ON THE FACTORS CONTROLLING THE STRUCTURAL SPECIFICITY AND STEREOSPECIFICITY OF THE L-LACTATE DEHYDROGENASE FROM BACILLUS STEAROTHERMOPHILUS - EFFECTS OF GLN102-]ARG AND ARG171-]TRP/TYR DOUBLE MUTATIONS

The factors determining the L-stereospecificity of the L-lactate dehydrogenase from Bacillus stearothermophilus have been probed by introducing Arg171Trp/Tyr and Gln102Arg mutations. These changes preclude normal 2-keto acid substrate binding via an Arg171-COO- electrostatic interaction and are positioned to induce a reversal of the natural substrate binding mode, thereby leading to D-2-hydroxy acid formation. However, the L-stereospecificities of the mutant enzymes remain unchanged, showing that there are important fail-safe stereospecificity determinants that take over when the key Arg171-COO-binding interaction is removed. The effects of the mutations on structural specificity are approximately additive, resulting in the broad 2-keto acid specificity of the wild-type enzyme being changed to give catalysts highly selective for the dicarboxylic substrate oxalacetate.