Different reactivity of mitochondrial and cytoplasmic aspartate aminotransferases toward an affinity labeling reagent analog of the coenzyme.
The two isoenzymes of aspartate aminotransferase from pig heart have been reacted with a derivative of the coenzyme, 4'-N-(2,4-dinitro-5-fluorophenyl) pyridoxamine-5'-phosphate, which is a potential affinity labeling reagent. The derivative has a great affinity for both isoapoenzymes. In the cytosolic isoenzyme, the reversible binding is followed by a covalent labeling of the epsilon amino group of lysine 258, which usually forms an aldimine bond with pyridoxal-5'-phosphate. In the mitochondrial isoenzyme, no labeling occurs at the active site. The different reactivity indicates that a small but definite difference exists in the geometry of the two active sites. In the cytosolic isoenzyme also a sulfhydryl group outside the active site region, namely cysteine 45, reacts, but not by an affinity labeling mechanism. In both isoenzymes, the reversibly bound reagent slowly undergoes a splitting reaction by which pyridoxal-5'-phosphate is regenerated and activity re-established; the rate of this reaction is not fast enough to impaire the labeling potential of the reagent.
D000345:Affinity Labels / Q000494:pharmacology*; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001219:Aspartate Aminotransferases / Q000378:metabolism*; D002918:Chymotrypsin; D003600:Cytosol / Q000201:enzymology; D007527:Isoenzymes / Q000378:metabolism; D008929:Mitochondria, Heart / Q000201:enzymology*; D009206:Myocardium / Q000201:enzymology*; D010446:Peptide Fragments / Q000032:analysis; D011733:Pyridoxamine / Q000031:analogs & derivatives* / Q000494:pharmacology; D013053:Spectrophotometry; D013552:Swine
C019355:4'-N-(2,4-dinitro-5-fluorophenyl)pyridoxamine 5'-phosphate; D000345:Affinity Labels; D000596:Amino Acids; D007527:Isoenzymes; D010446:Peptide Fragments; D011733:Pyridoxamine; D001219:Aspartate Aminotransferases; D002918:Chymotrypsin