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Increased drug resistance to anti-malarials highlights the need for the development of new therapeutics for the treatment of malaria. To this end, the lactate dehydrogenase (LDH) gene was cloned and sequenced from genomic DNA of Plasmodium vivax ( PvLDH) Belem strain. The 316 amino acid protein-coding region of the PvLDH gene was inserted into the prokaryotic expression vector pKK223-3 and a 34 kDa protein with LDH activity was expressed in E. coli. Structural differences between human LDHs and PfLDH make the latter an attractive target for inhibitors leading to novel anti-malarial drugs. The sequence similarity between PvLDH and PfLDH (90% residue identity and no insertions or deletions) indicate that the same approach could be applied to Plasmodium vivax, the most common human malaria parasite in the world.

An homology model of Candida methylica formate dehydrogenase (cm FDH) was constructed based on the Pseudomonas sp. 101 formate dehydrogenase (ps FDH) structure. In wild type cm FDH, Thr169 and Thr226 can form hydrogen bonds with each other. We measured the interaction energy between the two threonines independent of other interactions in the proteins by using a so-called double mutant cycle and assessing the protein stability from the concentration of guanidine hydrochloride needed to denature 50% of the molecules. We conclude that the hydrogen bonds stabilize the wild type protein by -4 kcal mol(-1).

The A245K mutant of Bacillus stearothermophilus L-lactate dehydrogenase has been expressed in Escherichia coli and purified. A qualitative change in the reaction mechanism prior to the hydride transfer step in the reverse direction in the mutant is revealed. Both transient and steady state characteristics of the mutant are presented and show in contrast to the wild-type enzyme where a rearrangement of an enzyme-NADH-pyruvate complex is rate-limiting that in the mutant the rearrangement is much faster and hydride transfer is the first slow step. The steady state is limited by a new second slower conformation change involving an NAD+ complex. The mutation may provide a valuable framework for inhibitor and drug design research.

Bacillus stearothermophillus lactate dehydrogenase (bsLDH) is activated in the presence of fructose 1,6 bisphosphate (FBP). The activator is expensive and representative of the sort of co-factor complications that are undesirable in industrial processes. Three rounds of random mutagenesis and screening produced a mutant (6A) which is almost fully activated in the absence of FBP. Wild-type bsLDH has a K(pyr)(M) of 5 mM in the absence of FBP but when activated (+FBP) the K(pyr)(M) drops to 0.05 mM. The mutant 6A has a K(pyr)(M) of 0.07 mM in the absence of FBP. 6A has three amino acid substitutions-R118C, Q203L and N307S-resulting in a 70-fold activation, none of the mutations are near the active site. The activation of wild type bsLDH is due to an FBP induced tetramerization of dimeric bsLDH bringing about a structural rearrangement of key active site residues. The most likely explanation for the activation of 6A is derived from the position of Q203L, which is at the dimer-dimer interface. The suggestion is that the hydrophilic to hydrophobic change has altered the dimer-tetramer equilibrium position towards that of the tetramer. What is significant is the activation of bsLDH by a subtle long range event produced by the 'blind' directed evolution approach.

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.

The occurrence of large domain motions associated with the mechanism of action of many proteins is well established. We present a general method of predicting domain closure applicable to proteins containing domains separated by an apparent hinge. The method attempts to allow for natural directional bias within the closing protein by repeatedly applying a weak pulling force over a short distance between pairs of atoms chosen at random in the two domains in question. Appropriate parameters governing the pulling function were determined empirically. The method was applied to the bi-lobal protein PGK and a closed-form activated ternary complex generated for Bacillus stearothermophilus PGK. This model was compared with the recently determined crystal structure of closed-form Trypanosoma brucei PGK. The model predicts the correct hinge regions, although the magnitude of movement at one hinge point was overestimated, and provides a reasonable representation of the closed-form ternary complex.

Current methods for reengineering enzyme substrate specificities rely heavily on the use of static x-ray crystallographic models. In this article we detail the use of a molecular mechanics approach for suggesting regions of Bacillus stearothermophilus L-lactate dehydrogenase (EC 1.1.1.27) involved in substrate specificity, and hence areas of interest for protein engineers. The approach combines molecular dynamics with energy minimization (MD/EM) to search the conformational space available to a 15-A sphere of the ternary complex centered on the catalytic histidine. The search is carried out by calculating a 30-ps dynamics trajectory at 300 K and minimizing structures at 1-ps intervals. The protocol has been performed on 14 systems containing different combinations of substrate and mutant/wt LDH. In order to discover which interactions are important in defining substrate specificity, eight conformational parameters representing substrate-active site interactions were measured in each of the 420 minimized structures. These parameters were then compared to the measured catalytic activity of the protein-substrate combinations. These comparisons show that arginine 109 orientation is a major determining factor in LDH specificity. Using this methodology it is possible to estimate the catalytic activity of proteins of varied sequence by computer simulation before synthesis.

Five residues involved in catalysis and coenzyme binding have been identified in D-2-hydroxy-4-methylvalerate dehydrogenase from Lactobacillus delbrueckii subsp. bulgaricus by using biochemical and genetical methods. Enzyme inactivation with diethylpyrocarbonate indicated that a single histidine residue was involved in catalysis. Since H296 is the only conserved histidine in the whole family of NAD-dependent D-2-hydroxyacid dehydrogenases, we constructed the H296Q and H296S mutants and showed that their catalytic efficiencies were reduced 10(5)-fold compared with the wild-type enzyme. This low residual activity was shown to be insensitive to diethylpyrocarbonate. Taken together these data demonstrate that H296 is responsible for proton exchange in the redox reaction. Two acidic residues (D259 and E264) were candidates for maintaining H296 in the protonated state and their roles were examined by mutagenesis. The D259N and E264Q mutant enzymes both showed similar and large reductions in their Kcat/K(m) ratios (200-800-fold, depending on pH), indicating that either D259 or E264 (or both) could partner H296. The conserved R235 residue was a candidate for binding the alpha-carboxyl group of the substrate and it was changed to lysine. The R235K mutant showed a 104-fold reduced Kcat/K(m) due to both an increased K(m) and a reduced Kcat for 2-oxo-4-methylvalerate. Thus R235 plays a role in binding the substrate carboxylate similar to R171 in the L-lactate dehydrogenases. Finally, we constructed the H205Q mutant to test the role of this partially conserved histidine residue (in 10/13 enzymes of the family). This mutant enzyme displayed a 7.7-fold increased Kcat and a doubled catalytic efficiency at pH 5, was as sensitive to diethylpyrocarbonate as the wild-type but showed a sevenfold increased K(m) for NADH and a 100-fold increase in Kd for NADH together with 10-30-fold lower substrate inhibition. The transient kinetic behaviour of the H205Q mutant is as predicted from our previous study on the enzymatic mechanism of D-2-hydroxy-4-methylvalerate dehydrogenase which showed that coenzyme binding is highly pH dependent and indicated that release of the oxidised coenzyme is a significant component of the rate-limiting processes in catalysis at pH 6.5.

The steady-state kinetics of D-2-hydroxy-4-methylvalerate dehydrogenase have been studied at pH 8.0 by initial velocity, product inhibition, and dead-end inhibition techniques. The mechanism is rapid-equilibrium ordered in the NAD+ plus D-2-hydroxy-4-methylvalerate direction, and steady-state ordered in the other direction. In both cases coenzyme is the first substrate added and both the E-NADH-D-2-hydroxy-4-methylvalerate and E-NAD+-2-oxo-4-methylvalerate give rise to abortive complexes which cause excess substrate inhibition. Steady-state measurements show that the rate-limiting step in both directions at pH 8.0 is between formation of the enzyme-coenzyme-substrate ternary complex and the release of the first product of the reaction. Transient kinetics combined with primary kinetic deuterium isotope effects show that in the NADH-->NAD+ direction there is a slow, rate-limiting rearrangement of the E-NADH-oxoacid complex while hydride transfer is very fast. The release of NAD+ at pH 8.0 is 200-times faster than Kcat (NADH-->NAD+) whereas the release of NADH is only 5-times faster than Kcat (NAD+-->NADH). The pH dependence of NADH binding depends upon the presence of two ionizable residues with a pKa of about 5.9. The pH dependence of kinetic parameters is explained by a third ionizable residue with pKa values 7.2 (in the E-NADH complex) and < or = 6.4 (in the E-NAD+ complex) which may be the proton donor and acceptor for the chemical reaction. At pH 6.5 the mechanism changes in the NADH-->NAD+ direction to be partly limited by the chemical step with a measured primary kinetic isotope effect of 5.7 and partly by an only slightly faster dissociation of NAD+. In addition the inhibition by excess oxo-4-methylvalerate is more pronounced. The mechanism implies that removing the positive charges created by the two groups which control coenzyme affinity could both enhance the catalytic rate at pH 6.5 and diminish excess substrate inhibition to provide an enzyme better suited to the bulk synthesis of D-2-hydroxyacids.

This paper describes the testing of a homology model of Plasmodium falciparum lactate dehydrogenase (pfLDH) by protein engineering. The model had been validated in structural terms. It suggests explanations of the unusual properties of pfLDH (compared with all other LDHs). These unusual features are a lack of substrate inhibition, high activity with the synthetic coenzyme 3-acetylpyridine adenine dinucleotide (APAD+) and changes in residues at previously conserved positions. pfLDH shows several amino acid insertions and deletions in an alignment with protein sequences from all other known LDHs. The most notable is a five amino acid insertion into the active-site loop. In addition, a conserved serine at position 163 is replaced by leucine. The results showed that when the unique pfLDH structural features were engineered into Bacillus stearothermophilus lactate dehydrogenase, the thermophilic enzyme acquired the properties previously uniquely associated with the malarial enzyme. We conclude that the homology model of the malarial enzyme is adequate for the prediction of successful redesigns and, in the regions tested, is accurate.

High concentrations of ketoacid substrates inhibit most natural hydroxyacid dehydrogenases due to the formation of an abortive enzyme-NAD+-ketoacid complex. It was postulated that this substrate inhibition could be eliminated from lactate dehydrogenases if the rate of NAD+ dissociation could be increased. An analysis of the crystal structure of mammalian LDHs showed that the amide of the nicotinamide cofactor formed a water-bridged hydrogen bond to S163. The LDH of Plasmodium falciparum is not inhibited by its substrate and, uniquely, in this enzyme the serine is replaced by a leucine. In the S163L mutant of human LDH-M4 pyruvate inhibition is, indeed, abolished and the enzyme retains high activity. However, the major contribution to this effect comes from a weakening of the interaction of pyruvate with the enzyme-coenzyme complex.

A gene library was constructed coding for all possible variants of two amino acids (101, 102) in a solvent-exposed surface return loop (alpha E-beta D) of Bacillus stearothermophilus L-lactate dehydrogenase (bsLDH). All but one of 38 enzyme variants examined were thermally stable and had native-like hydrodynamic properties. In this sample, there was no bias detected in either the DNA or amino acid sequences encoded. We argue that the alpha E-beta D surface loop sequence is unimportant for protein folding or stability and can be fully varied to select enzymes with new substrate specificities. The selection of NAD-dependent dehydrogenases with specificity for: malate, phenyllactate, hydroxyisocaproate and 4-phenyl-2-hydroxy-butanoate from two bsLDH libraries is described. This required a highly discriminatory screen for 2-hydroxy acid dehydrogenase activity to select enzymes which, in the absence of the natural allosteric activator fructose-1,6-bisphosphate (FBP), maintained high temperature stability and catalytic activity without substrate inhibition. In general the amino acid residues at positions 101 and 102 which determined substrate specificity were as expected from hydrophobic and ionic complementarity to the substrate. For example, a bsLDH variant with Asn101Va1102 is as efficient with phenylpyruvate as is the wild-type enzyme (Asn101Gln102) with pyruvate. Using molecular modelling, the valine at position 102 can be fitted into the active site without significant structural distortion caused by the aromatic side-chain of the substrate. Similarly, nine out of ten malate dehydrogenases (MDHs) selected had an arginine residue at position 102 to complement the negatively charged carboxyl group in oxaloacetate. One, Arg101Arg102 (Kcat/Kmoxaloacetate = 1.6 x 10(6) M-1 S-1) is 25% more active than the previous best synthetic MDH. There were surprises: present understanding would not have predicted the oxaloacetate transforming activity of Ser101Leu102 or the phenylpyruvate activity of Pro101Lys102. The former is about one-third as efficient as the best malate dehydrogenase selected, whilst the latter had about one-seventh of the best phenylpyruvate dehydrogenase activity.

Using the polymerase chain reaction, DNA encoding cytosolic malate dehydrogenase (cMDH) has been cloned from a pig heart cDNA library. Large amounts of the enzyme (30 mg per litre of original culture) have been produced in Escherichia coli using an inducible expression vector (pKK223-3) in which the 5'-non-coding region of the gene was replaced with the tac promoter. The complete nucleotide sequence of the DNA is reported for the first time. The recombinant cMDH purified was shown to be identical to the native enzyme according to: chromatographic behaviour, isoelectric point, N-terminal amino acid sequence, and physiochemical and catalytic properties.

NAD-dependent formate dehydrogenase (FDH) was isolated from Candida methylica (Cm) grown on 0.5% methanol. Its N-terminal amino acid (aa) sequence was determined, as was that of a commercial FDH from Candida boidinii. Degenerate oligodeoxyribonucleotides were made to the 5' region of the fdh gene from Cm using this information and to the 3' region using C-terminal aa sequence data from the methylotropic yeast, Hansenula polymorpha. An almost complete 1.1-kb fragment was amplified from Cm genomic DNA via the polymerase chain reaction (PCR). This fragment was cloned, sequenced and used to probe a Southern blot, from which a 3.4-kb EcoRI fragment containing the fdh open reading frame (ORF) was isolated. The complete nucleotide sequence of this fdh ORF was determined and corresponds to a protein of 364 aa (40,343 Da). The ORF of fdh was cloned into pKK223-3 using PCR and transformed into Escherichia coli. Enzymatically active FDH was produced to 15% of soluble E. coli protein. The deduced aa sequence of this FDH is compared to the aa sequences of four known FDH, from bacteria, yeast, fungi and plant mitochondria.

The NAD-dependent D-(-)-lactate dehydrogenase (D-LDH) from Lactobacillus delbrueckii subsp. bulgaricus (in short, L. bulgaricus) has been modified at position 175 by site-directed mutagenesis, changing a conserved aspartate residue into an alanine. The D175A mutant enzyme displays a 40-fold shift in coenzyme preference from NADH to NADPH. This demonstrates that D175 truly belongs to the amino acid consensus GXGXXGX(17)D (where X represents any residue) which is the signature of the coenzyme binding site of most NAD-dependent dehydrogenases.

A genomic library from Lactobacillus delbrueckii subsp. bulgaricus was used to complement an Escherichia coli mutant strain deficient for both lactate dehydrogenase and pyruvate formate lyase, and thus unable to grow anaerobically. One recombinant clone was found to display a broad specificity NAD(+)-dependent D-2-hydroxyacid dehydrogenase activity. The corresponding gene (named hdhD) was subcloned and sequenced. The deduced amino acid sequence of the encoded enzyme indicates a 333-residue protein closely related to D-2-hydroxyisocaproate (i.e. 2-hydroxy-4-methyl-pentanoate) dehydrogenase (D-HO-HxoDH) of Lactobacillus casei and other NAD(+)-dependent D-lactate dehydrogenases (D-LDH) from several other bacterial species. The hdhD gene was overexpressed under the control of the lambda phage PL promoter and the enzyme was purified with a two-step method. The L. delbrueckii subsp. bulgaricus enzyme, like that of L. casei, was shown to be active on a wide variety of 2-oxoacid substrates except those having a branched beta-carbon.

The X-ray structure of lactate dehydrogenase (LDH) shows the side-chain carboxylate group of Asp-143 to be buried in the hydrophobic interior of the enzyme, where it makes hydrogen-bonding interactions with both the side-chain hydroxyl group of Ser-273 and the main-chain amide group of His-195. This is an unusual environment for a carboxylate side-chain as hydrogen bonding normally occurs with water molecules at the surface of the protein. A charged hydrogen-bonding interaction in the interior of a protein would be expected to be much stronger than a similar interaction on the solvent-exposed exterior. In this respect the side-chain carboxylate group of Asp-143 appears to be important for maintaining tertiary structure by providing a common linkage point between three discontinuous elements of the secondary structure, alpha 1F, beta K and the beta-turn joining beta G and beta H. The contribution of the Asp-143 side-chain to the structure and function of Bacillus stearothermophilus LDH was assessed by creating a mutant enzyme containing Asn-143. The decreased thermal stability of both unactivated and fructose-1,6-diphosphate (Fru-1,6-P2)-activated forms of the mutant enzyme support a structural role for Asp-143. Furthermore, the difference in stability of the wild-type and mutant enzymes in guanidinium chloride suggested that the carboxylate group of Asp-143 contributes at least 22 kJ/mol to the conformational stability of the wild-type enzyme. However, there was no alteration in the amount of accessible tryptophan fluorescence in the mutant enzyme, indicating that the mutation caused a structural weakness rather than a gross conformational change. Comparison of the wild-type and mutant enzyme steady-state parameters for various 2-keto acid substrates showed the mutation to have a general effect on catalysis, with an average difference in binding energy of 11 kJ/mol for the transition-state complexes. The different effects of pH and Fru-1,6-P2 on the wild-type and mutant enzymes also confirmed a perturbation of the catalytic centre in the mutant enzyme. As the side-chain of Asp-143 is not sufficiently close to the active site to be directly involved in catalysis or substrate binding it is proposed that the effects on catalysis shown by the mutant enzyme are induced either by a structural change or by charge imbalance at the active site.(ABSTRACT TRUNCATED AT 400 WORDS)

mMDH and cMDH are structurally homologous enzymes which show very different responses to chaperonins during folding. The hydrophilic and stable cMDH is bound by cpn60 but released by Mg-ATP alone, while the hydrophobic and unstable mMDH requires both Mg-ATP and cpn10. Citrate equalises the stability of the native state of the two proteins but has no effect on the co-chaperonin requirement, implying that hydrophobicity, and not stability, is the determining factor. The yield and rate of folding of cMDH is unaffected while that of mMDH is markedly increased by the presence of cpn60, cpn10 and Mg-ATP. In 200 mM orthophosphate, chaperonins do not enhance the rate of folding of mMDH, but in low phosphate concentrations chaperonin-assisted folding is 3-4-times faster.

The crystal structure of a mutant Bacillus stearothermophilus lactate dehydrogenase, into which an additional loop has been engineered in order to prevent tetramerization of the enzyme, has been solved and refined at 2.4 A. The minimal repeat unit in the crystal is a dimer and the tetramer cannot be generated by any of the crystallographic symmetry operations in P2(1). The loop protrudes out into the solvent, stabilized by a good hydrogen bonding arrangement, and clearly sterically hinders tetramer formation. This is the first structure of B. stearothermophilus lactate dehydrogenase (bsLDH) in which the allosteric activator fructose, 1,6-bisphosphate (FBP) is not present. To investigate the mechanism of allosteric activation in this enzyme we have compared the structure with a ternary complex of B. stearothermophilus lactate dehydrogenase. Many of our observations confirm those reported from a comparison of FBP-bound ternary bsLDH complex with an FBP free LDH from another bacterial source, Bifidobacterium longum. Our results suggest that quaternary structural alterations may have less influence on the mechanism than previously reported. The differences in the quaternary structural behaviour of these two enzymes is discussed.

A prerequisite for the rational redesign of enzymes is that altering amino acids in an attempt to obtain new biological function does not unexpectedly alter the globular, natural framework of the native protein on which the design is being executed. The results of combinatorial-mutagenesis strategies suggest that random variation of amino acid sequence is most easily tolerated at the solvent-exposed surfaces of a protein. This review analyses effective redesigns of Bacillus stearothermophilus lactate dehydrogenase (bsLDH), in which all residue variations are at solvent-exposed surfaces. The majority of these variations were located within surface loops, which interconnect stable secondary structures traversing the globular core of the protein.

The Raman spectra of both the NAD-pyruvate and the pyridine aldehyde adenine dinucleotide (PAAD)-pyruvate bound to pig heart, pig muscle, and Bacillus stearothermophilus lactate dehydrogenases were measured and are nearly the same, which is consistent with the conserved shell of residues surrounding the active-site cavity in these enzymes. The symmetrical stretching mode of the pyruvate carboxylate group, found at 1398 cm-1, is shifted only slightly when complexed to these enzymes, which shows that the group remains ionized in the ion pair complex with Arg-171 on the enzyme. The vibrational mode for the carbonyl stretch of the bound pyruvate moiety is shifted about 35 cm-1 to a lower frequency than observed for the carbonyl of unliganded pyruvate in the bacterial enzyme because of polarization of the carbonyl bond. Thus, the bacterial enzyme shows the same substrate activation because of the C(+)-O- charge separation that was seen previously with the mammalian enzymes. On the basis of an empirical Badger-Bauer relationship between frequency shift and interaction enthalpy, this shift in frequency is equivalent to an approximately -14 to -17 kcal/mol interaction between the enzyme and the adduct C = O coordinate, a substantial part of which is an electrostatic interaction (hydrogen bond) between the C V O and the protonated His-195. Thus, while the C = O bond is polarized on the enzyme (which requires energy), the overall ground-state enthalpy of the carbonyl imidazolium part of the reaction coordinate is stability substantially relative to its value in solution, and this is the dominant enthalpic effect on the entire reaction coordinate since the other internal coordinates for the hydride transfer are not much affected during formation of the ternary complex.(ABSTRACT TRUNCATED AT 250 WORDS)

The Gln residue at amino acid position 102 of Bacillus stearothermophilus lactate dehydrogenase was replaced with Ser, Thr, Tyr, or Phe to investigate the effect on substrate recognition. The Q102S and Q102T mutant enzymes were found to have a broader range of substrate specificity (measured by kcat/Km) than the wild-type enzyme. However, it is evident that either Ser or Thr at position 102 are of a size able to accommodate a wide variety of substrates in the active site and substrate specificity appears to rely largely on size discrimination in these mutants. The Q102F and Q102Y mutant enzymes have low catalytic efficiency and do not show this relaxed substrate specificity. However, their activities are restored by the presence of an aromatic substrate. All of the enzymes have a very low catalytic efficiency with branched chain aliphatic substrates.

The reversible unfolding of two dissimilar proteins, phosphoglycerate kinase from Bacillus stearothermophilus (PGK) and Staphylococcus aureus nuclease (SAN), was induced with two denaturants, urea and guanidinium chloride (GuHCl). For each protein, structural transitions were monitored by intrinsic fluorescence intensity changes arising from a unique tryptophan residue. In the case of SAN the single, native tryptophan residue was used, whereas for PGK two versions, one with a tryptophan at position 315 and one at 379, were constructed genetically. The resultant folding curves were analyzed by considering the change in the solvation free energy of internal amino acid residues as the denaturant concentration was varied. We derive the following simple relationship: -RT ln K = delta Gw + n delta Gs,m[D]/Kden. + [D]) where K is the equilibrium constant describing the distribution of folded and unfolded forms at a given denaturant concentration [D], delta Gw is the free energy change for the transition in the absence of denaturant, and n is the number of internal side chains becoming exposed. delta Gs,m and Kden. are constants derived empirically from the solvation energies of model compounds and represent the behavior of an average internal side chain between 0 and 6 M GuHCl and 0 and 8 M urea. For proteins of known structure these values can easily be derived, and for others, average values in guanidinium chloride (delta Gs,m = 0.775 kcal/mol and Kden. = 5.4 M) or urea (delta Gs,m = 1.198 kcal/mol and Kden. = 25.25 M) can be used in the analysis. Results show that the parameters n and delta Gw are independent of the denaturant used for all 12 transitions studied. This supports the hypothesis that the unfolding activity of urea and GuHCl can be accounted for by their effect on the solvation energy of amino acid side chains which are buried in the folded but exposed in the unfolded protein. This simple analytical treatment allows the "cooperativity" of protein folding to be interpreted in terms of the number of side chains becoming exposed to the solvent in a given step and allows accurate estimation of the free energy irrespective of the denaturant concentration needed to induce the transition.

Cpn60 was labeled with pyrene maleimide in order to follow structural rearrangements in the protein triggered by the binding of nucleotides and cpn10. The conjugate binds ATP, AMP-PNP, and ADP(P(i)) with pyrene fluorescence enhancements of 60%, 60%, and 15%, respectively. In each case, binding is cooperative with half-saturation (K1/2) occurring at 10 microM, 290 microM, and 2500 microM and Hill constants (nH) of 4, 3, and 3, respectively. Inclusion of the co-protein, cpn10, tightens the binding of ATP, AMP-PNP, and ADP(P(i)) to give K1/2 values of 6 microM, 100 microM, and < 0.07 microM, respectively, and cooperativity is increased. Titration of the cpn60/ADP (14-mer) complex with cpn10 (7-mer) gives a stoichiometry of 14:7 with respect to subunits, confirming the molecular asymmetry shown by electron microscopy. Transient kinetics demonstrate that ATP initially forms a weak collision complex with cpn60 (Kd = 4 mM) which isomerizes to the strongly binding state at a rate of 180 s-1. We suggest that the slow structural rearrangement driven by ATP binding is the same event which lowers the affinity of the chaperonin for protein substrates; a suggestion reinforced by the loss of AMP-PNP binding affinity in the presence of an unstructured polypeptide. As such, this rearrangement of cpn60 is analogous to a force-generating step in energy transduction. Measurements of ATP hydrolysis (pH 7.5, 25 degrees C) show that it is slow (0.04 s-1) compared both with the structural rearrangement and with the dissociation of products. This defines the steady-state complex as cpn60/ATP, a form of the chaperonin which binds substrate proteins weakly. The rate of hydrolysis of ATP is stimulated 20-fold upon binding unfolded lactate dehydrogenase, and the yield of folded enzyme is increased even in the absence of cpn10. Addition of this co-protein inhibits hydrolysis on only half of the sites in cpn60 and leads to a faster release of folded LDH. A mechanism for the action of chaperonins is proposed which depends upon cpn60 being cycled between states which have, alternately, low and high affinity for unfolded proteins. This cycle is driven by the binding and hydrolysis of ATP.

X-ray crystallography predicts hydrogen-bonding interactions between the side chains of Thr198 and two other amino acid residues, Glu194 (adjacent to the catalytic His195) and Ser318 (on the alpha-H helix which rearranges on substrate binding). In order to investigate the contribution of this conserved amino acid residue, Thr198, two mutants of Bacillus stearothermophilus lactate dehydrogenase were created (Val198 and Ile198). The steady-state kinetic parameters for both mutant enzymes were very similar with increased substrate Km and reduced kcat when compared with the wild-type enzyme. The mutation Val198 allowed non-productive binding of pyruvate to the unprotonated form of His195. Steady-state kinetic parameters determined for the Val198 mutant enzyme in high solvent viscosity suggested both an altered rate-limiting step in catalysis and implicated Thr198 in allosteric activation by the effector fructose 1,6-bisphosphate (Fru1,6P2). A shift in the Fru1,6P2 activation constant for the Val198 mutant enzyme suggested that Thr198 stabilises the catalytically competent (Fru1,6P2-activated) form of the enzyme by 6.6 kJ/mol. However, Thr198 was not important for maintaining the thermal stability of the Fru1,6P2-activated form. Equilibrium unfolding in guanidinium chloride indicated that Thr198 contributes 17.2 kJ/mol subunits towards the tertiary structural stability. The results emphasise the importance of the side chain-hydroxyl group of Thr198 which is required for (a) productive substrate binding, (b) allosteric activation and (c) protein conformational stability. The characteristics of the B. stearothermophilus lactate dehydrogenase mutations reported here were significantly different from those of the same mutations made in the corresponding position of the analogous enzyme Thermus flavus malate dehydrogenase [Nishiyama, M., Shimada, K., Horinouchi, S., & Beppu, T. (1991) J. Biol. Chem. 266, 14294-14299].

The malate dehydrogenase from Escherichia coli has been specifically altered at a single amino acid residue by using site-directed mutagenesis. The conserved Arg residue at amino acid position 102 in the putative substrate binding site was replaced with a Gln residue. The result was the loss of the high degree of specificity for oxaloacetate. The difference in relative binding energy for oxaloacetate amounted to about 7 kcal/mol and a difference in specificity between oxaloacetate and pyruvate of 8 orders of magnitude between the wild-type and mutant enzymes. These differences may be explained by the large hydration potential of Arg and the formation of a salt bridge with a carboxylate group of oxaloacetate.

A mutant Bacillus stearothermophilus lactate dehydrogenase has been prepared in which all three tryptophan residues in the wild-type enzyme have been replaced by tyrosines. In addition, a tyrosine residue has been mutated to a tryptophan, which acts as a fluorescence probe to monitor protein folding. The mutant enzyme crystallizes in the same crystal form as the wild-type. The crystal structure of the mutant has been determined at 2.8 A resolution. Solution studies have suggested that there is little effect upon the mutant enzyme as judged by its kinetic properties. Comparison of the crystal structures of the mutant and wild-type enzymes confirms this conclusion, and reveals that alterations in structure in the region of these mutations are of a similar magnitude to those observed throughout the structure, and are not significant when compared with the errors in atomic positions expected for a structure at this resolution.

Restriction sites were introduced into the gene for Bacillus stearothermophilus lactate dehydrogenase which enabled a region of the gene to be excised which coded for a mobile surface loop of polypeptide (residues 98-110) which normally seals the active site vacuole from bulk solvent and is a major determinant of substrate specificity. Oligonucleotide-overlap extension (using the polymerase chain reaction) was used to obtain double-stranded DNA regions which coded for different length and sequence loops and which also contained the same restriction sites. The variable length and sequence loops were inserted into the cut gene and used to synthesize hydroxyacid dehydrogenases with altered substrate specificities. Loops which were longer and shorter than the original were made. The substrate specificities of enzymes with these new loops were considerably altered. For many poor enzyme-substrate pairs, the effect of fructose 1,6-bisphosphate on the steady-state kinetic parameters suggested that the substrate was mainly bound in a nonproductive mode. With one longer loop construction (BL1), activity with pyruvate was reduced one-million-fold but activity with phenylpyruvate was largely unaltered. A switch in specificity (kcat/KM) of 390,000-fold was achieved. The 1700:1 selectivity of enzyme BL1 for phenylpyruvate over pyruvate is that required in a phenyllactate dehydrogenase to be used in monitoring phenylpyruvate in the urine of patients with phenylketonuria consuming an apparently phenylalanine-free diet.

The proposal that the active site vacuole of NAD(+)-S-lactate dehydrogenase is unable to accommodate any imbalance in electrostatic charge was tested by genetically manipulating the cDNA coding for human muscle lactate dehydrogenase to make a protein with an aspartic acid introduced at position 140 instead of the wild-type asparagine. The Asn 140-Asp mutant enzyme has the same kcat as the wild type (Asn 140) at low pH (4.5), and at higher pH the Km for pyruvate increases 10-fold for each unit increase in pH up to pH 9. We conclude that the anion of Asp 140 is completely inactive and that it binds pyruvate with a Km that is over 1,000 times that of the Km of the neutral, protonated aspartic-140. Experimental results and molecular modeling studies indicate the pKa of the active site histidine-195 in the enzyme-NADH complex is raised to greater than 10 by the presence of the anion at position 140. Energy minimization and molecular dynamics studies over 36 ps suggest that the anion at position 140 promotes the opening of and the entry of mobile solvent beneath the polypeptide loop (98-110), which normally seals off the internal active site vacuole from external bulk solvent.

A cDNA for duck liver 'malic' enzyme (EC 1.1.1.40) was subcloned into pUC-8, and the active enzyme was expressed in Escherichia coli TG-2 cells as a fusion protein including a 15-residue N-terminal leader from beta-galactosidase coded by the lacZ' gene. C99S and R70Q mutants of the enzyme were generated by the M13 mismatch technique. The recombinant enzymes were purified to near homogeneity by a simple two-step procedure and characterized relative to the enzyme isolated from duck liver. The natural duck enzyme has a subunit molecular mass of approx. 65 kDa, and the following kinetic parameters for oxidative decarboxylation of L-malate at pH 7.0: Km NADP+ (4.6 microM); Km L-malate (73 microM); kcat (160 s-1); Ka (2.4 microM) and Ka' (270 microM), dissociation constants of Mn2+ at 'tight' (activating) and 'weak' metal sites; and substrate inhibition (51% of kcat. at 8 mM-L-malate). Properties of the E. coli-derived recombinant wild-type enzyme are indistinguishable from those of the natural duck enzyme. Kinetic parameters of the R70Q mutant are relatively unaltered, indicating that Arg-70 is not required for the reaction. The C99S mutant has unchanged Km for NADP+ and parameters for the 'weak' sites (i.e. inhibition by L-malate, Ka'); however, kcat. decreased 3-fold and Km for L-malate and Ka each increased 4-fold, resulting in a catalytic efficiency [kcat./(Km NADP+ x Km L-malate x Ka)] equal to 3.7% of the natural duck enzyme. These results suggest that the positioning of Cys-99 in the sequence is important for proper binding of L-malate and bivalent metal ions.

A model has been built for the plant NADP-malate dehydrogenase from Zea mays, a key enzyme in photosynthesis, which undergoes light-dependent regulation. The model was based on sequence and presumed structural homology to the known three-dimensional structure of mammalian porcine cytosolic NAD-malate dehydrogenase. A cystine-loop present in an extended C-terminal region of plant NADP-malate dehydrogenases was modelled using molecular mechanics and computer graphical methods, based on the assumption that a disulphide bridge exists in the inactive form of the enzyme between Cys351 and Cys363. The predicted conformation of the intact C-terminal cystine-loop suggests that the extended polypeptide will bind in the active centre and inhibit enzyme activity. Another ionizable cysteine residue in the active site is predicted to control the charge of the catalytic His215 and might be responsible for the uniquely tight binding of the positively charged nicotinamide ring of NADP+ in this and other C4 and C3 plant NADP-malate dehydrogenases.

We report the refined structure of a ternary complex of an allosterically activated lactate dehydrogenase, including the important active site loop. Eightfold non-crystallographic symmetry averaging was utilized to improve the density maps. Interactions between the protein and bound coenzyme and oxamate are described in relation to other studies using site-specific mutagenesis. Fructose 1,6-bisphosphate (FruP2) is bound to the enzyme across one of the 2-fold axes of the tetramer, with the two phosphate moieties interacting with two anion binding sites, one on each of two subunits, across this interface. However, because FruP2 binds at this special site, yet does not possess an internal 2-fold symmetry axis, the ligand is statistically disordered and binds to each site in two different orientations. Binding of FruP2 to the tetramer is signalled to the active site principally through two interactions with His188 and Arg173. His188 is connected to His195 (which binds the carbonyl group of the substrate) and Arg173 is connected to Arg171 (the residue that binds the carboxylate group of the substrate).

When Bacillus stearothermophilus LDH dimer is incubated with increasing concentrations of the denaturant guanidinium chloride, three distinct unfolded states of the molecule are observed at equilibrium [Smith, C. J., et al. (1991) Biochemistry 30, 1028-1036]. The kinetics of LDH refolding are consistent with an unbranched progression through these states. The Escherichia coli chaperonin, GroEL, binds with high affinity to the completely denatured form and more weakly to the earliest folding intermediate, thus retarding the refolding process. A later structurally defined folding intermediate, corresponding to a molten globule form, is not bound by GroEL; neither is the inactive monomer. The complex between GroEL and denatured LDH is destabilized by the binding of magnesium/ATP (Mg/ATP) or by the nonhydrolyzable analogue adenylyl imidodiphosphate (AMP-PNP). From our initial kinetic data, we propose that GroEL exists in two interconvertible forms, one of which is stabilized by the binding of Mg/ATP but associates weakly with the unfolded protein. The other is destabilized by Mg/ATP and associates strongly with unfolded LDH. The relevance of these findings to the role of GroEL in vivo is discussed.

New substrate specificities can be introduced into existing enzymes for the purpose of making them more suitable for the chemoenzymic synthesis of single compound drugs and other chiral compounds. The most productive route used in the past year has involved the utilization of the catalytic and substrate-binding properties from homologous enzymes found in nature, one example being the broadening of the substrate specificity of yeast alcohol dehydrogenase. Other highlights include the creation of thermostable dehydrogenases that will interconvert NADPH and NADH, and the design of mutant enzymes with improved catalytic rates compared with their wild-type counterparts.

Analysis of the mechanism and structure of lactate dehydrogenases is summarized in a map of the catalytic pathway. Chemical probes, single tryptophan residues inserted at specific sites and a crystal structure reveal slow movements of the protein framework that discriminate between closely related small substrates. Only small and correctly charged substrates allow the protein to engulf the substrate in an internal vacuole that is isolated from solvent protons, in which water is frozen and hydride transfer is rapid. The closed vacuole is very sensitive to the size and charge of the substrate and provides discrimination between small substrates that otherwise have too few functional groups to be distinguished at a solvated protein surface. This model was tested against its ability to successfully predict the design and synthesis of new enzymes such as L-hydroxyisocaproate dehydrogenase and fully active malate dehydrogenase. Solvent friction limits the rate of forming the vacuole and thus the maximum rate of catalysis.

L-Lactate dehydrogenase from Bacillus stearothermophilus was rebuilt by using site-directed mutagenesis to produce an enzymically active, tryptophan-less enzyme by replacing all the wild-type tryptophans (80, 150, and 203) by tyrosines. Nine single tryptophan-containing active enzymes were constructed from this enzyme by genetically replacing one of the tyrosines 36, 85, 147, 190, 203, 237, 248, 279, or 285 by tryptophan. The equilibrium and the time-resolved tryptophan fluorescence intensity and anisotropy were used to report unfolding events in guanidine hydrochloride (GHCl) monitored from these nine defined positions. Three structural transitions, half complete at 0.55, 1.7, and 2.8 M GHCl, were identified and defined four folding intermediates, I (native), II (expanded monomer 1), III (expanded monomer 2), and IV (random coil), stable at 0, 1, 2.2, and 4 M GHCl, respectively. Intermediate II is a globular monomer. All the probed alpha-helices and most of the beta-structure was intact. There was an increase in the rate but not the extent of the mobilities of six of the probed tryptophan side chains, indicating loss of tertiary structure. Circular dichroism (CD) showed all the secondary structure to be intact. Intermediate III is monomeric and still globular, but the tryptophan anisotropy indicated an increase mobility at positions 36, 85, 190, 203, 279, and 285. Helix alpha-B is further disrupted but helices alpha-1F, alpha-2G, and alpha 3G were still rigid. CD showed half the secondary structure to be still intact. Intermediate IV is a random coil in which all tryptophans have complete rotational freedom and the helix CD signal is lost.(ABSTRACT TRUNCATED AT 250 WORDS)

Variations have been made to the structure of the nicotinamide adenine dinucleotide (NAD) dependent L-lactate dehydrogenase from Bacillus stearothermophilus at regions of the enzyme that we believe determine specificity toward different alpha-hydroxy acids (RCHOHCOO-, R = CH3, C2H5, etc.). Two regions of LDH that border the active site (but are not involved in the catalytic reaction) were altered in order to accommodate substrates with hydrophobic side chains larger than that of the naturally preferred substrate, pyruvate (R = CH3). The mutations 102-105GlnLysPro----MetValSer and 236-237AlaAla----GlyGly were made to increase the tolerance for large hydrophobic substrate side chains. The triple and double mutants alone gave little improvement for branched-chain-substituted pyruvates. The five changes together produced a broader substrate specificity alpha-hydroxy acid dehydrogenase, with a 55-fold improved kcat for alpha-ketoisocaproate to a value about 1/14 that of the native enzyme for pyruvate. Rational protein engineering enabled coupled changes in enzyme structure to be obtained with greater probability of success than random mutagenesis.

The human LDH-A and LDH-B cDNAs, containing the coding regions for the L-lactate dehydrogenase A4 (M) and B4 (H) polypeptides respectively have been cloned into Escherichia coli to place the cDNAs under the control of hybrid E. coli/Bacillus stearothermophilus transcriptional and translational signals. Human A4- and B4-isoenzymes are produced in E. coli cells harbouring the expression plasmids pHLDHA22 and pHLDHB10 at levels of 6.5 and 1.5% of the soluble protein of the cell, respectively. The tac promoter of these vectors was not induced by isopropyl beta-D-thiogalactopyranoside. The A4 and B4 human isoenzymes synthesized in E. coli were purified to homogeneity and show the same properties as isoenzymes isolated from human tissue. The amino acid sequences of 12 N-terminal residues of the human isoenzymes synthesized in E. coli were determined to be identical to those deduced from the DNA sequence of the cloned cDNAs except that the N-terminal methionine was absent from both. However, in contrast to LDH made in human cells, acetylation of the N-terminal alanine does not take place in E. coli cells.

Using site-directed mutagenesis, the NADH-linked lactate dehydrogenase from Bacillus stearothermophilus has been specifically altered at a single residue to shift the coenzyme specificity towards NADPH. The single change is at position 53 in the amino acid sequence where a conserved aspartate has been replaced by a serine. This substitution was made to reduce steric hindrance on binding of the extra phosphate group of NADPH and to remove the negative charge of the aspartate group. The resultant mutant enzyme is 20 times more catalytically efficient than the wild-type enzyme with NADPH.

A site-directed mutant of Bacillus stearothermophilus lactate dehydrogenase (lactate:NAD+ oxidoreductase, EC 1.1.1.27) has been engineered in which the conserved hydrophobic residue isoleucine-250 has been replaced by the more hydrophilic residue asparagine. This isoleucine forms a large part of a water-accessible, hydrophobic surface in the active site of the apo-enzyme which is covered by the B-face of the nicotinamide ring when coenzymes are bound. Reduction in the area of this hydrophobic surface results in the mutant tetramer being more thermally stable than the wild-type enzyme.

Horse liver alcohol dehydrogenase specifically carboxymethylated on cysteine-46 (a ligand to the zinc in the active site) or acetimidylated on 25 of the 30 lysine residues per subunit (including residue 228) was studied. The tryptophan fluorescence of these enzymes decreased by 35% as pH was increased, with an apparent pKa of 9.8 +/- 0.2, identical with that of native enzyme. Native enzyme in the presence of 30mM-imidazole, which displaces a water molecule ligated to the zinc, also had a pKa of 9.8. The ionoizable group is thus neither the water molecule nor one of the modified groups. Binding of NAD+ shifted the pKa for the fluorescence transition to 7.6 with native enzyme and to 9.0 with acetimidylated enzyme, but did not shift the pKa of carboxymethylated enzyme. Binding of NAD+ and trifluoroethanol, an unreactive alcohol, gave maximal fluorescence quenching at pH7 with all three enzymes. The acetimidylated enzyme--NAD+--trifluoroethanol complex had an apparent pKa of 5.0, but the pK of the native enzyme complex was experimentally inaccessible. The results are interpreted in terms of coupled equilibria between two different conformational states. On binding of NAD+, the modified enzymes apparently change conformation less readily than does native enzyme, but binding of alcohol can drive the change to completion.