 150 mm KCl, 25 mm MgCl2, 150 lm ZnCl2,
0.25 lg of inorganic pyrophosphatase and 0.03 mg of
purine nucleotide phosphatase, were prepared Each
aliquot was then used in two sets of experiments, one set
with l-lysine (1–200 lm) and fixed ATP (25 mm), and the
other with ATP (0.1–20 mm) and fixed l-lysine (0.5 mm)
For each assay, the LysU reaction mixture with added
l-lysine and ATP was transferred to the cuvette and
equilibrated at 20C for 2 min, after which AMMPR
(400 lm final concentration) and LysU (20 nm final
con-centration) were added, the mixture was vigorously
agita-ted, and A360 was followed as a function of time After
background correction, these absorbance data were
con-verted into initial rate data These initial rate data were
then processed by standard Michaelis–Menten data-fitting
to give values of KM and kcat for ATP and lysine
conver-sion into lysyl-adenylate
Acknowledgements
MW would like to thank IC-Vec, and NB would like
to thank the Royal Thai Government for personal
sup-port We would also like to thank IC-Vec and the Mit-subishi Chemical Corporation for their support of the Imperial College Genetic Therapies Centre
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