J. John Holbrook

From analysis to synthesis: new ligand binding sites on the lactate dehydrogenase framework. Part II

In Part I of this article (published in the March issue of TIBS1), substrate-binding and catalysis in lactate dehydrogenase were examined by genetic modification of the protein structure and analysis of the functional consequences. In Part II, the conclusions are used in the design and synthesis of two modified forms of the enzyme; one in which the substrate specificity is shifted to produce a more effective malate dehydrogenase than that isolated from the host organism and one which no longer requires its allosteric activator (fructose 1,6-bisphosphate).

A single amino acid substitution in lactate dehydrogenase improves the catalytic efficiency with an alternative coenzyme

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 The single change is at position 53 in the amino acid sequence where a conserved aspartate has been replaced by a serine.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.

Approaches to the study of enzyme mechanisms lactate dehydrogenase

As the detailed description of chemical, structural and kinetic features of certain enzymes and their reactions becomes more and more sophisticated a review article should concern itself with the answers to quite specific questions. In the present discussion the aspect of enzyme reactions considered is the definition of the different enzyme intermediates and of the individual steps of enzyme-substrate combination and catalysis. Enzyme-substrate combination and the associated phenomenon of the formation of the “reactive” enzyme-substrate complex are here regarded as the specific and biological process. The subsequent catalytic process of the interconversion of the reactive complexes with substrates and products is more the concern of those who are interested in the physical-organic chemistry of reaction mechanisms and may be regarded as an open ended question. However, part of the exercise of defining the different enzyme-substrate complexes is to characterise the groups of the enzyme which play a direct role in the interconversion of the enzyme complexes. We are presenting the results of experiments with pig heart lactate dehydrogenase and liver alcohol dehydrogenase to illustrate the application of chemical,

Rational construction of a 2-hydroxyacid dehydrogenase with new substrate specificity

Using site-directed mutagenesis on the lactate dehydrogenase gene from Bacillus stearothermophilus, three amino acid substitutions have been made at sites in the enzyme which we suggest in part determine specificity toward different hydroxyacids (R-CHOH-COOH). To change the preferred substrates from the pyruvate/lactate pair (R = -CH3) to the oxaloacetate/malate pair (R = -CH2-COO-), the volume of the active site was increased (thr 246----gly), an acid was neutralized (asp-197----asn) and a base was introduced (gln-102 - greater than arg). The wild type enzyme has a catalytic specificity for pyruvate over oxaloacetate of 1000 whereas the triple mutant has a specificity for oxaloacetate over pyruvate of 500. Despite the severity and extent of these active site alterations, the malate dehydrogenase so produced retains a reasonably fast catalytic rate constant (20 s-1 for oxaloacetate reduction) and is still allosterically controlled by fructose-1,6-bisphosphate.

The stability and hydrophobicity of cytosolic and mitochondrial malate dehydrogenases and their relation to chaperonin-assisted folding

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 cpn 10. 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 use of a genetically engineered tryptophan to identify the movement of a domain of B. stearothermophilus lactate dehydrogenase with the process which limits the steady-state turnover of the enzyme

A general technique for monitoring the intramolecular motion of a protein is described. Genetic engineering is used to replace all the natural tryptophan residues with tyrosine. A single tryptophan residue is then inserted at a specific site within the protein where motion is then detected from the fluorescence characteristics of this fluorophore. This technique has been used in B. stearothermophilus lactate dehydrogenase mutant (W80Y, W150Y, W203Y, G106W) to correlate the slow closure of a surface loop of polypeptide (residues 98-110) with the maximum catalytic velocity of the enzyme.

A strong carboxylate-arginine interaction is important in substrate orientation and recognition in lactate dehydrogenase

Using site-directed mutagenesis, Arginine-171 at the substrate-binding site of Bacillus stearothermophilus, lactate dehydrogenase has been replaced by lysine. In the closely homologous eukaryotic lactate dehydrogenase, this residue binds the carboxylate group of the substrate by forming a planar bifurcated bond. The mutation diminishes the binding energy of pyruvate, alpha-ketobutyrate and alpha-ketovalerate (measured by kcat/Km) by the same amount (about 6 kcal/mol). For each additional methylene group on the substrate, there is a loss of about 1.5 kcal/mol of binding energy in both mutant and wild-type enzymes. From these parallel trends in the two forms of enzyme, we infer that the mode of productive substrate binding is identical in each, the only difference being the loss of a strong carboxylate-guanidinium interaction in the mutant. In contrast to this simple pattern in kcat/Km, the Km alone increases with substrate-size in the wild-type enzyme, but decreases in the mutant. These results can be most simply explained by the occurrence of relatively tight unproductive enzyme-substrate complexes in the mutant enzyme as the substrate alkyl chain is extended. This does not occur in the wild-type enzyme, because the strong orienting effect of Arg-171 maximizes the frequency of substrates binding in the correct alignment.

Changes in the state of subunit association of lactate dehydrogenase from Bacillus stearothermophilus.

Time-resolved measurements of the fluorescence anisotropy of an extrinsic dye-group attached to lactate dehydrogenase from B. stearothermophilus revealed that the rotational correlation time of the enzyme at low concentrations is 55 ns, while at high enzyme concentrations or in the presence of fructose 1,6-bisphosphate (Fru-1,6-P2) the correlation time increases to 95 ns. These correlation times are consistent with a change in Mr from 85 000 +/- 12 000 (dimer) to 150 000 +/- 22 000 (tetramer) and show that the tetrameric state can be induced either by raising the protein concentration or by the addition of the ligand. We have confirmed this change in molecular weight by gel-filtration experiments. In the ligand-induced tetramer, two Fru-1,6-P2 molecules are bound.

Removal of substrate inhibition in a lactate dehydrogenase from human muscle by a single residue change

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.

The assembly mechanism of the lactate dehydrogenase tetramer from Bacillus stearothermophilus; the equilibrium relationships between quaternary structure and the binding of fructose 1,6-biphosphate, NADH and oxamate

Abstract (1) Unliganded lactate dehydrogenase from B. stearothermophilus exists in two states of quaternary structure, a dimer and a tetramer. The K d for the dimer-dimer interaction is 33 μM (expressed as a concentration of dimer). (2) The tetrameric state is stabilized by the tight binding of two fructose 1,6-bisphosphate molecules with a microscopic K d of 6–8 μM. (3) The state of subunit association has no influence on the binding of NADH ( K d = 1.5 μ M) but has a marked effect on the affinity of the active site for oxamate; a close structural analogue of pyruvate and competitive inhibitor of the enzyme. (4) In the tetrameric state stabilized with fructose 1,6-bisphosphate, the K d for oxamate is 0.013 mM, whereas the dimer binds the molecule only weakly ( K d = 2.2 mM ). Even in the absence of this effector, oxamate binds more tightly to the tetramer than to the dimer. This demonstrates that dimer-dimer association alone is sufficient to increase the affinity of the enzyme for oxamate. The K d for the association of oxamate with the unstabilized tetramer is less than 0.25 mM. (5) In the presence of fructose 1,6-bisphosphate the properties of the enzyme closely resemble those of the intensively studied porcine lactate dehydrogenases in the following respects; (a) the K d for NADH, (b) the K d for oxamate, (c) the ordered nature of coenzyme and substrate binding, (d) the quench in tryptophan fluorescence on binding NADH, (e) the quench in bound NADH fluorescence when the active site is occupied with oxamate.