Reorientations of coenzyme in aspartate transaminase studied on single crystals of the enzyme by polarized-light spectrophotometry.
Investigation of polarized-light absorption spectra of single crystals of cytosolic aspartate transaminase (L-aspartate:2-oxoglutarate aminotransferase, EC 2.6.1.1) from chicken heart has revealed that the coenzyme's absorption bands at 430 and 360 nm are polarized in opposite directions, both in crystals of the free enzyme and in its complex with a quasi-substrate, 2-methylaspartate. The opposite signs of polarization of the 430 and 360 nm bands of the free enzyme indicate different orientation of the pyridine ring of pyridoxal 5'-phosphate in the protonated and non-protonated forms of the "internal" coenzyme-lysine aldimine. These data suggest that reorientation of the coenzyme ring occurs mainly in the first step of the catalytic reaction, associated with proton transfer from the NH+3 group of amino acid substrate to the coenzyme-lysine aldimine. Absorption bands at 333 and 430 nm are seen in the spectra upon soaking the crystals in solutions containing aspartate, glutamate or cysteinesulfinate. Both bands are polarized in the same direction as is the 430 nm absorption bands of the protonated internal aldimine. Soaking the crystals in solutions containing 2-oxoglutarate, glutarate or maleate reverses the sign of polarization of the 430 nm band. High concentrations of acetate induce the same effect. Thus, binding of dicarboxylate or acetate anions in the active site of aspartate transaminase appears to result in partial or complete return of the coenzyme ring to a position similar to that of the non-protonated internal aldimine.
D000085:Acetates / Q000378:metabolism; D000818:Animals; D001219:Aspartate Aminotransferases / Q000378:metabolism; D001224:Aspartic Acid / Q000031:analogs & derivatives / Q000378:metabolism; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D002645:Chickens; D003067:Coenzymes; D003460:Crystallization; D003998:Dicarboxylic Acids / Q000378:metabolism; D009206:Myocardium / Q000201:enzymology; D016202:N-Methylaspartate; D011048:Polarography; D011732:Pyridoxal Phosphate / Q000378:metabolism
D000085:Acetates; D003067:Coenzymes; D003998:Dicarboxylic Acids; D001224:Aspartic Acid; D011732:Pyridoxal Phosphate; D016202:N-Methylaspartate; D001219:Aspartate Aminotransferases