Inactivation of Escherichia coli L-aspartate aminotransferase by (S)-4-amino-4,5-dihydro-2-thiophenecarboxylic acid reveals "a tale of two mechanisms".
As a mechanism-based inactivator of PLP-enzymes, (S)-4-amino-4,5-dihydro-2-thiophenecarboxylic acid (SADTA) was cocrystallized with Escherichia coli aspartate aminotransferase (l-AspAT) at a series of pH values ranging from 6 to 8. Five structural models with high resolution (1.4-1.85 A) were obtained for l-AspAT-SADTA complexes at pH 6.0, 6.5, 7.0, 7.5, and 8.0. Electron densities of the models showed that two different adducts had formed in the active sites. One adduct was formed from SADTA covalently linked to pyridoxal 5'-phosphate (PLP) while the other adduct was formed with the inhibitor covalently linked to Lysine246,1 the active site lysine. Moreover, there is a strong indication based on the electron densities that the occurrence of the two adducts is pH dependent. We conclude that SADTA inactivates l-AspAT via two different mechanisms based on the binding direction of the inactivator. Additionally, the structural models also show pH dependence of the protein structure itself, which provided detailed mechanistic implications for l-AspAT.
Liu
Dali
D
Department of Biochemistry and Chemistry and Rosenstiel Basic Medical Sciences Research Center MS029, Brandeis University, Waltham, Massachusetts 02454-9110, USA.
Pozharski
Edwin
E
Lepore
Bryan W
BW
Fu
Mengmeng
M
Silverman
Richard B
RB
Petsko
Gregory A
GA
Ringe
Dagmar
D
D001219:Aspartate Aminotransferases / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D001665:Binding Sites; D018360:Crystallography, X-Ray; D019281:Dimerization; D004789:Enzyme Activation / Q000187:drug effects; D004926:Escherichia coli / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D008958:Models, Molecular; D017433:Protein Structure, Secondary; D011732:Pyridoxal Phosphate / Q000737:chemistry; D055672:Static Electricity; D013876:Thiophenes / Q000494:pharmacology*
C524002:(S)-4-amino-4,5-dihydro-2-thiophenecarboxylic acid; D013876:Thiophenes; D011732:Pyridoxal Phosphate; D001219:Aspartate Aminotransferases