Substrate inhibition Figure 1 shows the kcat value taken from the initial velocity of the reaction as a function of pyruvate concentration and near-saturating levels of NADH kcatis used
Trang 1enzyme from Plasmodium falciparum
Deborah K Shoemark1, Matthew J Cliff2, Richard B Sessions1and Anthony R Clarke1
1 Department of Biochemistry, University of Bristol, UK
2 Molecular Biology and Biotechnology Department, University of Sheffield, UK
The lactate dehydrogenase enzyme from the parasite
causing cerebral malaria, Plasmodium falciparum, is
currently the subject of efforts to find alternatives to
established drug regimens which suffer increasingly
from problems of resistance and side-effects [1] This
enzyme (PfLDH) catalyses the final step in the
glyco-lytic pathway upon which the parasite relies during its
anaerobic erythrocytic stages of development within
the human host The natural product gossypol, derived
from the cotton seed plant, is a known inhibitor of
dehydrogenases Inhibition of PfLDH by gossypol
derivatives has proved parasiticidal in vitro [2] and the
search for specific inhibitors is underway [1,3] The
enzyme PfLDH differs from the human isozymes in
several important structural and kinetic features,
among which is the possession of a five-residue
inser-tion in the substrate-specificity loop [4]
The fact that the enzyme has active-site properties
that differ substantially from those of human LDHs
implies that it might be possible to design selective inhibitors that could preferentially target the parasitic enzyme However, it is a considerable advantage in the selective targeting of an enzyme to have a firm grounding in both the structural and functional char-acteristics, the latter being useful in providing a basis for quantifying the effects of inhibitors on the enzyme To elucidate its functional characteristics, we have performed a mechanistic analysis using steady-state kinetics, equilibrium binding measurements and transient kinetic techniques It has been hitherto assumed that PfLDH follows the same kinetic mech-anism as other LDHs In these experiments, we define the steady-state kinetic mechanism and associ-ated rate constants in the forward and reverse direc-tions, the coenzyme binding affinities and the nature
of the rate-limiting step In addition, the effect of the unusual loop structure on substrate specificity is examined
Keywords
kinetic; lactate dehydrogenase; malaria;
mechanism; Plasmodium falciparum
Correspondence
D K Shoemark, Department of
Biochemistry, School of Medical Sciences,
University Walk, Clifton, Bristol BS8 1TD,
UK
Fax: +44 117 9288274
Tel: +44 117 9288595
E-mail: deb.shoemark@bris.ac.uk
(Received 24 January 2007, revised 13
March 2007, accepted 23 March 2007)
doi:10.1111/j.1742-4658.2007.05808.x
The lactate dehydrogenase enzyme from Plasmodium falciparum (PfLDH)
is a target for antimalarial compounds owing to structural and functional differences from the human isozymes The plasmodial enzyme possesses a five-residue insertion in the substrate-specificity loop and exhibits less marked substrate inhibition than its mammalian counterparts Here we provide a comprehensive kinetic analysis of the enzyme by steady-state and transient kinetic methods The mechanism deduced by product inhibition studies proves that PfLDH shares a common mechanism with the human LDHs, that of an ordered sequential bireactant system with coenzyme binding first Transient kinetic analysis reveals that the major rate-limiting step is the closure of the substrate-specificity loop prior to hydride transfer,
in line with other LDHs The five-residue insertion in this loop markedly increases substrate specificity compared with the human muscle and heart isoforms
Abbreviations
BsLDH, LDH enzyme from Bacillus stearothermophilus; FRET, fluorescence resonant energy transfer; LDH, lactate dehydrogenase; PfLDH, LDH enzyme from Plasmodium falciparum; TgLDH, LDH enzyme from Toxoplasma gondii.
Trang 2Substrate inhibition
Figure 1 shows the kcat value taken from the initial
velocity of the reaction as a function of pyruvate
concentration and near-saturating levels of NADH
(kcatis used as it is independent of enzyme
concentra-tion) The fact that there is a reduction in velocity at
high concentrations of pyruvate shows that the
enzyme, in common with most lactate and malate
de-hydrogenases, is prone to substrate inhibition,
although the magnitude of the effect is small The
data were fitted to Equation 1 (Experimental
pro-cedures) and reveal an inhibition constant (Ki) of
140 ± 18 mm, an apparent KM for pyruvate of
69 ± 4 lm and a catalytic rate constant of 96 s)1
This value of Ki is high compared with that for
human muscle LDH (4 mm) and the human heart
enzyme (0.8 mm) [5]
Product inhibition and binding order in the enzyme mechanism: determining the overall steady-state mechanism
An extensive steady-state analysis of the PfLDH reac-tion was performed to determine the basic mechanism, the catalytic rate constants for the forward and reverse reactions and KM values for pyruvate⁄ lactate and NADH⁄ NAD+, respectively Initially, a series of diag-nostic steady-state experiments were designed to assign the general kinetic mechanism In these enzyme assays, NADH and pyruvate were used as the substrates and NAD+or lactate as product inhibitors These studies were performed to test whether PfLDH has the char-acteristic mechanism for this class of dehydrogenases, i.e an ordered sequential binding system with NADH binding before pyruvate The manner in which prod-ucts cause inhibition, i.e competitive, mixed or uncom-petitive under certain experimental conditions are diagnostic of both the binding order and the extent
to which the system exhibits rapid-equilibrium charac-teristics, i.e whether off-rates are much faster than turnover
An initial set of four experiments used fixed, subsat-urating concentrations of either substrate or cofactor with varied concentrations of the other The experi-ments were performed at different, fixed concentrations
of either lactate or NAD+ A subsequent set of experi-ments was performed to see if saturating conditions could alleviate the effects on the apparent KMor kcat values An example of data from a steady-state prod-uct inhibition matrix is shown in Fig 2
The inhibition patterns found in these experiments are summarized in Table 1 They show that an ordered sequential bi-bi system in which NADH binds first is the appropriate mechanism for the enzyme The other six possible mechanisms are ruled out by the data in Table 1 [6]
Elucidation of steady-state kinetic constants The true KMvalue for pyruvate was determined using the secondary plot shown in Fig 3 Here the apparent
KMfor pyruvate is plotted as a function of the concen-tration of NADH and fitted to Equation 3 (see Experi-mental procedures) The plot shows a plateau at a value of about 55 ± 7 lm, the true KM for the sub-strate Fig 4 shows a secondary plot in which kcat val-ues for these data sets were plotted as a function of the NADH concentration and fitted to the Michaelis– Menten equation The plot yields a value for the max-imal catalytic rate constant of the reaction of 96 s)1 and a value for the KMfor NADH of 11 ± 2 lm
pyruvate (mM)
0
20
40
60
80
100
ktac
Fig 1 Secondary plot of steady-state reaction velocities plotted as
a function of pyruvate concentration Initial velocities of the enzyme
reaction were measured under steady-state conditions, in varied
concentrations of NADH and different fixed concentrations of
pyru-vate In these experiments, each initial rate measurement was
repeated five times and the values averaged These data were
fit-ted to the standard Michaelis–Menten equation to give values of
kcatat a series of fixed pyruvate concentrations The curve shows
these values for kcatversus pyruvate concentration fitted to Eqn 1
in Experimental procedures, yielding a K M for pyruvate 69 ± 4 lM,
Ki 140 ± 18 mM (kcat is used as it is independent of enzyme
concentration) Each point on the graph corresponds to five
repeated measurements of initial rates at five different NADH
con-centrations fitted to yield kcatvalues with the standard error shown.
As there are nine of these points, the data correspond to 225 rate
measurements.
Trang 3The kinetic constants describing the reaction in the
other direction, with NAD+and lactate as substrates,
were determined at a physiologically relevant pH
(pH 7.5) and all steady-state constants are given in
Table 2 In this study, for ease of purification and
sta-bility, a histidine-tagged version of PfLDH was used
(see Experimental procedures) [7] To assess any effect
of this tag on the catalytic function of the enzyme,
equivalent experiments to those described above were
performed with the wild-type enzyme The KM for
NADH, the KMand Kifor pyruvate and kcatfor both
wild-type and His-tagged enzymes were measured
These yielded the same constants, within error, as those
Table 1 Pattern of product inhibition in the steady state In order
to elucidate the basic kinetic mechanism for PfLDH, the pattern of
product inhibition was determined using NADH and pyruvate as
sub-strates and either NAD + or lactate as inhibitors For these diagnostic
purposes, the reactions were carried out at two set concentrations
of NADH, subsaturating (i.e KM· 1 ¼ 10 lM) and saturating (i.e.
KM· 20 ¼ 200 lM) The pattern of inhibitory behaviour shown in
the table is exactly that expected for an ordered bi bi kinetic
mech-anism with the coenzyme binding first [6].
Product
Subsaturating
substrate
Saturating substrate
Substrate varied
NADH (mM)
KM
0 0.02 0.04 0.06 0.08 0.1 0.12
Fig 3 The secondary plot of apparent KM for pyruvate plotted against NADH concentration showing the true KMfor pyruvate Ini-tial velocities were measured in five different NADH concentrations and varied pyruvate Each measurement was repeated five times and averaged; this graph represents 125 measurements From a standard Michaelis–Menten fit, the data revealed apparent K M val-ues for pyruvate for each NADH concentration (the error bars on the graph pertain to these fits) These apparent KMvalues for pyru-vate were then plotted against the corresponding NADH concentra-tion The data were fitted to Eqn 3 in Experimental procedures and show the true KMfor pyruvate; 55 ± 7 lM is found at infinite NADH concentrations seen here as the plateau This behaviour also indi-cates that the system is an ordered sequential bireactant system with pyruvate binding subsequently to NADH [6].
NADH (mM)
ktac
1- )
0 20 40 60 80 100
Fig 4 The secondary plot of steady-state reaction velocities plot-ted as a function of NADH concentration Data from the same set
of experiments as described for Fig 3 was used This time the sec-ondary plot shows steady-state values for the fitted k cat measured with varied pyruvate at different fixed NADH concentrations The re-plotted data were fitted to the Michaelis–Menten equation to yield the K M for NADH as 11 ± 2 lM and the k cat 96 s)1.
1/pyruvate (μM-1)
0
2
4
6
8
10
Fig 2 Example plot of data from the steady-state product inhibition
matrix, generating one piece of the information in Table 1 This
example shows a Lineweaver–Burk plot of rates under conditions
of subsaturating NADH and varied pyruvate in the presence of
dif-ferent fixed lactate concentrations s, zero lactate; j, 50 mM
lac-tate; n, 75 mM lactate; *, 100 mM lactate Each point on the graph
represents an average of five measurements.
Trang 4described above, hence the tag did not influence the
kin-etic behaviour of the enzyme to any measurable extent
Equilibrium binding affinity of NADH
The binding of NADH to the active site of
dehydro-genases is usually accompanied by a significant
alter-ation in its fluorescence properties resulting from
either a protection from solvent, collision quenching
and⁄ or a change in polarity of the environment of the
fluorophore In the case of PfLDH, the signal change
on binding to the active site was too small to be used
as a reliable reporter of the formation of the binary
complex As a result, fluorescent resonance energy
transfer (FRET) from the indole to the
dihydro-nico-tinamide groups was used to measure the affinity for
NADH The FRET data were fitted to Equation 5 (see
Experimental procedures) and are shown in Fig 5,
yielding a Kdof 4.0 ± 0.8 lm
pH dependence of substrate binding
A characteristic of this family of dehydrogenases is the
pH sensitivity of the KMvalues for pyruvate and
lac-tate [8] These parameters are controlled by the
proto-nation state of the active-site histidine residue, which
serves as a proton donor–acceptor in the redox
reac-tion Pyruvate binds only when the histidine is in the
protonated state and lactate only when it is
unproto-nated To investigate the pK of this residue, the KM
for pyruvate was determined as a function of pH and
the data are shown in Fig 6 The data was fitted to
Equation 6 (see Experimental procedures) and shows
that the KM is controlled by a single ionizing group
with a pK of 7.95 ± 0.08, similar to other lactate
dehydrogenases of this mechanistic family [9]
Transient kinetic properties of the enzyme: the
single-turnover reaction
Single-turnover experiments were carried out to help
elucidate the nature of the rate-limiting step In such
Table 2 Kinetic constants for the reduction of pyruvate and the
oxidation of lactate at pH 7.5.
Substrate ⁄ cofactor KM(lM) kcat(s)1)
k cat ⁄ K M
(s)1Æ M )1)
4 5 6
Fig 5 The fluorescent resonance energy transfer (FRET) titration
to establish the K d for NADH One micromolar additions of PfLDH were made to a cuvette containing 10 lM NADH in a SPEX Fluoro-max spectrophotometer The absorption wavelength for tryptophan
at 285 nm was used as the excitation wavelength and the emission wavelength of 450 nm for NADH was monitored Control experi-ments were carried out to correct for the inner filter effect of adding protein as described The data were fitted to the tight bind-ing equation (Eqn 5; see Experimental procedures) and the NADH concentration was allowed to float Results showed the Kd for NADH is 4 ± 0.8 lM and the fitted NADH concentration was 10.4 ± 0.9 lM (10 lM in cuvette).
[H+] (μΜ)
0.0 0.1
0.1 0.01
0.001 0.0001
1
1
Fig 6 A pH titration under steady-state conditions was carried out
to determine the pKa of the ionizable group at the active site The
K M for pyruvate was determined from rates measured for 8–10 dif-ferent pyruvate concentrations in 200 lM NADH in the pH range 6–10 (the enzyme was unstable below pH 5.5) Each rate was repeated three times and averaged Shown here is the log variation
in apparent KM(mM) for pyruvate versus log [H + ] NB In pH terms the x-axis reads left to right pH 10–6 A four buffer system was used to minimize variables other than pH (see Experimental procedures) The data has been fitted to the equation
KMapp.¼ K M (1 + Kh⁄ [H + ]) The Khfrom the graph was 11 ± 2 nM and equates to a pKa of 7.95 ± 0.08 for the ionizable group.
Trang 5an experiment, the enzyme is mixed with one
equival-ent of NADH to form a binary complex in one syringe
of the stopped-flow apparatus This solution is then
challenged with pyruvate and the first-order,
on-enzyme conversion of NADH to NAD+ is recorded
by monitoring the loss of absorbance at 340 nm In
this way, the reaction is simplified as it is only the
hydride-transfer chemistry itself, or a preceding
con-formational rearrangement that can limit the recorded
rate constant The single-turnover data are shown in
Fig 7, where the observed rate constant is plotted
against the varied concentration of pyruvate The
data are fitted to the Michaelis–Menten equation
giving a maximum rate constant 130 s)1 and an
apparent KM of 240 lm The maximum rate constant
is significantly higher than the catalytic rate constant
measured in the steady state, suggesting that some
other process is partially limiting the steady-state
reaction rate
The experiment at 2 mm pyruvate was repeated,
reversing the mixing order In this case, 75 lm enzyme
was challenged with 4 mm pyruvate and 75 lm NADH
giving a single turnover rate for 2 mm pyruvate
post-mix of 116 s)1 (data not shown) This is a similar rate
to that seen in the previous experiment, with a
pre-equilibrated binary complex challenged with 2 mm
(postmix) pyruvate This result rules out the possibility that there is a rate-limiting, or partly rate-limiting, step that occurs after the binding of NADH and before the association of pyruvate, i.e a structural rearrangement
of the E–NADH binary complex These transient kinetic results therefore demonstrate that the major rate-limiting step or steps occur after the binding of pyruvate
Rapid kinetics of the multiple-turnover reaction The result of a multiple-turnover experiment in which
200 lm NADH was mixed with 35 lm enzyme at a pyruvate concentration of 1 mm is shown in Fig 8 The reaction trace (Fig 8A) shows curvature in the initial milliseconds of the experiment, followed by a steady-state rate of about 75 s)1 per active site as shown by the linear regression The first turnover was
pyruvate (mM)
0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 2
1- )
0
20
40
60
80
100
120
Fig 7 The secondary plot of single turnover rates as a function of
pyruvate concentration fitted to the Michaelis–Menten equation.
The KMvalue for pyruvate under transient conditions was 240 lM,
five times weaker binding than in the steady state and the k cat was
faster, 130 s)1compared to 96 s)1in the steady state Each single
turnover rate was measured under transient kinetic conditions with
equimolar enzyme and NADH in one syringe challenged with
increasing concentrations of pyruvate in the other Each of the
measurements was repeated 10 times, averaged and fitted to a
single exponential giving the rate constant at each concentration of
pyruvate.
A
0.15 0.1
0.05 0
0.3
0.2
0.1
0
Time (s)
0.04 0.02
0
0.06
0.04
0.02
0
Time (s)
B
Fig 8 Multiple turnovers measured in the stopped flow apparatus NADH (200 lM) was mixed with enzyme (35 lM) and mixed with pyruvate (1 mM) The change in absorbance at 340 nm was meas-ured An average of five transients was used for the fitting (A) shows the averaged data from the experiment with a linear fit to the steady-state rate of 75 s)1 (B) shows the initial 0.04 s of the data after subtracting the steady-state rate These data were fitted
to a single exponential to give an initial rate of 134 s)1 in the approach to the steady-state rate.
Trang 6fitted to a single exponential with slope for subsequent
turnovers removed (Fig 8B) This gave a first-order
rate constant of 134 s)1 This experiment shows that
there must be a process following hydride transfer that
partially limits the steady-state catalytic rate
Primary deuterium isotope effect
Figure 9 shows a comparison of the single-turnover
reaction carried out with NADH and with 4R-NADD
The observed kinetic isotope effect, KIE(obs), was
approximately 1.2 (given by the ratio of the first-order
rate constants) Previous data for this class of
dehy-drogenase enzymes show that the intrinsic kinetic
iso-tope effect [KIE(int)] should be close to 2.7 [10] for a
reaction in which hydride transfer is completely
rate-limiting This value was extrapolated from data on a
series of LDH mutants [10] A plot of kcat versus the
observed kinetic isotope effect showed that as kcat
ten-ded to zero the KIE tenten-ded to 2.7 The value of 2.7
was taken to represent the maximal KIE for an LDH
limited by hydride transfer Here, the observed value
of 1.2 indicates that while there is a small component
from hydride transfer in the rate limiting process (the
value is greater than 1), there must also be a major
contribution from a conformational change Rate
con-stants for hydride transfer (k3H) and conformational
change (k3C) were calculated using Equations 7 and 8
(Experimental procedures) and found to be 2000 s)1 and 160 s)1, respectively
A likely candidate for this conformational change is movement of the substrate-specificity loop, as observed
in other lactate dehydrogenases [10] This will be con-sidered in more detail, in the context of crystal struc-tures, in the discussion
Alternative substrates The fact that there is a unique five-residue insertion in the active-site loop of the PfLDH enzyme raises the possibility that substrate specificity is different from the LDHs thus far investigated in detail, both eukaryotic and prokaryotic To investigate this possibility, the activity of the enzyme was tested with alternative sub-strates for comparison with other well-characterized LDHs; Table 3 shows a summary of the results There was an approximately 10-fold decrease in PfLDH effi-ciency between pyruvate and a-ketobutyrate The pres-ence of the extra methylene group of a-ketobutyrate results in a 10-fold increase in KM However, the pres-ence of two extra methylene groups, compared with pyruvate, in a-ketovalerate results in a catastrophic decrease in enzyme efficiency For this substrate the
KM is increased 2000-fold and the kcat decreased 200-fold compared with a 130-fold increase in KMfor a-ketovalerate in BsLDH, which had just a five-fold decrease in kcat [10] The ability of the enzyme to reduce phenylpyruvate was also assessed Surprisingly, and unlike the case of other LDH enzymes of this fam-ily, we could detect no catalytic activity at all with this substrate
Testing for malate dehydrogenase activity One of the more striking sequence differences between PfLDH and other LDHs of the same fold is the presence of a lysine residue at position 102 The pres-ence of a positive charge in this position in the sequence is a possible characteristic of an enzyme that has malate dehydrogenase activity [11] Indeed, appar-ent activity is seen under standard steady-state condi-tions when oxaloacetate is used as the substrate in place of pyruvate In neutral solutions, oxaloacetate
Time (s)
-0.05
-0.04
-0.03
-0.02
-0.01
0
Fig 9 The kinetic primary isotope effect measured in the stopped
flow apparatus In this experiment 75 lM enzyme was challenged
with 75 lM 4R-NADD (top trace) and 75 lM NADH (bottom trace).
The ratio of rates of the two single turnover events is 1.2 The ratio
expected (observed kinetic isotope effect) for this class of enzymes
in a process that is wholly rate-limited by hydride transfer is 2.7
[10] The rate of conformational change was calculated as 160 s)1
and the rate of hydride transfer as 2000 s)1using the equations
described in the primary deuterium isotope effect section of
Experi-mental procedures Each transient is an average of 10.
Table 3 Kinetic constants for pyruvate, a -ketobutyrate and a-ket-ovalerate (values in parentheses are taken from reference [16]) Substrate KM(mM) kcat(s)1) kcat⁄ K M (s)1ÆM)1)
a-ketobutyrate 0.6 (0.47) 80 (180) 1.3 · 10 5
(3.8 · 10 5
)
Trang 7decarboxylates rapidly to pyruvate, even in the absence
of an enzyme We used proton NMR to determine the
actual substrate responsible for activity Over a period
of hours, peaks for NADH and oxaloacetate were
replaced by those corresponding to NAD+, lactate
and pyruvate At no time were peaks corresponding to
malate observed This indicates that oxaloacetate
de-carboxylates rapidly in the presence of PfLDH under
these conditions and the observed activity at pH 7.2 is
due to turnover of the resulting pyruvate
Discussion
The general reaction mechanism of PfLDH is, by and
large, similar to those of other LDHs of the
nicotina-mide-dependent type The reaction follows an ordered
bi-bi kinetic pattern [6] with coenzyme binding first
(see Fig 10) In addition, the steady-state constants
(see Table 2) are very similar to those measured for
structurally related counterparts with KM values for
NADH and pyruvate being typically in the 10)5 and
10)4m ranges, respectively, and those for NAD+ and
lactate being in the 10)4 and 10)2m ranges Similarly
the steady-state catalytic rate constants in each
direc-tion are in keeping with other LDHs
With regard to the nature of the rate-determining
steps, conformational rearrangement is the
predomin-ant kinetic barrier in the single-turnover reaction, i.e
in a process that can only be limited by a
rearrange-ment of the ternary collision complex or by the rate
of hydride transfer, the latter must be the more rapid,
as shown by the relatively small primary kinetic
isotope effect The rate-limiting conformational
rearrangement in other LDHs is identified as the
closure of an active-site loop triggered by substrate
binding The function of this change in structure is to
remove solvent from the catalytic site and bring the
positive charge of Arg-109 into proximity, so that the
carbonyl group of pyruvate can be strongly polarized
Additionally, loop-closure enhances substrate
selectiv-ity by engulfing the pyruvate within a catalytic
vacuole to maximize contact between substrate and enzyme The steady-state catalytic rate constant is slightly slower than that recorded for the single-turn-over reaction, showing that some process that follows hydride transfer partially limits the steady-state reac-tion cycle This process must be a product-release step, either a rate of dissociation of lactate or NAD+
or the rate of loop opening after the hydride transfer reaction
A consequence of this partial rate-limiting process is that the apparent Michaelis constant for pyruvate in the single-turnover reaction is higher than that recor-ded in the steady-state This phenomenon is due to a relatively slow product off-rate in the system as des-cribed above To illustrate this, if the binding of pyru-vate to the E–NADH complex is a rapid equilibrium process, then the measured KM (KM¢) in the single-turnover reaction is simply equal to the Kdfor the for-mation of the encounter complex However, in the steady state all the partially rate-limiting steps come into play and the true KMis given by:
KM¼ KM 0=ð1 þ k3C=k3Hþ k3C=k4Þ where k3c represents the rate of the conformational change, k3H the hydride transfer and k4 the rate of the product-off step Hence, in these circumstances, the steady-state KM is expected to be smaller than the apparent KMmeasured in the single turnovers
Furthermore, it is interesting to note that the fact that all three of the above rate constants are partially rate-limiting shows that the enzyme obeys the
‘Knowlesian’ principle that biological catalysts should evolve to have no single, dominant energy barrier [12] Rather, there is an evolutionary advantage in equalizing the energies of intermediate and transition states in the on-enzyme reaction pathway
A major aim of these experiments was to elucidate unusual features of the enzyme that might distinguish
it from other LDHs, particularly those of human origin, having confirmed the lack of malate dehydro-genase activity The principal differences in the kinetic
Fig 10 Simplified schematic of the mech-anism of PfLDH where k3Hand k3C repre-sent the rate constants for hydride transfer and conformational change, respectively, as calculated from isotopic effects.
Trang 8constants of PfLDH compared with human LDHs
(with native substrate and cofactor) are twofold
Firstly, substrate inhibition of PfLDH (140 mm; in
the direction pyruvate to lactate) is much weaker
than that shown by the human heart and muscle
iso-forms by around 175- and 35-fold, respectively [5]
Second, the binding of NADH to PfLDH is some
10-fold weaker than that shown by the human
iso-forms, hence Kd for PfLDH is 4 ± 0.8 lm compared
with 0.5 and 0.6 lm for the human heart and muscle
enzymes, respectively [5] Both of these differences
(raised Kd and Ki) appear to be largely attributable
to the presence of leucine at 163 in PfLDH, a residue
which is serine in all known LDHs that do not
pos-sess the extra five residues in the substrate-specificity
loop Crystal structures of holo-LDHs with serine at
position 163 show that the hydroxy group of the
ser-ine side-chain is hydrogen bonded to the nicotinamide
amide group of NADH, often via a water molecule
Site-directed mutagenesis has been used to make the
S163L variants of both human heart and muscle
LDH isoforms [5] In both cases, substrate inhibition
was removed (Ki> 500 mm) and the Kd for NADH
was raised about 10-fold compared with the
wild-types Structural studies of ternary complexes of
plasmodial LDHs [13,14] all show a displacement in
the position of the nicotinamide ring when compared
with all other ternary LDH structures, which is
con-sistent with the presence of leucine rather than serine
at position 163 Whilst the human S163L mutants
show rather similar kinetic and binding parameters
for NADH, the KM for pyruvate is raised by 40- to
200-fold We may speculate that the five-residue
inser-tion in the PfLDH substrate-specificity loop
compen-sates for the deleterious effect on the pyruvate
binding site due to the S163L change (since the
reac-tion mechanism is ordered bi-bi, the pyruvate binding
site is only fully formed after NADH binding) Some
supporting evidence for this hypothesis comes from a
kinetic study in which the substrate-specificity-loop
sequences from the broad-specificity ketoacid
reduc-tase, l-hydroxyisocaproate dehydrogenase (l-hicDH),
and PfLDH were engineered into Bacillus
stearother-mophilus LDH (BsLDH), replacing the wild-type loop
[15] The BsLDH construct containing the l-hicDH
loop (a four-residue insertion compared with typical
LDHs, e.g those from human and bacillus) had a
KMfor pyruvate of 42 mm, raised some 670-fold over
wild-type BsLDH By contrast, the BsLDH construct
possessing the substrate-specificity loop from PfLDH
had a KMfor pyruvate raised only 13-fold to 0.8 mm,
despite this corresponding to a five-residue loop
inser-tion with respect to wild-type BsLDH
A simple method to explore the size of the substrate binding site in a functional enzyme is to measure its ability to turn over larger substrate-like molecules This
is straightforward in the case of LDHs as many com-pounds R-CO.CO2H (R¼ methyl in pyruvate) are readily available The data in Table 3 clearly show that,
in the case of PfLDH, extending R from ethyl (i.e a-ketobutyrate) to n-propyl (i.e a-ketovalerate) causes
a catastrophic fall off in the catalytic efficiency (kcat⁄ KM) of nearly six orders of magnitude In the case
of wild-type BsLDH, this change causes a loss in cata-lytic efficiency closer to three orders of magnitude com-pared with pyruvate Even more striking are the relative activities of this pair of enzymes towards phe-nylpyruvate This bulky substrate is turned over by BsLDH with a reasonable catalytic efficiency of 1.8· 104m)1Æs)1 [15], whilst no activity at all was detected with PfLDH either in this study (data not shown) and elsewhere [16] This behaviour may be con-trasted with that of two lactate dehydrogenases present
in the parasite Toxoplasma gondii that turn over phe-nylpyruvate at a comparable rate to pyruvate Recent structural work [17] has shown that TgLDH1 has a very similar structure to PfLDH, including the long substrate-specificity loop Both TgLDH enzymes con-tain another loop insertion (of two residues) between helices a-G2 and a-G3 and other changes in residue types lining the active site, any or all of these factors may be responsible for the activity shown by TgLDHs towards phenylpyruvate
Consequences for drug design The intolerance of PfLDH towards larger substrates limits the possibilities for inhibitor design based
on substrate or product (i.e pyruvate or lactate) analogues This observation is borne out by the recent development of a series of azole-based lactate analogues which are strong inhibitors of the oxidized binary complex of PfLDH and NAD+ [3] Attempts
to elaborate these compounds to improve binding and specificity were unsuccessful, presumably due to the precise conformational requirements of the closed substrate-specificity loop The bi-bi mechanism, demon-strated in this paper, requires binding of NADH before substrate As both the NADH and the ordered substrate-specificity loop comprise part of the sub-strate-binding site, substrate analogues are not expec-ted to bind tightly to the apoenzyme However, an inhibitor that competes with endogenous NADH will firstly benefit from the 10-fold weaker affinity of NADH for PfLDH compared with the human LDH enzymes Additionally, the differences in residues
Trang 9lining the NADH binding site such as the switch of
Ser to Leu at position 163 should be exploitable in
drug design Finally, with respect to improving
affin-ity, compounds could be targeted to the apoenzyme
[18] Binding compounds across the substrate and
coenzyme sites could increase the scope for elaboration
The large surface exposed when the substrate-specificity
loop is disordered, as seen in the apoenzyme
crystal structure, affords the opportunity to design
inhibitors that are not restricted by the limited space
available in the closed-loop conformation of the
protein
Experimental procedures
Expression and purification
Six histidines were added to the C-terminus of the PfLDH
gene by PCR without linker or cleavage sites The modified
gene was inserted into the pKK vector and cloned into
JM105 strain of Escherichia coli [4] Cells were harvested
from overnight culture in 2xYT (yeast tryptone media)
without the need for isopropyl-b-d-thiogalactopyranoside
induction [7] Following sonication, cell debris was
separ-ated by centrifugation at 5000 g for 30 min
The supernatant was then applied to a Nickel-NTA
agarose column (Qiagen, Crawley, UK) The enzyme was
eluted in 250 mm imidazole, concentrated against polyethylene
glycol 20K and dialysed into phosphate buffered saline
(NaCl⁄ Pi), 10% glycerol, 5 mm EDTA and 10 mm
dithio-threitol This protocol yielded pure enzyme at an average
of 80 mg cellsẳL)1 Aliquots (100 lL) of enzyme were snap
frozen in liquid nitrogen and stored at)80 C The activity
of the enzyme stored under these conditions remained
con-stant within the time-scale of the experiments The
concen-tration of enzyme used was assessed by Bradford assay and
by absorbance at 280 nm where 1 mgẳmL)1corresponds to
an absorbance of 0.42 for a 1 cm path length Enzyme
pur-ity was assessed as the only visible band by SDS⁄ PAGE
Where used for comparison with the his-tagged enzyme,
wild-type PfLDH was expressed from a pKK vector in
JM105 cells incubated overnight in 2xYT and purified on
an oxamate affinity column and eluted with NADH
Con-centration, dialysis and storage methods were the same as
for the his-tagged enzyme
Steady-state kinetics
Enzyme assays were carried out at 25C using a Perkin
Elmer spectrophotometer with a perfused cuvette block
Grade I NADH and NAD+were purchased from
Boehrin-ger Mannheim (Mannheim, Germany; now Roche) and the
buffers and substrates from Sigma (Gillingham, UK) The
data were analysed using grafit 4 software
To assess substrate inhibition, the data for experiments
in which pyruvate was varied were fitted to the following equation:
vỬ Vmax:S=ơS ợ KMợ đS2=Kiỡ đ1ỡ where v is the initial steady-state reaction velocity, S is sub-strate concentration, KMis the Michaelis constant for pyru-vate and Kiis the substrate-inhibition constant
At substrate concentrations well below Ki, this equation reduces to the standard MichaelisỜMenten equation All experiments to elucidate the steady-state mechanism were performed at pyruvate concentrations at least 50-fold lower than Ki This equates to a reduction in rate of less than 2% hence the following rate equations do not account for inhi-bition by substrate
Steady-state rate equations The steady-state rate equation for an ordered bi-bi reaction
in the absence of reaction products is shown below v=E0Ử đk1k2k3ơNơPỡ=đC0ợ ơNơPCNPợ ơPCPợ ơNCNỡ
đ2ỡ where k1, k2and k3 are the forward rate constants for the binding of NADH, the binding of pyruvate and the catalytic rate constant, respectively; the concentrations of NADH and pyruvate are [N] and [P], respectively; the coefficients CO,
CNP, CPand CNrepresent the groups of rate constants that are dependent on the subscripted substrates, e.g Coare those that are independent of substrate and CNPthose dependent
on both coenzyme and substrate, etc The component rate constants are as follows: CoỬ k-1ẳk-3+ k-1ẳk-2, CNPỬ k1ẳk2,
CPỬ k2ẳk3, and CNỬ k1ẳk-2+ k1ẳk3
To determine the Michaelis constants for pyruvate in the steady state, velocities were determined at a series of coen-zyme and substrate concentrations and the apparent KMfor pyruvate [KM.pyr.(app)] was determined as a function of NADH concentration ([N]) Using Eqn 2 as the parent equation, the secondary data were then fitted to the following relationship:
KM:pyr:đappỡỬ đCO=CNPợ ơNCN=CNPỡ=đơN ợ CN=CNPỡ đ3ỡ where the y-value at infinite [N] is CN⁄ CNP, which translates
to (k3+ k-2)⁄ k2and represents the true KMfor pyruvate The apparent kcat of the system [kcat(app)] was deter-mined using pyruvate as the varied reactant The value of
kcat.pyr.(app)was then determined at a series of fixed NADH concentrations ([N]) Again using Eqn 2 as the parent equa-tion, the secondary data were fitted to the following derived relationship:
kcat:pyr:đappỡỬ đk1k2k3=CNPỡơN=đCP=CNPợ ơNỡ đ4ỡ where k1ẳk2ẳk3⁄ CNP Ử k3 and CP⁄CNPỬ k3⁄ k1 The former
is the true kcatand the latter the true KMfor NADH Steady-state reactions were carried out at 25C in
50 mm tris⁄ 50 mm KCl buffer at pH 7.5
Trang 10Proton NMR analysis of reaction products
For the alternative substrates in addition to
spectrophoto-metric assays, 1H NMR was used to assign the products
formed in the presence of oxaloacetate and NADH As
oxaloacetate undergoes decarboxylation to pyruvate, NMR
was used to determine whether the activity seen was due to
the turnover of oxaloacetate to malate or pyruvate to
lac-tate Pyruvate formation, due to spontaneous oxaloacetate
decarboxylation at low pH, was minimized by adding two
molar equivalents of NaOH to ice-cold buffer prior to the
addition of solid oxaloacetic acid In this manner, a
prepar-ative reaction was set up with PfLDH (3 lm), oxaloacetate
(5 mm) and NADH (5 mm) in NaCl⁄ Pi⁄ D2O (pH 7.2) and
1
H NMR was used to follow product formation
Transient kinetics
Transient kinetic data were collected using an SX.18 mV
apparatus supplied by Applied Photophysics
For the stopped-flow reactions, buffers comprised 10%
glycerol, 50 mm phosphate, 150 mm NaCl, with 5 mm
EDTA and 10 mm dithiothreitol at pH 7.5 Reactions were
carried out at 25C Enzyme solutions were made up as
stocks in this buffer and lost no activity during 48 h
Gly-cerol is known to slow the rate of loop closure for other
LDHs so the difference between steady-state rates in each
of the buffers was assessed There was a 5–10% reduction
in kcatin the presence of 10% glycerol so that, within error,
direct comparisons could be made between stopped-flow
data and the steady state
All NADH solutions were diluted in buffer from freshly
thawed 0.25 m stocks made up in water and stored at
minus 80C NADH or 4R-NADD was added to enzyme
immediately before rates were measured
Equilibrium fluorescence
FRET reactions were measured in a Spex Fluoromax
spec-trophotometer Over a 1500-s time-base, additions of 1 lm
enzyme were made to a solution of 10 lm NADH The
excitation wavelength was 285 nm and emission monitored
at 450 nm Adding protein to the cuvette causes an inner
filter effect To compensate, an identical experiment was set
up using N-acetyltryptophanamide in place of enzyme The
PfLDH data were then divided by the resulting linear
change in fluorescence for N-acetyltryptophanamide Data
were fitted to a tight binding equation with floating
[NADH]:
Signal¼ initial þ ðfðE þ N0þ KdÞ ½ðE þ N0þ KdÞ2
4N0E0:5g=ð2N0ÞÞamp ð5Þ
where ‘initial’ is the starting fluorescence, ‘amp’ the
ampli-tude of change, E is the concentration of LDH added and
N0is total concentration of NADH in the titration
pH dependence The pH titration experiments at 25C were carried as for other steady-state assays A four buffer system was used comprising 20 mm each of potassium acetate, 2-(cyclohexylamino)-ethanesulfonic acid (CHES), 2-amino-2-(hydroxymethyl)-1,3-propanediol (Tris), 2,2-bis(hydroxy-methyl)-2,2¢,2¢-nitrilotriethanol (Bis Tris) The pH of the buffer was adjusted by the addition of either HCl or NaOH
to produce a range of pH values from 5 to 10 Measure-ments were made in saturating NADH and varied pyruvate
to determine the KM for pyruvate at different pHs The pKa of the ionizable group was determined by fitting data
to Eqn 6
KMðappÞ¼ KMð1 þ Kh=½HþÞ (6) The primary deuterium isotope effect was measured in the SX.18 mV to determine the difference in single turnover rates achieved in the presence of NADH or 4R-NADD Mono-deuterated cofactor was enzymically produced by formate dehydrogenase (from Candida methylica) in the presence of NAD+ and deuterated formic acid (kindly donated by C M Eszes, University of Bristol, UK) Formate dehydrogenase catalyses the addition of hydride (deuteride) to the A face of NAD+giving 4R-NADD This
is the same hydride (deuteride) that is transferred from the
A face of the cofactor to pyruvate during reduction to lactate catalysed by LDH [19] PfLDH (75 lm) with 75 lm cofactor was mixed with 4 mm pyruvate (premix concentra-tions) From these measurements the rates of conformational change and hydride transfer were calculated using the equa-tions below
kh¼ ½kobs;NADHkobs;NADDð1=R 1Þ=ðkobs;NADD=R
kc¼ ðkobs;NADHkhÞ=ðkh kobs;NADHÞ ð8Þ where kobsNADH¼ observed rate constant with NADH ¼ (kcÆkh)⁄ (kc+ kh); kobs,NADD¼ observed rate constant with 4R-NADD¼ [kcÆ(kh⁄ R)] ⁄ kc(kh⁄ R); R ¼ 2.7 (the basis for R-value explained in results section for the primary deuter-ium isotope effect [7]); kh¼ rate constant for hydride trans-fer; and kc¼ rate constant for conformational change
References
1 Mehlin C (2005) Structure-based drug design for Plasmodium falciparum Comb Chem High T Scr 8, 5–14
2 Royer RE, Deck LM, Campos NM, Hunsaker LA & Vander Jagt DL (1986) Biologically active derivatives of gossypol: synthesis and antimalarial activities of peri-acylated gossylic nitriles J Med Chem 29, 1799–1801
3 Cameron A, Read JA, Tranter R, Winter VJ, Sessions
RB, Brady RL, Vivas L, Easton A, Kendrick H, Croft