J. Biochem, 2003, Vol. 134, No. 2 277-285
© 2003 Japanese Biochemical Society
BIOCHEMISTRY |
Reaction of Aspartate Aminotransferase with C5-Dicarboxylic Acids: Comparison with the Reaction with C4-Dicarboxylic Acids
Department of Biochemistry, Osaka Medical College, 2-7 Daigakumachi, Takatsuki 569-8686
The reaction of Escherichia coli aspartate aminotransferase (AspAT) with glutamate and other C5-dicarboxylates was analyzed in order to compare its mechanism of action toward C5 substrates with that toward C4 substrates, which had been extensively characterized. The association of the amino-group protonated and unprotonated forms of glutamate (SH+ and S, respectively) with the Schiff-base protonated and unprotonated forms of the enzyme (ELH+ and EL, respectively) yields at least three forms of the Michaelis complex, whereas in the case of aspartate, only two species of this complex exist, EL·SH+ and ELH+·S. The reaction of AspAT with 2-methylglutamate can be explained only when we consider all the protonation states of the Michaelis complex. Based on the previous crystallographic studies [Miyahara et al. (1994) J. Biochem. 116, 1001–1012], we consider that glutamate binds to the open form of AspAT and takes an extended conformation in the Michaelis complex, with the 
-amino group of glutamate oriented in the opposite direction to the Schiff base. This is in contrast to the Michaelis complex of aspartate, in which a strong interaction of the -amino group of aspartate and the Schiff base excludes the presence of the species ELH+·SH+. It is concluded that AspAT recognizes the two types of dicarboxylates with different chain lengths by changing the gross conformation of the enzyme protein.
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