The separate effects of coenzyme components may not be additive. Roles of pyridoxal and inorganic phosphate in aspartate aminotransferase apoenzymes.
Both cytosolic and mitochondrial aspartate transaminase can be resolved of pyridoxal phosphate. The resulting apoenzymes still bind individual structural components of the coenzyme. The separate contributions of coenzyme components to protein thermal stability have been independently assessed for phosphate ions (Pi) and for the pyridoxal or pyridoxamine components of the coenzyme. 31P NMR and differential scanning calorimetry reveal that the thermodynamic contributions of binding are not additive and are dissimilar for the two isozymes. High and low affinity sites for Pi binding are present in both apoenzymes with only the low affinity site being present in the holoenzyme forms. The contribution of both bound phosphates to increasing temperatures (Tm) and enthalpies (delta Hd) of denaturation differ between the isozymes and within sites. In either isozyme occupancy of the high affinity site by Pi produces only a 4- or 5- degree increase in the Tm value with respect to Pi-free apoenzyme. By contrast, in the mitochondrial apoenzyme, the presence of Pi at the second low affinity site increases the calorimetric parameters from Tm = 47 degrees C and delta Hd = 4.7 cal g-1 to Tm = 62 degrees C and delta Hd = 7 cal g-1. For cytosolic apoenzyme the respective changes are from 66 to 69.5 degrees C and 5.2 to 5.8 cal g-1. Addition of pyridoxal, but not pyridoxamine, displaces the high affinity Pi in both apoenzymes. This shows that the pyridine ring and Pi groups of pyridoxal-P bind exclusive of each other when they are not covalently linked as an ester, as in the coenzyme. The observation has been exploited as a method to prepare completely dephosphorylated mitochondrial apoenzyme. Electrostatic effects, structural differences in the phosphate binding pockets, and steric effects can be invoked to account for the Pi and pyridine binding behavior in the two proteins.
D000818:Animals; D001051:Apoenzymes / Q000378:metabolism*; D001059:Apoproteins / Q000378:metabolism*; D001219:Aspartate Aminotransferases / Q000378:metabolism*; D002152:Calorimetry, Differential Scanning; D003600:Cytosol / Q000201:enzymology; D006863:Hydrogen-Ion Concentration; D007527:Isoenzymes / Q000378:metabolism; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008929:Mitochondria, Heart / Q000201:enzymology*; D009206:Myocardium / Q000201:enzymology*; D010710:Phosphates / Q000494:pharmacology*; D011730:Pyridoxal / Q000494:pharmacology*; D013552:Swine
D001051:Apoenzymes; D001059:Apoproteins; D007527:Isoenzymes; D010710:Phosphates; D011730:Pyridoxal; D001219:Aspartate Aminotransferases