title: Substrate recognition mechanism of thermophilic dual-substrate enzyme.
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.
authors: Department of Biology, Graduate School of Science, Osaka University, Machikaneyama-cho, Toyonaka, Osaka 560-0043, Japan.
authors: H
authors: H
authors: Ura
authors: T
authors: T
authors: Nakai
authors: S I
authors: SI
authors: Kawaguchi
authors: I
authors: I
authors: Miyahara
authors: K
authors: K
authors: Hirotsu
authors: S
authors: S
authors: Kuramitsu
mesh_terms: D001120:Arginine / Q000235:genetics / Q000378:metabolism; D001219:Aspartate Aminotransferases / Q000737:chemistry / Q000378:metabolism*; D001665:Binding Sites; D018360:Crystallography, X-Ray; D004795:Enzyme Stability; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation / Q000235:genetics; D013379:Substrate Specificity; D013816:Thermodynamics; D016963:Thermus thermophilus / Q000201:enzymology*
chemical_list: D001120:Arginine; D001219:Aspartate Aminotransferases