Keith W. Hart

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 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mutagenesis, Arginine-171 at the substrate-binding site of Bacillus stearothermophilus, lactate dehydrogenase has been replaced by lysine.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.

The importance of arginine 171 in substrate binding by Bacillus stearothermophilus lactate dehydrogenase.

A variant of lactate dehydrogenase from Bacillus stearothermophilus has been engineered by site-directed mutagenesis in which an active-site arginine residue at position 171 in the protein sequence is replaced by lysine. Replacement of this arginine by lysine has no effect on co-enzyme binding, a relatively small effect on the rate of turnover of the enzyme, but causes a 2000-fold increase in the Michaelis constant for pyruvate, a 6000-fold increase in the dissociation constant for oxamate and results in a Michaelis constant for lactate which is too high to measure. The decrease in binding energy for these carboxylate-containing substrates caused by this mutation is very large, around 5.5 kcal.mol-1 and in part, is explained by the small increase in the distance of a lysine-substrate carboxylate interaction at this site and the absence of the additional hydrogen bond from a two-point arginine-carboxylate interaction. Consistent with this last observation, the ability of this mutant enzyme to stabilize an NAD+-sulphite compound in its active site (an alternative enzyme-substrate complex which does not involve bifurcated bonding to arginine) is only reduced 14-fold.

The rates of defined changes in protein structure during the catalytic cycle of lactate dehydrogenase

Rapid mixing, kinetic experiments were performed on native and modified [Tyr(3NO2)237)] porcine H4 lactate dehydrogenase at low temperatures in a medium containing 30% dimethyl sulphoxide. In the temperature range -16 to +8 degrees C, the modified enzyme-NADH complex, when mixed with 1 mM pyruvate, is converted to enzyme, NAD+ and lactate at two distinctly different rates. At -16 degrees C the more rapid process occurs at a rate of 40 s-1 and the slower at 3 s-1. The slower rate is identical to that assigned to the steady-state turnover of the enzyme in these conditions and therefore reflects the slow, rate-limiting rearrangement of protein structure which has been inferred from previous kinetic experiments. The fast phase of NADH oxidation, however, proceeds at a rate which coincides with that of the closure of a loop of polypeptide over the active site of the enzyme (sensed by the nitrotyrosine group, which protonates in response to the approach of glutamate 107, a residue situated on this mobile loop). We explain these results by proposing that: (i) both the slow and fast changes in protein structure must occur before the enzyme can accomplish the redox step, (ii) the enzyme-NADH (binary) complex exists in two, slowly interconverting forms, (iii) the structural change giving rise to this slow conformational equilibrium can also occur in the ternary (enzyme-NADH-pyruvate) complex and (iv) it is this step which limits the rate of the steady-state reaction. Both of the binary forms are able to bind pyruvate, but the rate of NADH oxidation in one of the forms is rapid, since it has already undergone this slow rearrangement. In this rapidly reacting form, it is the closure of the loop (not transfer of the hydride ion) which limits the rate at which the coenzyme is oxidized, while the slowly reacting form must undergo both loop-closure and the slow structural conversion before the redox reaction can occur.

The use of site-directed mutagenesis and time-resolved fluorescence spectroscopy to assign the fluorescence contributions of individual tryptophan residues in Bacillus stearothermophilus lactate dehydrogenase

Site-directed mutagenesis has been used to generate two mutant Bacillus stearothermophilus lactate dehydrogenases: in one, Trp-150 has been replaced with a tyrosine residue and, in the other, both Trp-150 and -80 are replaced with tyrosines. Both enzymes are fully catalytically active and their affinities for substrates and coenzymes, and thermal stabilities are very similar to those of the native enzyme. Time-resolved fluorescence measurements using a synchrotron source have shown that all three tryptophans in the native enzyme fluoresce. By comparing the mutant and native enzymes it was possible, for the first time, to assign, unambiguously, lifetimes to the individual tryptophans: Trp-203 (7.4 ns), Trp-80 (2.35 ns) and Trp-150 (less than 0.3 ns). Trp-203 is responsible for 75-80% of the steady-state fluorescence emission, Trp-80 for 20%, and Trp-150 for less than 2%.

The greater strength of arginine: Carboxylate over lysine carboxylate ion pairs implications for the design of novel enzymes and drugs

The rational design of enzyme catalysts for chiral chemistry and of drugs which bind to proteins would be facilitated if rules for the recognition of one partner by the other could be formulated. This communication suggests and tests one generalization: arginine forms a tighter ion pair with a carboxylate group than does lysine and is always used for ion-pairs which are not broken during turnover in naturally-occurring enzymes.

On the effect on specificity of Thr246→Gly mutation in L-lactate dehydrogenase of Bacillus stearothermophilus

The function of the amino acid Thr246 in L-lactate dehydrogenase from Bacillus stearothermophilus has been investigated by site-directed replacement with glycine. Kinetic experiments with a number of 2-oxo acids showed strongly reduced activity for the mutated enzyme. However, the mutant enzyme shows a relative preference for the large hydrophobic sidechains of alpha-keto acids and an even higher specific activity than the wild-type lactate dehydrogenase for the polar oxaloacetate substrate. Graphic analyses indicate that the loss of one hydrogen bond, or intrusion of water into the active site, might be responsible for the reduced activity. The kinetic results suggest that the binding modes of bulky hydrophobic or polar substrates compensate to some degree for the partially disrupted active site.