Determinants of substrate specificity in the superfamily of amino acid dehydrogenases.
The subunit of the enzyme glutamate dehydrogenase comprises two domains separated by a cleft harboring the active site. One domain is responsible for dinucleotide binding and the other carries the majority of residues which bind the substrate. During the catalytic cycle a large movement between the two domains occurs, closing the cleft and bringing the C4 of the nicotinamide ring and the Calpha of the substrate into the correct positioning for hydride transfer. In the active site, two residues, K89 and S380, make interactions with the gamma-carboxyl group of the glutamate substrate. In leucine dehydrogenase, an enzyme belonging to the same superfamily, the equivalent residues are L40 and V294, which create a more hydrophobic specificity pocket and provide an explanation for their differential substrate specificity. In an attempt to change the substrate specificity of glutamate dehydrogenase toward that of leucine dehydrogenase, a double mutant, K89L,S380V, of glutamate dehydrogenase has been constructed. Far from having a high specificity for leucine, this mutant appears to be devoid of any catalytic activity over a wide range of substrates tested. Determination of the three-dimensional structure of the mutant enzyme has shown that the loss of function is related to a disordering of residues linking the enzyme's two domains, probably arising from a steric clash between the valine side chain, introduced at position 380 in the mutant, and a conserved threonine residue, T193. In leucine dehydrogenase the steric clash between the equivalent valine and threonine side chains (V294, T134) does not occur owing to shifts of the main chain to which these side chains are attached. Thus, the differential substrate specificity seen in the amino acid dehydrogenase superfamily arises from both the introduction of simple point mutations and the fine tuning of the active site pocket defined by small but significant main chain rearrangements.