Catalytic activity in crystals of mitochondrial aspartate aminotransferase as detected by microspectrophotometry.

Triclinic single crystals of mitochondrial aspartate aminotransferase from chicken were examined by microspectrophotometry using nonpolarized light. The crystalline enzyme shows the same protein and coenzyme absorption bands in the 250 to 500 nm region as the enzyme in solution. The spectrum of the pyridoxal form (A,,, 355 nm) changes into that of the pyridoxamine form (X,,, 333 nm) when excess cysteine sulfinate is added to the medium (20% (w/v) polyethylene glycol 6000/50 mM sodium phosphate (pH 7.5)). Addition of excess oxalacetate reverses the spectral change. In either direction, the conversion is complete, indicating that both active sites of the enzyme dimer are catalytically competent in the crystal lattice. As determined by spectrophotometric pH titration of crystals, the apparent p& of enzyme-bound pyridoxal-5’-P is 6.1 to 6.2, i.e. about the same as in solution. The changes of the absorption spectrum brought about by the addition of substrates and substrate analogs essentially correspond to those observed in solution. Thus, addition of 2-oxoglutarate to the pyridoxal form of the crystalline enzyme at pH 7.5 generates the 430 nm absorption band ascribed to a nonproductive adsorption complex. On addition of the substrate analog 3-hydroxyaspartate, the crystals turn red due to the intense absorption maximum at 495 nm of the semiquinoid intermediate. The different absorption bands of the enzyme substrate intermediates generated in the presence of 2-methylaspartate or of the transaminating substrate pair aspartate and oxalacetate (X,,, 340, 362 (sh), 430 nm) differ in their relative intensities from those observed in solution.