An evaluation of the substrate specificity, and of its modification by site-directed mutagenesis, of the cloned L-lactate dehydrogenase from Bacillus stearothermophilus

The L-lactate dehydrogenase of Bacillus stearothermophilus (BSLDH) is a stable, thermophilic oxidoreductase. In this study, the specificity of BSLDH toward representative alpha -keto acids possessing straight- and branched-alkyl, cycloalkyl, or aromatic side chains has been evaluated. The results show that substrates that are sterically bulky in the region of the alpha -keto group to be reduced are poorly accepted by the enzyme. Graphics analyses indicated that the low activities of these hindered substrates might be partly due to a bad interaction of the active site residue Gln102 with large or branched substituents adjacent to the alpha -keto group. Accordingly, Gln102 has been replaced by the smaller Asn residue by site-directed mutagenesis in an attempt to expand the active site volume available to receive substrates larger than the natural pyruvate. However, the kinetic data show that bulky alpha -keto acids are only marginally better accommodated by the Gln102 arrow right Asn mutant than by the wild-type enzyme.