Selective tryptic cleavage of native cytoplasmic aspartate transaminase holoenzyme.
The cytoplasmic isozyme of aspartate transaminase is inactivated by trypsin due to loss of a 19-residue peptide from the NH2-terminal region. A second peptide bond at Arg-25 is then cleaved by trypsin leaving a residual core protein, transaminase 26-412. Inactivation by trypsin resembles that for the mitochondrial enzyme (Sandmeier, E., and Christen, P. (1980) J. Biol. Chem. 255, 10284-10289), yet occurs 10 times faster for the cytoplasmic isozyme. In the mitochondrial enzyme, trypsin cleavage produces equal concentrations of proteins missing the first 26 and 31 amino acids. Sequence variation in the NH2-terminal regions can explain such differences. Specifically, the mitochondrial NH2 terminus has no trypsin-susceptible residue at position 19 and is stabilized by an electrostatic interaction between Asp-15 and Arg-292, whereas position 15 is a valyl residue in the cytoplasmic enzyme. Calorimetric data reveal both a decreased transition temperature (Td) and enthalpy (delta Hd) of denaturation in transaminases 20-412 and 26-412. Interaction of substrates with the active site chromophore and differential scanning calorimetry (DSC) reveal that catalytically inactive transaminases 20-412 and 26-412 can bind amino acid substrates and produce spectroscopically detectable conversion of the pyridoxal to the pyridoxamine form of the protein. By contrast, substrate analogs only form enzymatic Michaelis-type complexes.
D000595:Amino Acid Sequence; D000818:Animals; D001219:Aspartate Aminotransferases / Q000378:metabolism*; D001224:Aspartic Acid / Q000031:analogs & derivatives / Q000378:metabolism; D002152:Calorimetry, Differential Scanning; D002851:Chromatography, High Pressure Liquid; D003593:Cytoplasm / Q000201:enzymology; D007527:Isoenzymes / Q000378:metabolism*; D009206:Myocardium / Q000201:enzymology; D016202:N-Methylaspartate; D013552:Swine; D014357:Trypsin / Q000378:metabolism*
D007527:Isoenzymes; D001224:Aspartic Acid; D016202:N-Methylaspartate; D001219:Aspartate Aminotransferases; D014357:Trypsin