Kinetic analysis of phenylalanine dehydrogenase mutants designed for aliphatic amino acid dehydrogenase activity with guidance from homology-based modelling

Through comparison with the high-resolution structure of Clostridium symbiosum glutamate dehydrogenase, the different substrate specificities of the homologous enzymes phenylalanine dehydrogenase and leucine dehydrogenase were attributed to two residues, glycine 124 and leucine 307, in Bacillus sphaericus phenylalanine dehydrogenase, which are replaced with alanine and valine in leucine dehydrogenases [Britton, K.L., Baker, P.J., Engel, P.C., Rice, D.W. & Stillman, T.J. (1993) J. Mol. Biol.234, 938-945]. As predicted, making these substitutions in phenylalanine dehydrogenase decreased the specific activity towards aromatic substrates and enhanced the activity towards some aliphatic amino acids in standard assays with fixed concentrations of both substrates [Seah, S.Y.K., Britton, K.L., Baker, P.J., Rice, D.W., Asano, Y. & Engel, P.C. (1995) FEBS Lett.370, 93-96]. This study did not, however, distinguish effects on affinity from those on maximum catalytic rate. A fuller kinetic characterization of the single- and double-mutant enzymes now reveals that the extent of the shift in specificity was underestimated in the earlier study. The maximum catalytic rates for aromatic substrates are reduced for all the mutants, but, in addition, the apparent K-m values are higher for the single-mutant G124A and double-mutant G124A/L307V compared with the wild-type enzyme. Conversely, specificity constants (k(cat)/K-m) for the nonpolar aliphatic amino acids and the corresponding 2-oxoacids for the mutants are all markedly higher than for the wild type, with up to a 40-fold increase for L-norvaline and a 100-fold increase for its 2-oxoacid in the double mutant. In some cases a favourable change in K-m was found to outweigh a smaller negative change in k(cat). These results emphasize the risk of misjudging the outcome of protein engineering experiments through too superficial an analysis. Overall, however, the success of the predictions from molecular modelling indicates the usefulness of this strategy for engineering new specificities, even in advance of more detailed 3D structural information.