The relative hydrolytic reactivities of pyrophosphites and pyrophosphates

Article abstract of DOI:10.1039/c3ob40755a

The pH-rate profiles for the hydrolysis of pyrophosphate (PP(v)) and pyrophosphite (PP(iii), pyro-di-H-phosphonate) are a complex function of pH, reflecting the different ionic species and their relative reactivities. PP(iii) is more reactive than PP(v) at all pHs and only PP(iii) shows a hydroxide-ion reaction at high pH, so it is 10¹⁰-fold more reactive than PP(v) in 0.1 M NaOH. The pK_a2 of PP(iii) ～0.44, so the dominant species at pH's > 1 is the di-anion PP(iii)^2-. Although there is no observable (NMR or ITC) binding of Mg²⁺ to the PP(iii) di-anion there is a modest increase in the rate of hydrolysis of PP(iii) by Mg²⁺. PP(iii) is neither a substrate nor an inhibitor of pyrophosphatase, the enzyme that efficiently catalyses the hydrolysis of PP(v).

Full text of DOI:10.1039/c3ob40755a

Organic &
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PAPER
The relative hydrolytic reactivities of pyrophosphites
and pyrophosphates†
Cite this: Org. Biomol. Chem., 2013, 11,
5727
Dharmit Mistry and Nicholas Powles*
The pH-rate profiles for the hydrolysis of pyrophosphate (PP(V)) and pyrophosphite (PP(III), pyro-
di-H-phosphonate) are a complex function of pH, reflecting the different ionic species and their relative
reactivities. PP(III) is more reactive than PP(V) at all pHs and only PP(III) shows a hydroxide-ion reaction at high
pH, so it is 1010-fold more reactive than PP(V) in 0.1 M NaOH. The pKa2 of PP(III) ∼0.44, so the dominant
species at pH’s > 1 is the di-anion PP(III)2−. Although there is no observable (NMR or ITC) binding of Mg2+
to the PP(III) di-anion there is a modest increase in the rate of hydrolysis of PP(III) by Mg2+. PP(III) is neither a
substrate nor an inhibitor of pyrophosphatase, the enzyme that efficiently catalyses the hydrolysis of PP(V).
Received 16th April 2013,
Accepted 16th July 2013
DOI: 10.1039/c3ob40755a
www.rsc.org/obc
Introduction
The reactions and stabilities of phosphate mono- and di-esters
underpin most life processes such as the storage and manifes-
tation of genetic information, energy transduction, signalling,
regulation, differentiation, compartmentalisation, substrate
modification to facilitate chemical reactions, and as structural
components.1,2 One of the amazing contrasts within this list is
the remarkable variation from extreme stability to high reacti-
vity: from half-lives of million of years for the spontaneous
hydrolyses of some phosphate esters to milli-second turnovers
of their enzyme catalysed reactions.3 A fundamental property
of phosphate mono- and di-esters is their negative charge over
the normal pH range encountered in living systems, which in
turn contributes to their versatility.1 Phosphate mono- and
di-esters show remarkable resistance to spontaneous hydrolysis
under normal physiological conditions, hence their contri-
bution to the stability of genes. By contrast enzyme-catalysed
phosphoryl group transfer reactions are highly efficient and
show some of the largest enzymatic rate enhancements4 – up
to 1020. The key to understanding enzyme catalysis is the
charge and geometric complementarity between substrate and
enzyme expressed in the transition state, so a major feature of
enzymes catalysing phosphoryl transfer is the neutralisation of
the substrate negative charge.5,6
Scheme 1
during energy metabolism is an essential part of the synthesis
of proteins, DNA and RNA.8 The group of enzymes controlling
levels of PP(V) are pyrophosphatases (PPases).9 There are two
classes of PPases which have been identified – Type I PPases
are found in eukaryotes and Escherichia coli and differ from
the Type II PPases found in some bacteria.10 These enzymes
have remarkably similar active sites but Type I PPases require
three Mg2+ ions for activity,11 whereas Type II PPases require
four Mn2+ ions.12
The P(III) pyrophosphite analogue of pyrophosphate exists
as pyro-di-H-phosphonate (PP(III))13 but has only two ionisable
protons compared with the four in PP(V) (Scheme 1). We have
been interested in H-phosphonates as phosphorylating agents,
in particular diethyl pyro-di-H-phosphonate (1).14 Pyrophos-
phite has also been suggested as a phosphorylating agent in
pre-biotic chemistry.15 Herein we report on a comparison
of the relative reactivities of the P(III) pyrophosphite (pyro-
di-H-phosphonate) and P(V) pyrophosphates towards hydrolysis
(Scheme 1) over a wide range of pH and their relative inter-
actions with PPases. The main factor controlling reactivity is
the state of ionisation of both species at different pHs.
Pyrophosphate (PP(V)) is the anhydride of two phosphate
ions (Scheme 1) and is important in intermediary metabolism
and cell growth.7 The control of intracellular levels of PP(V)
IPOS, The Page Laboratories, Department of Chemical and Biological Sciences,
The University of Huddersfield, Queensgate, Huddersfield, HD1 3DH, UK.
E-mail: n.t.powles@hud.ac.uk
†Electronic supplementary information (ESI)
available. See DOI:
10.1039/c3ob40755a
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Results and discussion
pH-rate profiles for the hydrolysis of PP(V) and PP(III)
The rates of hydrolysis of pyrophosphate and pyro-di-H-phos-
phonate were determined by 31P{H} NMR as a function of pH
at 25 °C and ionic strength I = 1.0 M and by auto-titration. The
observed first order rate constants were determined at con-
stant pH with different buffer concentrations and the buffer
independent rate constant obtained by extrapolation to zero
buffer concentration (Fig. 1).
The pKa’s of pyrophosphate are known16 but not those for
the two ionisable protons of pyro-di-H-phosphonate, which are
presumed to be low. However the 31P{H} NMR chemical shift
is unchanged from pH 2 to 13.5, suggesting that both pKa’s are
<1.0 and that the dominant species over this pH range is the
di-anion PP(III)2−. However, rapid scanning of 31P{H} NMR
spectra at different pH’s shows that the chemical shift of
Fig. 3 pH rate-profile for the hydrolysis of PP(III) (+) and PP(V) (×) at 25 °C.
PP(III)2− at δ −5.08 ppm does change below pH 2.0 (Fig. 2). pH and it is not possible to measure the chemical shift below
Unfortunately PP(III) undergoes acid catalysed hydrolysis at low pH 0.6. Nonetheless, if it is assumed that the observed data is
part of a normal sigmoidal titration, it can be fitted to a pKa =
0.44 0.1 as indicated by the solid line (Fig. 2). This value is
compatible with analogous systems, which show that P(III)
derivatives are generally more acidic than the corresponding
P(V) compound. For example, a similar 31P{H} NMR titration
with pH for ethyl H-phosphonate (3) yields a pKa = 0.47 which
may be compared with that for ethyl phosphate = 1.60.17 The
first pKa of P(III) phosphorous acid is 1.0716 and a calculated
value is quoted as 0.918 compared with 2.0 for P(V) phosphoric
acid.16 The first pKa of isohypophosphoric acid which contains
a P(III) and a P(V) phosphorous atom bridged by an oxygen has
been estimated to be 0.6.19 A value <0.5 for the pKa of PP(III) is
also consistent with the kinetic pH-rate profile reported later.
The pH-rate profile for the hydrolysis of pyrophosphate and
pyro-di-H-phosphonate is a complex function of pH (Fig. 3)
reflecting the different ionic species and their relative reactiv-
ities. PP(III) is more reactive than PP(V) at all pHs and only the
former shows a hydroxide-ion reaction at high pH. The rate of
Fig. 1 Plot of the observed first-order rate constants for the hydrolysis of PP(III)
hydrolysis of pyrophosphate PP(V) decreases with increasing
as a function of MOPS buffer concentration at pH 8.0 at I = 1.0 M (KCl) and
pH and the rate constants for the tri- and tetra-anion (Fig. 3) at
25 °C.
higher pH are calculated from a recent report.20 The results
reported here for the neutral, mono- and di-anionic forms of
PP(V) are from our observations using 31P NMR.
The observed pseudo first order rate constant for the hydro-
lysis of PP(III) shows a first order dependence on H+ concen-
tration between pH 2 and 4, where the dominant species in
solution is the di-anion. However, below pH 2 this changes to
a second order dependence on H+ concentration, best seen by
the enlarged Fig. 4 and changes in values of kobs/[H+] as a func-
tion of pH (ESI†). In addition there is a pH independent term
for hydrolysis of the PP(III) di-anion between about pH 5 and 8
followed by a rate term that is dependent on hydroxide-ion
concentration at higher pH. The observed rate law for the
hydrolysis of PP(III) is thus given by eqn (1), where k1, k2, k0
and kOH all refer to the rate constants for the hydrolysis of
PP(III) dianion and k2 is that which is first order in H+ con-
centration, k0 is the pH independent hydrolysis and kOH is the
Fig. 2 31P{H} NMR chemical shift of PP(III) against pH with external reference to
diphenyl phosphate at I = 1.0 M, 25 °C.
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Scheme 2
constant for the hydrolysis of the undissociated PP(III) is
>0.073 s−1. Presumably, the undissociated PP(III) is much more
reactive than its mono- and di-anions so that between pH 1
and 2 it is the reactive hydrolytic species even though its con-
centration is small and falls off with a second order depen-
dence on [H+]. Similarly, between about pH 2 and 5 where the
rate of hydrolysis shows a first order dependence on [H+] and
the rate law is dominated by the k2 term in eqn (1), hydrolysis
is actually occurring through the mono-anion of PP(III) even
though, again, the dominant species in solution is the di-
anion. The calculated first order rate constant for the hydro-
Fig. 4 pH rate profile enlarged from Fig. 3 to show the change from first order
dependence on H+ (dashed line is a continuation of a first order dependence on
H+) to second order dependence on H+.
Table 1 Rate constants (eqn (1)) for the hydrolysis of PP(III)2− at 25 °C
lysis of the mono-anion PP(III)− is given by k2Ka1 = 3.41 × 10−3 s−1
.
Rate constants (eqn (1))
k1
k2
k0
2.00 × 10−1 (M−2 s−1
9.40 × 10−3 (M−1 s−1
)
)
The rate constant for the spontaneous hydrolysis of the PP(III)
di-anion is 1.20 × 10−7 s−1, so the relative hydrolytic reacti-
vities of the neutral, anionic and di-anionic PP(III) are approxi-
mately: 6 × 105 : 3 × 104 : 1.0, respectively. The rates of
hydrolysis of the neutral and mono-anionic PP(III) differ by a
factor of 20 and presumably both involve nucleophilic attack
by water on the more electrophilic neutral P centre although
they require the expulsion of different leaving groups, the
phosphite mono- and di-anions, respectively (Scheme 2).
These relative reactivities are compared later with the hydro-
lysis of ethyl-H-phosphonate (3) to generate a simple linear
free-energy relationship. The much reduced activity of the di-
anion of PP(III) reflects the less favourable ease of nucleophilic
attack by water on the relatively negatively charged P centre
and expulsion of the di-anionic phosphite.
In contrast to that for the hydrolysis of PP(III), the observed
pseudo first order rate constant for the hydrolysis of PP(V) con-
tinues to decrease with increasing pH (Fig. 3) as the state of
ionisation increases and forms anions of increasingly lower
reactivity. The rate law for the hydrolysis of PP(V) is given by
eqn (2). The constants for the tri- and tetra-anions of PP(V) are
taken from the recent literature obtained by extrapolation from
determinations at elevated temperatures20 and are compatible
with other reports.21 The known pKa values (0.79 (I = 1.0 M),
1.72 (I = 1.0 M), 6.6 and 9.4) of PP(V)16 and the rate constants
obtained in this work at lower pH (Table 2) have been used to
generate the overall pH-rate-profile (Fig. 3).
1.20 × 10−7 (s−1
)
kOH
1.35 × 10−1 (M−1 s−1
)
hydroxide-ion catalysed hydrolysis of PP(III)2−. Experimentally,
there is no substantial levelling off of the observed pseudo
first-order rate constants at low pH or a reversion to a first-
order dependence on [H+] which may be expected if the PP(III)
mono-anion or undissociated acid become significant species.
This is compatible with the 31P{H} NMR data indicating a pKa
= 0.44. Consequently, it appears that the k1 term (eqn (1))
refers to a reaction involving PP(III) dianion and [H+]2 or its
kinetic equivalent.
Rate=½PPðiiiÞtotꢀ ¼ kobs
2
¼ k1½Hþꢀ þ k2½Hþꢀ þ k0 þ kOH½OHꢁꢀ
ð1Þ
The line in Fig. 3 is generated from the values of the rate
constants given in Table 1. The first two terms in the rate law,
k1 and k2 (eqn (1)) have kinetically equivalent expressions, and
so, for example, the k1 term could represent the spontaneous
hydrolysis of neutral PP(III), and the k2 term could actually
reflect either the hydrolysis of the mono-anionic species PP
(III)− or even hydroxide-ion attack upon neutral PP(III), although
the calculated rate constant for the latter k2Ka1Ka2/Kw is greater
than the diffusion controlled rate and so can be excluded.
The calculation of these kinetically equivalent mechanisms
requires a knowledge of the two pKa’s of PP(III). The estimated
value of pKa2 = 0.44 is subject to error and there is no simple
way to estimate pKa1. Nonetheless it is a worthwhile exercise to
enable a comparison with the PP(V) analogues. The third order
rate constant k1 is kinetically equivalent to the spontaneous
Rate ¼ kH½PPðvÞꢀ½Hþꢀ þ k0½PPðvÞꢀ þ k1ꢁ½PPðvÞꢁꢀ
þ k2ꢁ½PPðvÞ2ꢁꢀ þ k3ꢁ½PPðvÞ3ꢁꢀ þ k4ꢁ½PPðvÞ4ꢁꢀ
ð2Þ
There is no base-catalysed hydrolysis observed in the hydro-
hydrolysis of the undissociated acid with a first order rate con- lysis of the PP(V) anions in alkaline solutions. This is unlike
stant equal to k1Ka1Ka2. Based on the pH dependence of the the behaviour of di-anionic PP(III) which, despite its negative
31P NMR chemical shifts (Fig. 2) and the pH-rate profile charge, does undergo a hydroxide-ion catalysed hydrolysis
(Fig. 3) the product Ka1Ka2 is >10−1. The calculated rate and, consequently PP(III) is much more reactive than PP(V) at
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The Mg2+ catalysed hydrolysis of PP(V) at neutral pH is
assumed20 to be that of the tetra-anion, i.e. the overall di-
anionic MgPP2− is the reactive species with an estimated rate
constant at 25 °C of 2.8 × 10−10 s−1, three orders of magnitude
greater than that of PP(V)4−. The dissociative or associative
nature of phosphoryl transfer reactions is defined by the
extent of bond formation between phosphorus and the incom-
ing nucleophile and that between phosphorus and the leaving
group in the transition state.3 The processes of phosphoryl-
ation and dephosphorylation that occur by associative type
mechanisms usually have a penta-coordinate trigonal-bipyra-
midal geometry in the transition state, whereas in the case of
the dissociative pathway it effectively involves a trigonal planar
metaphosphate anionic species, PO3−. The 3 orders of magni-
tude rate enhancement of the hydrolysis of PP(V) by Mg2+ is
presumably due to the metal-ion binding to the tetra-anion
and so neutralising some of the negative charge and facilitat-
ing nucleophilic attack by water. However, the hydrolysis of
phosphate monoesters proceeding through the dissociative
pathway is not sensitive to the presence of Mg(II) ions in
aqueous solution.22 Although metal-ion coordination may
enhance the electrophilicity of the phosphorus it will disfavour
electron-donation from the non-bridging oxygens thus redu-
cing their ability to assist in expelling the leaving group, which
is essential in the dissociative pathway. Similarly, Mg2+ only
enhances the rate of hydrolysis of ATP4− by 3-fold and binds
preferentially between the β and γ phosphates, making the di-
anionic γ phosphate less effective at expelling the ADP leav-
ing group, but does not facilitate an alternative associative
pathway.23 An associative transition state can be sensitive to
stabilization by Mg2+ ions if coordination occurs to the leaving
group.24
Table 2 Rate constants (eqn (2)) for the hydrolysis of PP(V) at 25 °C
Rate constants (eqn (2))
kH
k0 (I = 1.0 M)
1.30 × 10−5 (M−1 s−1
)
1.00 × 10−7 (s−1
7.00 × 10−8 (s−1
3.50 × 10−8 (s−1
)
)
)
k
k
1− (I = 1.0 M)
2− (I = 1.0 M)
k3−
k4−
1.07 × 10−9 (s−1 a
)
8.59 × 10−13 (s−1 a
)
a From ref. 20.
high pH (Fig. 3) e.g. it is 1010-fold more reactive in 0.1 M
NaOH.
Of course, there are kinetically equivalent processes for the
hydrolysis of PP(V). For example, if the reported rate constant
for the spontaneous hydrolysis of PP(V) tri-anion20 actually
reflects the kinetically equivalent hydroxide-ion catalysed
hydrolysis of the di-anion, then the corresponding second-
order rate constant would be Ka3k3−/Kw = 1.7 × 10−3 M−1 s−1
which can be compared with kOH for PP(III)2− = 1.35 × 10−1 M−2 s−1
.
This 79-fold higher rate for the pyro-di-H-phosphonate
compared with pyrophosphate is of the order expected, so the
hydrolysis of the PP(V) tri-anion probably does occur through a
mechanism involving hydroxide-ion attack on the di-anion.
Mg2+ catalysed hydrolysis of PP(III)
Many enzymes that catalyse phosphate ester hydrolysis and
transesterification require one or more metal-ions as cofactors.
It is interesting to note that using 31P{H} NMR or ITC tech-
niques there is no observable binding of Mg2+ to the PP(III) di-
anion. Nonetheless, there is a modest increase in the rate of
hydrolysis of PP(III) by Mg2+ (Fig. 5) giving a second order rate
constant for this process = 2.20 × 10−5 M−1 s−1. If it is assumed
that Mg2+ catalysis is due to initial complexation of Mg2+ to
PP(III) di-anion followed by nucleophilic attack by water and
that metal-ion binding has a weak association constant of
<10−2, then the calculated rate constant for water attack on
this complex (2) is >2 × 10−3 s−1 which is 4-orders of magni-
tude greater than that for the uncomplexed PP(III) di-anion.
Hydrolysis of other P(III) derivatives
The mechanisms of hydrolysis of pyrophosphate mono- and
di-esters resemble those for phosphate monoesters,25 except
that a phosphate ester or salt is expelled rather than an
alcohol. For comparison, the hydrolytic activity of diethyl pyro-
di-H-phosphonate (1) H-phosphonate mono- and di-ethyl
esters (3 and 4) were investigated.
The rate of hydrolysis of diethyl pyro-di-H-phosphonate (1) is
too fast to measure by 31P{H} NMR and has a rate constant at
Fig. 5 Pseudo first-order rate versus MgCl2 concentration of PP(III) at fixed
buffer concentration (0.8 M), pH 7, I = 1.0 M at 25 °C.
pH 7 > 0.1 s−1
.
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Diethyl-H-phosphonate (4) undergoes hydrolysis to give
ethyl-H-phosphonate (3) and shows an acid catalysed hydro-
lysis with a second order rate constant kH+ = 1.21 × 10−4 M−1 s−1
at 25 °C (I = 1.0 M). It also undergoes a hydroxide-ion cata-
lysed hydrolysis with a second order rate constant kOH
=
81.3 M−1 s−1 at 25 °C (I = 1.0 M), obtained from observed
pseudo first-order rate constants at constant pH with differ-
ent buffer concentrations and the buffer independent rate
constant obtained by extrapolation to zero buffer concen-
tration.
Ethyl-H-phosphonate (3) is a strong acid and exists pre-
dominantly as its mono-anion above pH 1. Its hydrolysis to give
phosphorous acid and ethanol at 25 °C (I = 1.0 M) shows a
much slower hydroxide-ion catalysed reaction than the di-ester (4)
The phosphite mono-anion is a better leaving group with a
pKa of its conjugate acid being 1.1 compared with 6.6 for that
of the di-anion.16 Perhaps surprisingly, the expulsion of the
poor leaving group ethoxide fits well on the Brønsted plot,
which, although only based on three points, does cover a pKa
range of 14 to generate a Brønsted βlg = −0.27. This relatively
small value is indicative of little or no P–O bond cleavage in
the transition state with the development of only a small
change in the effective charge on oxygen, relative to that in the
reactant.26 This is consistent with rate limiting formation of a
trigonal-bipyramidal intermediate (6).
with a second order rate constant kOH = 1.55 × 10−5 M−1 s−1
.
This corresponds to a rate decrease of 5 × 106 fold due to the
additional negative charge and repulsion against the negatively
charged nucleophile.
The acid catalysed hydrolysis of the mono-anion shows a
second order rate constant kH+ = 6.90 × 10−6 M−1 s−1. The
latter reaction is kinetically equivalent to the hydrolysis of the
Pyrophosphatase catalysed hydrolysis of PP(III)
neutral (3) and if it is assumed that the pKa of (3) is ∼0, then Inorganic pyrophosphate (PP(V)) or pyrophosphite (PP(III)) may
the corresponding first order rate constant would be 6.90 × have acted as a phosphoryl group donor in primitive biological
10−6 s−1. This can then be combined with the data reported systems.15,27 In modern organisms, the enzyme-catalysed
earlier for the hydrolysis of other derivatives involving water hydrolysis of PP(V) is an important part of the metabolism to
attack on the neutral H-phosphonates expelling phosphites remove PP(V) that is generated by biosynthetic reactions.7,28
and ethanol to generate a simple Brønsted plot (Fig. 6) as a There are two classes of inorganic pyrophosphatase (PPase)
function of the pKa of the leaving group XH (5) using the data that catalyse the hydrolysis of PP(V), both of which have an
summarised in Table 3.
unusual activity dependence on metal ions but similar active
sites.10 Type I PPases (found in eukaryotes and E. coli) require
three Mg2+ ions for activity11 and differ significantly from
those of Type II PPases (found in some bacteria) which require
four Mn2+ ions.12 In both enzymes PP(V) tetra-anion is co-ordi-
nated to metal-ions so that the extremely polar active site neu-
tralizes the negative charges on substrate to facilitate
nucleophilic attack by water.29 The metal ions probably also
coordinate to the attacking water so decreasing its pKa and
providing an activated nucleophile if the Brønsted βnuc for
attack by nucleophiles is <1.0.30
It is difficult to follow the pyrophosphatase catalysed hydro-
lysis of PP(V) by 31P{H} NMR because of limited solubility of
the substrate. However, the use of isothermal titration calori-
metry (ITC) provides a suitably sensitive continuous assay.
Experiments were conducted at pH 8.4 and 7.55 as hydrolysis
of PP(V) is the slowest step at the higher pH, rather than sub-
strate binding or product release.31 ITC can be used to follow
the kinetics of an enzyme catalysed reaction because the
amount of heat released is directly proportional to the amount
of substrate that has reacted. The instrument output reflects
the energy required to balance the heat generated by the reac-
tion to maintain constant temperature (Fig. 7). The initial posi-
tive spike represents the endothermic heat of injection/
dilution. The exothermic hydrolysis reaction gives a negative
displacement from the baseline and as substrate is consumed,
the rate at which heat is generated decreases and the output
Fig. 6 Brønsted plot for the spontaneous pH independent hydrolysis of
H-phosphonate derivatives (HO)PH-X at 25 °C as a function of the pKa of the
leaving group XH.
Table 3 Summary kinetic data calculated for the spontaneous pH independent
hydrolysis of H-phosphonate derivatives (HO)PH-X at 25 °C
X
pKa XH
k0 (s−1
)
H2PO3
HPO3
EtO
1.1
6.6
15.9
0.073
3.41 × 10−3
−
6.90 × 10−6 trace returns to the baseline.
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so the dominant species at pH’s > 1 is the di-anion PP(III)2−
which shows a hydroxide-ion reaction at high pH, so it is 1010-
fold more reactive than PP(V) in 0.1 M NaOH. Although there is
no observable (NMR or ITC) binding of Mg2+ to the PP(III) di-
anion there is a modest increase in the rate of hydrolysis of
PP(III) by Mg2+. PP(III) is neither a substrate nor an inhibitor of
pyrophosphatase, the enzyme that efficiently catalyses the
hydrolysis of PP(V).
Experimental
Materials
PP(III) – samples of PP(III) were initially and kindly provided by
T. Kee (University of Leeds) and prepared by dissolving phos-
phorous acid (16.4 g) and sodium hydroxide (8.0 g) in 100 ml
water. The water was removed under reduced pressure. The
resulting mixture was heated in a tube furnace with N2 flowing
through at 200 °C for three days.
Fig. 7 ITC trace of the enzymatic hydrolysis of pyrophosphate (PP(V)) with
1 × 10−9 M E. coli PPase, 3 mM MgCl2, initiated with PP(V) to give a final concen-
tration of 4.11 × 10−5 M at 25 °C. The endothermic peak represents the heat of
dilution followed by PP(V) consumption until the heat generated with respect to
time (dQ/dt) returns to the baseline.
Diethyl pyro-di-H-phosphonate (1) and H-phosphonate
monoethyl esters (3) were prepared as previously described.14
H-phosphonate di-ethyl ester (>99%) (4) and E. coli pyro-
phosphatase (lyophilized powder ≥90%, ≥800 units per mg
protein) were purchased from Sigma.
The total heat released by hydrolysis is represented by the
well area under the dashed line and dQ/dt is the difference
between the baseline and the heat released at any time t
(Fig. 7). The concentration of PP(V) at any time t can be used to
give the Michaelis–Menten kinetic parameters kcat and Km as
previously described.20
Kinetics
Ionic strength of solutions were maintained using KCl. Reac-
tions monitored by 31P{1H} NMR at 25 °C used a 500 MHz
Bruker Avance I, and a D2O insert was used for a lock signal
and diphenylphosphate as an internal standard. Where auto-
titration was used to follow reactions these were carried out
using a Metrohm 859.
Auto-titration: a jacketed titration vessel aspirated with N2
was used to maintain a constant temperature. The vessel was
charged with 50 ml HCl or NaOH at I = 1.0 M and titrated with
an appropriate solution to maintain the pH. The titration was
initiated by addition of 1.0 mmole PP(III) to the vessel.
It is of interest to determine if PP(III) is a substrate of PPase
and, if not, whether it binds sufficiently to act as an inhibitor
of the enzyme. Using the PPase from E. coli (2 × 10−7 M) there
was no discernible rate difference from the background hydro-
lysis of PP(III) at pH 7.55 (300 mM buffer, 50 mM Mg2+, I =
1.0 M), indicating that PP(III) is not a substrate and that kcat/Km
is <6 M−1 s−1. This may be due to PP(III) showing no significant
binding to Mg2+ and/or having insufficient negative charge to
bind to the positively charged active site of PPase. To investi-
gate whether PP(III) binds sufficiently to act as an inhibitor of
PPase, the rates of the enzyme catalysed hydrolysis of PP(V)
were measured in the absence and presence of PP(III). Above
saturation, the kcat values measured for PP(V) at 25 °C at pH
8.40 and 7.55 were 208 s−1 and 82 s−1, respectively. This is in
good agreement with a value of kcat = 290 s−1 at pH 8.4 which
has been reported.31 The second order rate constant for the
enzyme catalysed hydrolysis kcat/Km = 3.68 × 107 M−1 s−1 and
2.89 × 107 M−1 s−1 again at 25 °C, pH 8.40 and 7.55, respect-
ively. These parameters decreased by less than 27% with
1 × 10−3 M PP(III) indicative that the binding of PP(III) to PPase
is weak and Ki must be >1 × 10−3 M.
Effect of Mg2+ on the hydrolysis of PP(III): samples were run
in 0.8 M MOPS buffer at pH 7 and 1.0 M ionic strength con-
taining 10% D2O for a D2O lock and 20 mM diphenylphos-
phate as an internal standard. Magnesium concentrations
were varied by the addition of 0.001 M, 0.005 M and 0.01 M
magnesium chloride (MgCl2), to 37.6 mM PP(III).
E. coli Pyrophosphatase catalysed reactions using VP- ITC:
the enzyme (1 × 10−9 M) was prepared in 20 mM HEPES buffer
pH 8.40 or 20 mM MOPS buffer pH 7.55, with 3 mM mag-
nesium chloride. 10 mM PP(V) in the same buffer, excluding
PPase and magnesium chloride was used to initiate the reac-
tion to give a final concentration of 0.0411 mM or 0.0211 mM,
I = 0.1 M at 25 °C. Both solutions were degassed for
15 minutes to prevent bubble formation during the exper-
Conclusions
The pH-rate profiles for the hydrolysis of pyrophosphate iment. The same experiment was repeated but with the
(PP(V)) and pyrophosphite (PP(III), pyro-di-H-phosphonate) are addition of 1 mM PP(III) as a potential inhibitor.
a complex function of pH. Increasing ionisation with increasing
The pKa of PP(III) was monitored by 31P{H} NMR which con-
pH steadily decreases the rate of hydrolysis of PP(V). PP(III) is tained an insert with diphenylphosphate in MOPS buffer, pH
more reactive than PP(V) at all pHs. The pKa2 of PP(III) = 0.44, 7, I = 1.0 M as an external standard.
5732 | Org. Biomol. Chem., 2013, 11, 5727–5733
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Paper
14 N. T. Powles, J. H. Atherton and M. I. Page, Org. Biomol.
Chem., 2012, 10, 5940.
Acknowledgements
We thank T. P. Kee for partly initiating this work and for pro- 15 D. E. Bryant, K. E. R. Marriott, S. A. Macgregor,
viding an initial sample of pyrophosphite and M. I. Page for
helpful discussions.
C. W. G. Fishwick, M. A. Pasek and T. P. Kee, Chem.
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