Substrate recognition mechanism of thermophilic dual-substrate enzyme.
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Ura H, Nakai T, Kawaguchi SI, Miyahara I, Hirotsu K, Kuramitsu S
Substrate recognition mechanism of thermophilic dual-substrate enzyme.
J Biochem. 2001 Jul;130(1):89-98.
- PubMed ID
- 11432784 [ View in PubMed]
- Abstract
Aspartate aminotransferase from an extremely thermophilic bacterium, Thermus thermophilus HB8 (ttAspAT), has been believed to be specific for an acidic substrate. However, stepwise introduction of mutations in the active-site residues finally changed its substrate specificity to that of a dual-substrate enzyme. The final mutant, [S15D, T17V, K109S, S292R] ttAspAT, is active toward both acidic and hydrophobic substrates. During the course of stepwise mutation, the activities toward acidic and hydrophobic substrates changed 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 confirmed by X-ray crystallography. This work together with previous studies on various enzymes suggest that this unique "dual-substrate recognition" mechanism is a feature of not only aminotransferases but also other enzymes.