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mutagenesis, Arginine-171 at the substrate-binding site of Bacillus stearothermophilus, lactate dehydrogenase has been replaced by lysine.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