Stabilization of Quinonoid Intermediate E-Q by Glu32 of D-Amino Acid Transaminase

The stable anchorage of pyridoxal 5'-phosphate (PLP) in the active site of D-amino acid transaminase (D-AT) is crucial for the enzyme catalysis. The three-dimensional structure of D-AT revealed that Glu32 is one of the active site groups that may playa role in PLP binding. To prove the role of Glu32 in PLP stability, we firstly checked the rate of the potential rate-limiting step. The kinetic analysis showed that the rate of the ${\alpha}$-deprotonation step reduced to 26-folds in E32A mutant enzyme. Spectral analyses of the reaction of D-AT with D-serine revealed that the E32A mutant enzyme failed to stabilize the key enzyme-substrate intermediate, namely a quinonoid intermediate (E-Q). Finally, analysis of circular dichroism (CD) on the wild-type and E32A mutant enzymes showed that the optical activity of PLP in the enzyme active site was lost by the removal of the carboxylic group, proving that Glu32 is indeed involved in the cofactor anchorage. The results suggested that the electrostatic interaction network through the groups from PLP, Glu32, His47, and Arg50, which was observed from the three-dimensional structure of the enzyme, plays a crucial role in the stable anchorage of the cofactor to give necessary torsion to the plane of the cofactor-substrate complex.