Modulation of activity and substrate specificity by modifying the backbone length of the distant interdomain loop of D-amino acid aminotransferase.
The activity and substrate specificity of D-amino acid aminotransferase (D-AAT) (EC 2.6.1.21) can be rationally modulated by replacing the loop core (P119-R120-P121) with glycine chains of different lengths: 1, 3, or 5 glycines. The mutant enzymes were much more active than the wild-type enzyme in the overall reactions between various amino acids and pyruvate. The presteady-state kinetic analyses of half-reactions revealed that the 5-glycine mutant has the highest affinity (Kd) among all mutant enzymes and the wild-type enzyme towards various amino acids except D-aspartate. The 5-glycine mutant was much more efficient as a catalyst than the wild-type enzyme because the mutant enzyme showed the highest value of specificity constant (kmax/Kd) for all amino acids except D-aspartate and D-glutamate. The kmax/Kd values of the three mutants decreased with decrease in glycine chain length for each amino acid examined. Our findings may provide a new approach to rational modulation of enzymes.
Gutierrez
A
A
Institute for Chemical Research, Kyoto University, Uji, Kyoto-Fu, Japan.
Yoshimura
T
T
Fuchikami
Y
Y
Esaki
N
N
D000410:Alanine Transaminase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D051263:D-Alanine Transaminase; D017931:DNA Primers; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D013379:Substrate Specificity
D017931:DNA Primers; D000410:Alanine Transaminase; D051263:D-Alanine Transaminase