Substrate recognition mechanism of thermophilic dual-substrate enzyme

Aspartate aminotransferase from an extremely thermophilic bacterium, Thermu s thermophilus HB8 (ttAspAT), has been believed to be specific for an acidi c substrate. However, stepwise introduction of mutations in the active-site residues finally changed its substrate specificity to that of a dual-subst rate enzyme. The final mutant, [S15D, T17V, K109S, S292R] ttAspAT, is activ e toward both acidic and hydrophobic substrates, During the course of stepw ise mutation, the activities toward acidic and hydrophobic substrates chang ed independently, The introduction of a mobile Arg292* residue into ttAspAT was the key step in the change to a "dual-substrate" enzyme. The substrate recognition mechanism of this thermostable "dual-substrate" enzyme was con firmed by Xray crystallography, This work together with previous studies on various enzymes suggest that this unique "dual-substrate recognition" mech anism is a feature of not only aminotransferases but also other enzymes.