Specific labeling of the active site of cytosolic aspartate aminotransferase through the use of a cofactor analogue, N-(Bromoacetyl)pyridoxamine.
The cofactor analogue N-(bromoacetyl)pyridoxamine (BAPM) has been employed to inactivate the cytosolic isozyme of apo-aspartate aminotransferase. Inactivation is the result of covalent bond formation in the (bromoacetyl)-pyridoxamine-transferase complex, via the epsilon-amino group of a lysyl residue at the active site. The stoichiometry of this inactivation is one molecule of (bromoacetyl)pyridoxamine per subunit of the transaminase dimer. Trace amounts of inorganic phosphate protect the enzyme from BAPM inactivation. In the absence of phosphate, inactivation demonstrates time, concentration, and pH dependence with an apparent pK for the target group of about 8.5 or higher. A tryptic peptide from the alpha subform has been obtained containing the carboxymethyl derivative of lysine-258, identifying this particular residue as the reactive group in the region of cofactor binding. Evidence is presented indicating that the pK of Lys-258 appears to be highly dependent upon the electrostatic state of neighboring groups in the active site region. Hence, experimentally obtained values vary according to the chemical nature and charge of the modifying agent or probe.
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001219:Aspartate Aminotransferases / Q000378:metabolism*; D001665:Binding Sites; D004791:Enzyme Inhibitors; D006863:Hydrogen-Ion Concentration; D007527:Isoenzymes / Q000037:antagonists & inhibitors; D010710:Phosphates / Q000378:metabolism; D011733:Pyridoxamine / Q000031:analogs & derivatives* / Q000378:metabolism; D013552:Swine; D014357:Trypsin / Q000378:metabolism
D000596:Amino Acids; D004791:Enzyme Inhibitors; D007527:Isoenzymes; D010710:Phosphates; C017086:bromoacetylpyridoxamine; D011733:Pyridoxamine; D001219:Aspartate Aminotransferases; D014357:Trypsin