Substrate recognition mechanism of thermophilic dual-substrate enzyme.
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.
Ura
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H
Department of Biology, Graduate School of Science, Osaka University, Machikaneyama-cho, Toyonaka, Osaka 560-0043, Japan.
Nakai
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T
Kawaguchi
S I
SI
Miyahara
I
I
Hirotsu
K
K
Kuramitsu
S
S
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*
D001120:Arginine; D001219:Aspartate Aminotransferases