Modulation of activity and substrate specificity by modifying the backbone length of the distant interdomain loop of Dâamino acid aminotransferase
2000
The activity and substrate specificity of dâamino acid aminotransferase ( dâAAT) (ECâ2.6.1.21) can be rationally modulated by replacing the loop core (P119âR120âP121) with glycine chains of different lengths: 1, 3, or 5 glycines. The mutant enzymes were much more active than the wildâtype enzyme in the overall reactions between various amino acids and pyruvate. The presteadyâstate kinetic analyses of halfâreactions revealed that the 5âglycine mutant has the highest affinity (Kd) among all mutant enzymes and the wildâtype enzyme towards various amino acids except dâaspartate. The 5âglycine mutant was much more efficient as a catalyst than the wildâtype enzyme because the mutant enzyme showed the highest value of specificity constant (kmax/Kd) for all amino acids except dâaspartate and dâglutamate. 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.
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