Reaction of aspartate aminotransferase with C5-dicarboxylic acids: comparison with the reaction with C4-dicarboxylic acids.
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 (E(L)H(+) and E(L), respectively) yields at least three forms of the Michaelis complex, whereas in the case of aspartate, only two species of this complex exist, E(L).SH(+) and E(L)H(+).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 alpha-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 alpha-amino group of aspartate and the Schiff base excludes the presence of the species E(L)H(+).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.
Islam
Mohammad Mainul
MM
Department of Biochemistry, Osaka Medical College, 2-7 Daigakumachi, Takatsuki 569-8686.
Hayashi
Hideyuki
H
Kagamiyama
Hiroyuki
H
D001219:Aspartate Aminotransferases / Q000737:chemistry / Q000378:metabolism*; D001224:Aspartic Acid / Q000737:chemistry / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology; D018698:Glutamic Acid / Q000737:chemistry / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008958:Models, Molecular; D011485:Protein Binding; D013053:Spectrophotometry / Q000379:methods; D013379:Substrate Specificity; D013816:Thermodynamics
D001224:Aspartic Acid; D018698:Glutamic Acid; D001219:Aspartate Aminotransferases