CONTRIBUTION OF A BURIED ASPARTATE RESIDUE TOWARDS THE CATALYTIC EFFICIENCY AND STRUCTURAL STABILITY OF BACILLUS-STEAROTHERMOPHILUS LACTATE-DEHYDROGENASE

The X-ray structure of lactate dehydrogenase (LDH) shows the side-chai n carboxylate group of Asp-143 to be buried in the hydrophobic interio r of the enzyme, where it makes hydrogen-bonding interactions with bot h the side-chain hydroxyl group of Ser-273 and the main-chain amide gr oup 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 sim ilar interaction on the solvent-exposed exterior. In this respect the side-chain carboxylate group of Asp-143 appears to be important for ma intaining tertiary structure by providing a common linkage point betwe en three discontinuous elements of the secondary structure, alpha 1F, beta K and the beta-turn joining beta G and beta H. The contribution o f the Asp-143 side-chain to the structure and function of Bacillus ste arothermophilus LDH was assessed by creating a mutant enzyme containin g Asn-143. The decreased thermal stability of both unactivated and fru ctose-1,6-diphosphate (Fru-1,6-P-2)-activated forms of the mutant enzy me support a structural role for Asp-143. Furthermore, the difference in stability of the wild-type and mutant enzymes in guanidinium chlori de suggested that the carboxylate group of Asp-143 contributes at leas t 22 kJ/mol to the conformational stability of the wild-type enzyme. H owever, there was no alteration in the amount of accessible tryptophan fluorescence in the mutant enzyme, indicating that the mutation cause d a structural weakness rather than a gross conformational change. Com parison 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 1 1 kJ/mol for the transition-state complexes. The different effects of pH and Fru-1,6-P-2 on the wild-type and mutant enzymes also confirmed a perturbation of the catalytic centre in the mutant enzyme. As the si de-chain of Asp-143 is not sufficiently close to the active site to be directly involved in catalysis or substrate binding it is proposed th at the effects on catalysis shown by the mutant enzyme are induced eit her by a structural change or by charge imbalance at the active site. As Asp-143, His-195 and Ser-273 are absolutely conserved in all known LDH sequences this charged interaction is an important and general fea ture of the structure/function relationship in LDH.