A general method for relieving substrate inhibition in lactate dehydrogenases.

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.

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.

Guided evolution of enzymes with new substrate specificities.

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.

Engineering surface loops of proteins--a preferred strategy for obtaining new enzyme function.

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.

Allosteric activation in Bacillus stearothermophilus lactate dehydrogenase investigated by an X-ray crystallographic analysis of a mutant designed to prevent tetramerization of the enzyme.

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.

The structural consequences of exchanging tryptophan and tyrosine residues in B. stearothermophilus lactate dehydrogenase.

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.

Design of a specific phenyllactate dehydrogenase by peptide loop exchange on the Bacillus stearothermophilus lactate dehydrogenase framework.

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.

Construction of a stable dimer of Bacillus stearothermophilus lactate dehydrogenase.

A molecular graphics analysis of the features which prevent cytosolic malate dehydrogenase dimers from forming tetramers was evaluated by its success in predicting the synthesis of a version of the LDH framework which is a stable dimer. Surface residues responsible for malate dehydrogenases being dimers were revealed by superimposing the structures of two dimers of pig cytosolic malate dehydrogenase on one homologous tetramer of L-lactate dehydrogenase from Bacillus stearothermophilus. Four regions were identified as composing the P-axis dimer-dimer interface. Two regions of the dimer were surface loops that collided when built as a tetramer: a large loop (residues 203-207, KNOBI) and a small loop (residues 264-269, KNOBII), and these were candidates to explain the dimeric character of malate dehydrogenase. The analysis was tested by constructing a synthetic B. stearothermophilus lactate dehydrogenase (KNOBI) containing the large malate dehydrogenase loop (residues 203-207 being AYIKLQAKE, and extra four amino acids). The new construct was thermotolerant (90 degrees C) and enzymically active with kcat and KM (pyruvate) values similar to those of the wild-type enzyme. However, whereas the allosteric activator fructose 1,6-bisphosphate decreased KM 100 times for wild type, it had no influence on KNOBI. The molecular volumes of 1-120 microM concentrations of the construct were measured by time-resolved decay of tryptophan fluorescence anisotropy and by gel filtration. Both methods showed the molecular weight of wild type increased from dimer to tetramer with Kd about 20 microM dimer. KNOBI remained a dimer under these conditions.(ABSTRACT TRUNCATED AT 250 WORDS)