title: Inorganic phosphate binding and electrostatic effects in the active center of aspartate aminotransferase apoenzyme.
abstract: The ionization state of the phosphate group bound at the aspartate aminotransferase apoenzyme's active site has been investigated utilizing Fourier-transform infrared spectroscopy following the band corresponding to the symmetric stretching of the dianionic phosphate. Unlike free phosphate, when inorganic phosphate is bound at the enzyme's active site, the integrated intensity value of the dianionic band does not change with pH within the studied range, and this value is similar to that for free dianionic phosphate at pH 8.3. From these results, we propose a dianionic state for the phosphate ion bound to cytosolic aspartate aminotransferase throughout the pH range of 5.7-8.3. The presence of other anions such as acetate and chloride or the substrate aspartate and its analogues produces a pH-dependent phosphate removal from the active site which is favored at low pH values. Elimination of the charged primary amine at the active-site Lys-258, through formation of a Schiff base with pyridoxal or chemical modification by carbamylation, also produces a pH-independent phosphate release. These results are interpreted as Lys-258 together with the active-site alpha-helix and other residues may be involved in stabilizing phosphate as a dianion in the apoenzyme phosphate pocket which anchors the phosphate ester of pyridoxal phosphate in the holoenzyme. It is proposed that the dianionic phosphate contributes to the apoenzyme's thermal stability through formation of strong hydrogen bond and salt bridges with the amino acid residues forming the phosphate binding pocket with assistance of Lys-258, and other active-site cationic components.
authors.affiliation: School of Basic Life Sciences, University of Missouri-Kansas City 64110-2499.
authors.forename: J H
authors.initials: JH
authors.lastname: Martinez-Liarte
authors.forename: A
authors.initials: A
authors.lastname: Iriarte
authors.forename: M
authors.initials: M
authors.lastname: Martinez-Carrion
mesh_terms: D000818:Animals; D000838:Anions; D001051:Apoenzymes / Q000378:metabolism*; D001219:Aspartate Aminotransferases / Q000378:metabolism*; D001665:Binding Sites; D004560:Electricity; D004795:Enzyme Stability; D005583:Fourier Analysis; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D009206:Myocardium / Q000201:enzymology; D010710:Phosphates / Q000378:metabolism*; D011732:Pyridoxal Phosphate / Q000378:metabolism; D011733:Pyridoxamine / Q000378:metabolism; D013055:Spectrophotometry, Infrared; D013552:Swine
chemical_list: D000838:Anions; D001051:Apoenzymes; D010710:Phosphates; D011732:Pyridoxal Phosphate; D011733:Pyridoxamine; D001219:Aspartate Aminotransferases