DETERMINANTS OF SUBSTRATE-SPECIFICITY IN THE SUPERFAMILY OF AMINO-ACID DEHYDROGENASES

The subunit of the enzyme glutamate dehydrogenase comprises two domain s separated by a cleft harboring the active site. One domain is respon sible for dinucleotide binding and the other carries the majority of r esidues which bind the substrate. During the catalytic cycle a large m ovement between the two domains occurs, closing the cleft and bringing the C4 of the nicotinamide ring and the C alpha 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 gro up of the glutamate substrate, In leucine dehydrogenase, an enzyme bel onging to the same superfamily, the equivalent residues are L40 and V2 94, which create a more hydrophobic specificity pocket and provide an explanation for their differential substrate specificity. In an attemp t to change the substrate specificity of glutamate dehydrogenase towar d that of leucine dehydrogenase, a double mutant, K89L,S380V, of gluta mate dehydrogenase has been constructed. Far from having a high specif icity for leucine, this mutant appears to be devoid of any catalytic a ctivity over a wide range of substrates tested. Determination of the t hree-dimensional structure of the mutant enzyme has shown that the los s of function is related to a disordering of residues linking the enzy me's two domains, probably arising from a steric clash between the val ine side chain, introduced at position 380 in the mutant, and a conser ved 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 c hains are attached. Thus, the differential substrate specificity seen in the amino acid dehydrogenase superfamily arises from both the intro duction of simple point mutations and the fine tuning of the active si te pocket defined by small but significant main chain rearrangements.