[Study of the reorientation of coenzyme in active sites of aspartate-aminotransferase isoenzymes using linear dichroism method].
Cytosolic and mitochondrial pig aspartate aminotransferases (cAAT and mAAT) and chicken cAAT were oriented in a compressed slab of polyacrylamide gel. Linear dichroism (LD) spectra of the pyridoxal and pyridoxamine forms of AATs and of complexes of the pyridoxal form with substrate analogues have been recorded. The tilt angles of the coenzyme at the intermediary steps of the transamination reaction have been calculated on the basis of reduced LD values (delta A/A), atomic coordinates of the coenzyme and directions of the transition dipole moments in the coenzyme ring. It was assumed that rotation of the coenzyme ring occurs around the C2-C5 axis in all cases except the enzyme complex with glutarate: in the latter case the direction N1-C4 was assumed to be a rotation axis. It has been found that formation of the enzyme complex with glutarate and protonation of the internal aldimine induce dissimilar reorientations of the coenzyme. As a result of protonation, the coenzyme tilts by 27 degrees in cAAT and 13 degrees in mAAT. Formation of the external aldimine with 2-methylaspartate is accompanied by tilting of the coenzyme ring by 44 degrees in cAAT and 39 degrees in mAAT. For the quinonoid complex with erythro-3-hydroxyaspartate, the tilt angles were found to be 63 degrees in cAAT and 53 degrees in mAAT. It was inferred that the basic features of the active site dynamics are similar in three AATs studied. The differences in the coenzyme tilt angles between cAAT and mAAT might be linked to catalytic peculiarities of the isoenzymes.
Rozenberg
M V
MV
Makarov
V L
VL
Torchinskiĭ
Iu M
IuM
D000818:Animals; D001219:Aspartate Aminotransferases / Q000032:analysis*; D001665:Binding Sites; D002384:Catalysis; D002645:Chickens; D003067:Coenzymes / Q000032:analysis*; D003600:Cytosol / Q000201:enzymology; D004591:Electrophoresis, Polyacrylamide Gel; D007527:Isoenzymes / Q000032:analysis*; D008929:Mitochondria, Heart / Q000201:enzymology; D013053:Spectrophotometry; D013379:Substrate Specificity; D013552:Swine
D003067:Coenzymes; D007527:Isoenzymes; D001219:Aspartate Aminotransferases
Issledovanie reorientatsii kofermenta v aktivnykh tsentrakh izofermentov aspartat-aminotransferazy pri pomoshchi metoda lineĭnogo dikhroizma.