A general method for relieving substrate inhibition in lactate dehydrogenases

C.O. Hewitt, C.M. Eszes, R.B. Sessions1, K.M. Moreton, T.R. Dafforn2, J. Takei, C.E. Dempsey, A.R. Clarke and J.J. Holbrook

Molecular Recognition Centre and Department of Biochemistry,School of Medical Sciences, University Walk, Bristol BS8 1TD and 2 Department of Haematology, University of Cambridge, Hills Road,Cambridge CB2 2QH, UK


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The mutation S163L in human heart lactate dehydrogenase removes substrate inhibition while only modestly reducing the turnover rate for pyruvate. Since this is the third enzyme to show this behaviour, we suggest that the S163L mutation is a general method for the removal of substrate inhibition in L-LDH enzymes. Engineering such enzymatic properties has clear industrial applications in the use of these enzymes to produce enantiomerically pure {alpha}-hydroxy acids. The mutation leads to two principal effects. (1) Substrate inhibition is caused by the formation of a covalent adduct between pyruvate and the oxidized form of the cofactor. The inability of S163L mutants to catalyse the formation of this inhibitory adduct is demonstrated. However, NMR experiments show that the orientation of the nicotinamide ring in the mutant NAD+ binary complex is not perturbed. (2) The mutation also leads to a large increase in the KM for pyruvate. The kinetic and binding properties of S163L LDH mutants are accounted for by a mechanism which invokes a non-productive, bound form of the cofactor. Molecular modelling suggests a structure for this non-productive enzyme–NADH complex.

Keywords: lactate dehydrogenase/mutagenesis/protein engineering/substrate inhibition


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Mammalian and bacterial lactate dehydrogenases show inhibition by high concentrations of substrate during the reduction of pyruvate to lactate. This inhibition is a consequence of the formation of a covalent adduct between pyruvate and the oxidized cofactor, NAD+, before it is released from the enzyme (Fromm, 1961Go; Gutfreund et al., 1968Go; Coulson and Rabin, 1969Go). The enzyme catalyses the formation of this adduct which then binds tightly to the protein, inhibiting further redox catalysis (Everse et al., 1971Go) (Figure 1Go). Apart from purely scientific interest, the ability to remove substrate inhibition in enzymes by appropriate mutagenesis has applications in the industrial context, allowing the production of large quantities of enantiomerically pure pharmaceutical intermediates.



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Fig. 1. Steady-state rates of the wild-type hH4LDH (•) and the S163L mutant ({circ}) proteins are plotted against the logarithm of the pyruvate concentration to emphasize the differences in KM and Ki between these enzymes. The inset is a repetition of the S163L rate data between 0.1 and 1 M pyruvate, plotted against ionic strength, compared with the effect of adding increasing amounts of NaCl to the 0.1 M pyruvate reaction (x– – –x).

 
We have shown previously that the mutation S163L in the lactate dehydrogenases from Bacillus stearothermophilus (bsLDH) and human muscle (hM4LDH) removes substrate inhibition (Eszes et al., 1996Go; Hewitt et al., 1997Go). In this paper, we show that this mutation is also effective in the human heart isoform (hH4LDH), the most strongly substrate-inhibited LDH known. Kinetic and binding data are presented for all three enzymes which support a mechanism involving productive and non-productive binding states of the S163L enzyme–cofactor complexes. Molecular modelling studies are used to suggest a structure for this non-productive complex.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Plasmids, bacterial strains and microbiological media

Bacterial strain Escherichia coli TG2 {supE hsd{Delta}5 thi{Delta} (lac-proAB) {Delta} (srl-recA) (306::Tn10) (tetr) F'[traD36 proAB+ lacIq lacZ{Delta}M15]} was used as a host to prepare all dsDNA for mutagenesis and sequencing in plasmid pUC-18 (Pharmacia Biotech, Uppsala, Sweden), containing the hH4LDH gene. The same E.coli strain was also used as a host for transformation and expression of hH4LDH proteins in pKK223–3 (Pharmacia Biotech). We prepared 2xYT medium as described previously (Sambrook et al., 1989Go).

Mutagenesis to construct S163L mutant LDH-B

Mutants were made by directed mutagenesis using the overlap extension polymerase chain reaction (PCR), incorporating the thermostable recombinant pfu polymerase (Stratagene Cloning Systems). The PCR strategy had two stages. First, mutant oligonucleotides together with commercially available M13–20 and M13 reverse sequencing primers (Stratagene Cloning Systems) were used to generate two overlapping LDH-B gene fragments (20 cycles of 94°C 1.5 min, 52°C 1.5 min, 72°C 5 min). Second, these were joined by overlap extension after purification from a 0.8% agarose gel (seven cycles of 94°C 2 min, 52°C 1.5 min, 72°C 5 min), prior to amplification (20 cycles of 94°C 1.5 min, 52°C 1.5 min, 72°C 3.5 min). All PCR reactions were carried out in the presence of 0.2 mM each dNTP, 0.5 µM each primer, 20 mM Tris–HCl (pH 8.5), 10 mM (NH4)2SO4, 2 mM MgCl2, 5 µg BSA (bovine serum albumin), 0.1% Triton X100 and 2.5 units Pfu polymerase. Mutant double stranded hH4LDH genes were digested using EcoR1 and were ligated into similarly digested pUC-18 following purification from a 0.8% agarose gel. Mutant dsDNA was sequenced in the region of the mutation to check for correct amino acid codon insertion using a Sequenase Version 2.0 DNA sequencing kit (United States Biochemicals, Cleveland, OH). Mutant genes were ligated into EcoR1-digested pKK223–3 for transformation into calcium chloride competent cells and expression.

All DNA-modifying enzymes were obtained from New England Biolabs or Boehringer Mannheim Biochimica and were used under conditions advised by the manufacturer.

Two 24 base pair oligonucleotides were designed to replace serine 163 in hH4LDH by leucine and were synthesized using the Millipore Expedite Nucleic Acid Synthesis System. The oligonucleotide sequences were as follows:

hH4LDH S163L coding strand 5'C GTG ATT GGA TTA GGA TGT AAT CTG 3'

hH4LDH S163L non-coding strand 5'CAG ATT ACA TCC TAA TCC AAT CAC G 3'

Purification of wild-type and S163L hH4LDH enzymes

Transformed cells were grown overnight in 2xYT broth (containing ampicillin) at 37°C. Cells were harvested by centrifugation and were then resuspended in a minimum volume of 50 mM triethanolamine (pH 6.0). The cells were broken open by sonication on ice (XL2010 ultrasonic processor from Heat Systems, using standard probe and 5x30 s bursts). Wild-type hH4LDH was purified by affinity chromatography on a column of oxamate-Sepharose (O'Carra and Barry, 1972Go; Dafforn, 1996Go), pre-washed with 0.5 mM NADH in 0.05 M TEA buffer (pH 6.0). The hH4LDH was added to the column in 10 mM NADH and eluted with 0.05 M TEA (pH 9.0) containing 1 M NaCl. Eluate containing LDH was precipitated using 430 g/l ammonium sulphate. S163L hH4LDH did not bind the oxamate-Sepharose affinity column. Instead, a two-column procedure was used. First, S163L hH4LDH was eluted from a Q-Sepharose column by a 0–1 M NaCl gradient in 0.05 M TEA (pH 7.5). The final step of the purification was a blue Sepharose-F3GA column (Dafforn, 1996Go). LDH was loaded on to the column in 10 mM TEA (pH 6.0) and eluted in 10 mM TEA (pH 6.0) with a 0–1 M NaCl gradient. Fractions containing LDH were pooled and protein precipitated using 430 g/l ammonium sulphate. SDS–PAGE and Coomassie Brilliant Blue staining were used to estimate the protein content after the described purification. Human muscle LDH, hM4LDH and the S163L mutant on this template were prepared as described previously (Eszes et al., 1996Go).

Steady-state kinetics

Initial rates of decrease in NADH concentration were followed at 340 nm (366 nm for pyruvate concentrations above 50 mM) at 25°C. The buffer used was 20 mM Bis-Tris (pH 6.0) with 50 mM KCl. Addition of pyruvate was used to initiate catalysis. Data were fitted to the Michaelis–Menten equation (corrected for substrate inhibition) using the following equation and the the non-linear regression facility in Grafit 3.0: v/vmax = S/[S + KM + (S2/Ki)], where S = substrate concentration and v = velocity at substrate concentration S (Leatherbarrow, 1992Go). The coenzyme concentration was 0.2 mM for wild-type hH4LDH and 0.4 mM for S163L hH4LDH. Pyruvate concentration ranges were 5 µM–1 M for wild-type and 10 µM–1 M for S163L hH4LDH. The protein concentration used was 10 nM.

Calculations of kcat depended upon the protein concentration as measured at 280 nm using an extinction coefficient of 1.16 mg/ml.cm for both wild-type and S163L hH4LDH proteins [the extinction coefficient for the hM4LDH proteins was 1.2 mg/ml.cm (Barstow et al., 1990Go)].

Study of coenzyme binding by changes in fluorescence intensity

A Perkin-Elmer L550B luminescence spectrofluorimeter was used to follow the decrease in protein fluorescence upon titration with reduced coenzyme. The buffer used was 20 mM Bis-Tris (pH 6.0) with 50 mM KCl. The excitation wavelength was 295 nm and emission was measured at 340 nm. Appropriate corrections were made for the inner filter effect by using a NATA (N-acetyl-L-tryptophanamide) control and for the amount of added ligand bound to the protein. Fits were made to appropriate ligand binding equations using Grafit 3.0 (Leatherbarrow, 1992Go). The protein concentration was 2 µM and the NADH concentration range for wild-type hH4LDH was 0–5 µM and for S163L was 0–50 µM. Reduced coenzyme binding was followed directly; oxidized coenzyme binding was followed indirectly in competition studies, using Kd (apparent) = Kd (NADH){1 + [NAD+]/Kd (NAD+)}.

Oxamate binding reported by changes in fluorescence intensity

Formation of the ternary complex (enzyme with reduced coenzyme and substrate analogue bound) was determined by following the decrease in NADH fluorescence in the binary complex (enzyme with coenzyme bound) upon titration with oxamate. The buffer used was 20 mM Bis-Tris (pH 6.0) containing 50 mM KCl. The excitation wavelength was 340 nm and emission was observed at 445 nm. The protein and NADH concentrations were 2 µM for hH4LDH and 7.6 µM for S163L hH4LDH. Oxamate was added in 2 µM aliquots for wild-type and 2 mM aliquots for S163L hH4LDHs.

APAD+ utilization

Relative rates were compared for hH4LDH and, hM4LDH in both wild-type and S163L mutant forms in the lactate to pyruvate direction using both NAD+ and APAD+ (3-acetylpyridinium adenine dinucleotide). A linked reaction was used to pull the reaction in this thermodynamically unfavourable direction employing NBT (nitro blue tetrazolium) and PES (phenazine ethosulphate). This resulted in the formation of a blue formazan dye measured at 548 nm [{varepsilon}548 formazan = 10.2 mM–1 cm–1 (A.Cortes, personal communication)]. Three lactate concentrations were used, 0.06, 0.3 and 0.6 M. The reaction buffer was Tris–HCl (pH 8.0) at 25°C.

Abortive adduct formation (enzyme–NAD+–pyruvate complex)

Time courses for adduct formation were followed. Measurements of absorbance at 327 nm were made at regular intervals between 0 and 15 min. A plot of A327 against time shows that the initial increase in A327 and the subsequent plateau represent the formation and stabilization of the enzyme–NAD+–pyruvate complex. The buffer used was 67 mM phosphate (pH 7.2) at 25°C. The proteins were used at a concentration of 15 µM in each case, the NAD+ concentration was 1 or 6 mM and pyruvate was then added to give a concentration of 5 or 30 mM to begin the reaction.

Transferred NOE NMR of NAD+ bound to wild-type and S163L hH4LDH

Protein samples were dialysed overnight into 0.05 M TEA (pH 6.0). Transfer into 0.02 M phosphate (pD 6.0) was accomplished by repeatedly (3–4 times) concentrating in a Centricon centrifugation device (molecular weight cut-off 10 kDa) and resuspending in the desired buffer. Proton magnetic resonance spectra were recorded using a 500 MHz Jeol {alpha} spectrometer. The sample volume was 0.5 ml and the temperature was 25°C. The protein concentration was 84 µM and the concentration of NAD+ was 4 mM. The residual water peak was used for reference and was taken to be 4.7 p.p.m. The mixing time (75–100 ms) was chosen to allow NOEs to build up in the bound state. 2D data sets were generated by collecting 340 T1 increments into 2K data points and Fourier transformation of FIDs after applying shifted sine-bell window functions in both dimensions. All NMR processing was carried out using FELIX 2.3.

Computer modelling NADH bound to wild-type and S163L hH4LDH

Hydrogen atoms were added to the crystal structure (Q-axis dimer) of pig muscle LDH (PDB code 9LDT) consistent with a pH of 7. All protein residues with an atom within 15 Å of the NADH bound to subunit A were selected and the rest discarded. Residues in this subset were mutated to the human heart LDH sequence as appropriate and the side chains repacked automatically where necessary. All residues with an atom within 10 Å of NADH were allowed to move during the simulations including all residues in the substrate specificity loop and helix {alpha}-1G, while the remainder were fixed in space. Layers of water molecules were added to a depth of 30 Å around NADH and to a depth of 15 Å around G106 to ensure adequate solvation of the substrate specificity loop. Water molecules at the surface of the solvent (529 of 1059) were tethered to their initial positions by a weak constraint (0.5 kcal/Å) applied to the oxygen atom. The mutant structure was generated from these wild-type coordinates by changing serine 163 to leucine.

During the calculations a 12 Å non-bonded cut-off was used and interactions smoothly reduced to zero between 10 and 12 Å with a switching function. Both systems were relaxed by 2000 steps of conjugate gradient energy minimization and molecular dynamics initiated by assigning random velocities to the atoms consistent with a temperature of 300 K. A 1 fs time step was used for the molecular dynamics integration. Two 100 ps trajectories were generated for each system: first the temperature was maintained at 300 K; second, the temperature was ramped at 10 K per 10 ps to a final temperature of 390 K. In the second protocol the solvent is retained by the layer of tethered waters until the final temperature when the unconstrained water starts to boil off.

Structures were viewed and manipulated using InsightII (95) and calculations were carried out using Discover (2.97) (MSI/Biosym) and the CVFF force field.


    Results
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 Abstract
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 Materials and methods
 Results
 Discussion
 References
 
Kinetics and binding

Exchange of serine for leucine at position 163 in hH4LDH apparently results in the complete removal of substrate inhibition at measurable pyruvate concentrations (Figure 1Go). The reduction in rate observed when the pyruvate concentration is above 100 mM is similar to the effect of sodium chloride at these concentrations, and hence is most likely due to a general ionic strength effect (Figure 1Go, inset). However, the onset of substrate inhibition at these elevated concentrations cannot be ruled out. Nevertheless, it is possible to state that the Ki for pyruvate, which is 0.8 mM in the wild-type protein, is increased to >500 mM in the S163L mutant hH4LDH. The pyruvate KM was also dramatically increased in the mutant protein (from 41 µM to 12 mM). The S163L mutant hH4LDH had reduced maximum activity (~50%) when compared with the wild-type protein.

Equilibrium binding constants for NADH to wild-type and mutant proteins showed that binding of NADH to the S163L mutant was eight times weaker than in wild-type hH4LDH. The binding of NAD+ was moderately weakened in the S163L mutant (165 to 230 µM). Values of Kd for oxamate binding to the enzyme–NADH complex were 9 µM for the wild-type and 6.7 mM for the S163L mutant protein, which is in accordance with the raised KM for pyruvate in the mutant. This is also consistent with the fact that the S163L mutant did not bind to the oxamate affinity column. Kinetic and equilibrium binding constant measurements made on hH4LDH and S163L proteins are summarized in Table IGo.


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Table I. Comparison of kinetic and equilibrium binding data for hH4LDH, hM4LDH, bsLDH and their respective S163L mutants
 
It is interesting that the LDH from Plasmodium falciparum (pfLDH), which is the only natural LDH to have leucine rather than serine at position 163 (Dunn et al., 1996Go), shows an enhanced reactivity with the non-natural cofactor APAD+. Indeed, this reactivity is exploited in a particular test for malarial parasitaemia (Makler and Hinricks, 1993Go). The human S163L mutants behave in a similar fashion and the enhancement of APAD+ reactivity in the S163L mutants compared with the wild-type proteins is given by the rate ratios shown in Table IIGo. These results are similar to those previously observed for bsLDH (Hewitt et al., 1997Go).


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Table II. Comparison of the relative rates of lactate oxidation by wild-type hH4LDH, hM4LDH and S163L mutants using APAD+ and NAD+ ([coenzyme] = 3 mM)
 
Modelling of enzyme–NADH binary complexes:

The results above show that, although the mutation is in the coenzyme binding site, the main effect is on the enzyme–pyruvate interaction. Since the coenzyme and substrate bind in an obligatory order, it appears likely that the binary complex in the case of the mutant enzyme has become less able to accept pyruvate. Molecular modelling was used to explore alternative binding conformations of NADH which might correspond to a non-productive binary complex [E.NADH]np, in turn explaining the high KM for pyruvate as illustrated in Scheme 2. The torsion angle between the dihydronicotinamide and the ribose ring of the coenzyme {Phi}NR (Figure 3Go) determines the syn or anti relationship of these groups. During all four molecular dynamics trajectories the values of {Phi}NR remain in the initial anti conformation, fluctuating around 180 ± 40°. Despite the persistence of the anti conformation, a significant conformational transition occurs elsewhere in the NADH of the S163L mutant. This event occurs in both the room-temperature and the heated trajectories and is revealed by monitoring the distance between the C{alpha} atom of residue 163 and the amide carbon of the dihydronicotinamide amide group (C7, Figure 3Go). This distance in the wild-type complex fluctuates around 7.5 Å in both trajectories for the full 100 ps. By contrast, this distance in the S163L mutant, while starting off around 7.4 Å, extends to 10 Å within the first 20 ps of both the room-temperature and heated simulations. Representative structures before and after this transition are shown in Figure 4Go. The transition corresponds approximately to a +30° rotation around the ribose C5–OP ester linkage (while actually being made up of several smaller torsional changes in this region of the NADH). The net effect of this transition is to translate the dihydronicotinamide ring from its normal binding pocket so that it intrudes into the substrate (pyruvate) binding pocket.



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Fig. 3. The anti and syn conformations of NADH–NAD+. The nicotinamide–ribose torsion angle {Phi}NR is defined by atoms C6–N1–C1–H1. The four hydrogen atoms shown are those monitored in the transfer NOE experiments.

 




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Fig. 4. Representative structures from the S163L–NADH room-temperature MD trajectory (before and after the conformational transition). The dotted lines represent the position where pyruvate would bind (taken from the pig LDH crystal structure). (a) After 5 ps; (b) after 55 ps. Scheme 1 Scheme 2

 
Inhibitory adduct formation

The formation of the `on-enzyme' inhibitory adduct between pyruvate and NAD+ can be detected by monitoring its UV absorption (Gutfreund et al., 1968Go). Figure 2Go shows time courses of such reactions catalysed by wild-type human LDHs at two different concentrations of NAD+ and pyruvate. The final absorbance values indicate that the proteins are saturated under these conditions. When the same reactions are carried out with the S163L mutants, little inhibitory adduct is formed. This shows that the loss of substrate inhibition in these mutants is due to a reduced ability to form the `on-enzyme' inhibitory adduct.



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Fig. 2. Inhibitory adduct formation (enzyme–NAD+–pyruvate). The reaction was followed by the increase in aborbance at 327 nm. Conditions: [protein] = 15 µM; WTA = hM4LDH; WTB = hH4LDH; SLA = S163L hM4LDH; SLB = S163L hH4LDH; • = WTA, 6 mM NAD+, 30 mM pyr; {circ} = WTB, 6 mM NAD+, 30 mM pyr; {blacksquare} = SLA, 6 mM NAD+, 30 mM pyr; {square} = SLB, 6 mM NAD+, 30 mM pyr; {blacktriangleup} =WTA, 1 mM NAD+, 5 mM pyr; {triangleup} = WTB, 1 mM NAD+, 5 mM pyr; {blacktriangledown} = SLA, 1 mM NAD+, 5 mM pyr; {triangledown} = SLB, 1 mM NAD+, 5 mM pyr.

 
NMR of enzyme–NAD+ binary complexes:

The binding conformations of NAD+ to both wild-type and mutant hH4LDHs were determined in order to investigate the inability of the S163L [E.NAD+] binary complex to form the inhibitory adduct with NAD+. The NMR experiments show the build-up of NOEs between the proton pairs H2–H1 and H6–H2r (Figure 3Go), indicating that these protons are in close proximity and thus the nicotinamide–ribose glycosidic bond is in an anti conformation. The orientation about this bond is the same in both the wild-type and the S163L hH4LDHs. This contrasts with both bsLDH (J.J.Holbrook, unpublished results) and dogfish LDH (Vincent et al., 1997Go), where mixtures of syn and anti conformers were seen. However, the result cannot explain the lack of reactivity of the mutant–NAD+ complex with pyruvate since the anti conformation corresponds to the redox-active conformation of the cofactor.


    Discussion
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 Abstract
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 Materials and methods
 Results
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 References
 
Kinetic and binding data for the hH4LDH wild-type and mutant proteins are shown in Table IGo and compared with previously determined values for hM4LDH and bsLDH. The removal of substrate inhibition in the hH4LDH S163L mutant is dramatic, with the wild-type Ki value of 0.8 mM rising to >500 mM in the mutant. The reduction in rate above 100 mM pyruvate is consistent with an ionic strength effect as discussed in the Results section. While the kcat for pyruvate reduction is lower in the mutant, the effect is modest, with the kcat being about half of that for the wild-type. More striking is the degree to which the KM for pyruvate is raised, almost 300 times higher than wild-type. Likewise, the Kd for binding of oxamate (a substrate analogue) to the mutant NADH binary complex is about 800 times higher than to the wild-type binary complex. By contrast, the Kd for NADH binding is only eight times higher in the mutant than the wild-type, despite the mutation being made in the nicotinamide rather than the pyruvate binding pocket. This paradoxical behaviour is resolved by the fact that binding of substrate and cofactor to LDH is an ordered process: NADH is required to bind before pyruvate (Scheme 1). Hence a perturbation in the orientation of the bound dihydronicotinamide can affect subsequent binding of pyruvate. However, the nature of this perturbation must also be reconciled with the fact that the kcat for pyruvate reduction is only modestly compromised in the mutant. A model which accounts for these observations, invokes a productive and a non-productive binding mode for the NADH (Eszes et al., 1996Go) (Scheme 2). Hence a small equilibrium concentration of the productive [E.NADH]p binds pyruvate tightly and gives a ternary complex with only slightly perturbed geometry, and hence a reasonable kcat. The major, non-productive, [E.NADH]np complex cannot bind pyruvate, giving rise to the large apparent KM for pyruvate because [E.NADH]p and [E.NADH]np are in equilibrium and substrate binding has to displace the position of this equilibrium.

Molecular modelling gives a possible structure for the [E.NADH]np complex. During both molecular dynamics simulations (room-temperature and heated) of the wild-type NADH binary complex, the NADH remained in a conformation close to that observed in the crystal structure of known LDH–NADH complexes. In contrast, during both simulations of the S163L mutant binary complex, the nicotinamide–ribose group underwent a conformational transition to a new, persistent, binding geometry within 20 ps. This model of the non-productive binary complex retains the relative orientation of the dihydronicotinamide and ribose rings but moves the nicotinamide ring away from residue 163 into the pyruvate binding pocket (Figure 4Go). Such a binary complex would be unable to bind pyruvate in the normal manner.

Substrate inhibition occurs in LDHs as a consequence of the oxidized substrate (pyruvate) reacting with the oxidized cofactor (NAD+) on enzyme to form a covalent adduct which binds tightly to the active site. Removal of substrate inhibition in the S163L mutant can be accounted for if it is assumed that the nicotinamide group of NAD+ has a similar non-productive binding complex to that invoked for the dihydronicotinamide of NADH above. Such an [E.NAD+]np complex would not be expected to react with pyruvate and form the inhibitory adduct. This behaviour is observed when pyruvate is added to the [LDH.NAD+] binary complex and the UV absorbance at 327 nm (characteristic of the inhibitory adduct) is monitored. Figure 2Go shows the build-up of adduct with both wild-type human proteins (hH4LDH and hM4LDH), whereas only ~10% of the inhibitory adduct is formed by the corresponding S163L mutants under the same conditions.

The Kd of NAD+ binding to the S163L mutants is only modestly raised (about 1.5-fold for hH4LDH), but this is consistent with the fact that the nicotinamide ring typically contributes little or nothing to the overall binding of NAD+ to LDH (Eszes et al., 1996Go). A structural probe for the binding conformation of NAD+ to the hH4LDH is afforded by the transfer NOE experiments. These show that both wild-type hH4LDH and the S163L mutant bind NAD+ in an anti conformation about the nicotinamide–ribose linkage as is always observed in crystal structures of coenzyme bound to LDH. Hence lack of adduct formation cannot be explained by an antisyn reversal of the coenzyme conformation.

With respect to enzyme–coenzyme interactions in general, it is worthy of note that the only other naturally occurring LDH with a leucine at position 163 is that from Plasmodium falciparum. This enzyme shows enhanced reactivity with the non-natural cofactor APAD+. This behaviour is mirrored by the S163L mutants and was investigated by driving the reaction in the reverse direction (lactate and oxidized cofactor as substrates) and comparing the relative activity with NAD+ and APAD+. The cofactor APAD+ differs from NAD+ only in that the nicotinamide CONH2 group is replaced with COCH3. This less polar group is better able to pack against the leucine side chain in the S163L mutants. Consequently, the ratios of activities (APAD+:NAD+) are between two and three orders of magnitude greater for both the hH4LDH and hM4LDH mutants (Table IIGo).

Conclusions

The S163L mutation in hH4LDH removes substrate inhibition while kcat for pyruvate is only modestly reduced. This kinetic behaviour is similar to that previously observed for the same mutation in hM4LDH and bsLDH. Detailed studies of these three enzymes shows that substrate inhibition is ameliorated by perturbing binding of the dihydronicotinamide/nicotinamide ring of the cofactor which is, in turn, propagated into binding of the substrate. Hence we suggest that this mutation is a general method of removing substrate inhibition without drastically compromising the turnover rate of the enzyme. As shown in Figure 1Go, the reduced inhibition means that the mutant hH4LDH is a more effective catalyst at pyruvate concentrations >10 mM. Since malate dehydrogenases are inhibited by a similar mechanism, this approach may be extended to this class of dehydrogenases (C.M.Eszes, unpublished results).


    Acknowledgments
 
C.O.H is a recipient of a Wellcome Trust Prize studentship. C.M.E. thanks the European Union for a TEMPUS studentship. A.R.C. is a Fellow of the Lister Institute.


    Notes
 
1 To whom correspondence should be addressed.E-mail: r.sessions{at}bristol.ac.uk Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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Vincent,S.J.F., Zwahlen,C., Post,C.B., Burgner,J.W. and Bodenhausen,G. (1997) Proc. Natl Acad. Sci. USA, 94, 4383–4388.[Abstract/Free Full Text]

Received August 28, 1998; revised February 5, 1999; accepted February 19, 1999.