Correlation of polarized absorption spectroscopic and X-ray diffraction studies of crystalline cytosolic aspartate aminotransferase of pig hearts.

Absorption spectra of large, well-formed crystals of cytosolic aspartate aminotransferase have been recorded using plane polarized light. Making use of measurements of crystal thickness we have calculated extinction coefficients with the electric vector of the light parallel to both the a and c axes of the crystals of the enzyme in space group P212121. The spectra have been resolved into components with lognormal distribution curves and the resulting integrated intensities have been used to calculate the ca polarization ratios for the absorption bands of the bound co-enzyme pyridoxal 5′-phosphate. From the polarization ratio and the co-ordinates of the co-enzyme ring atoms, provided by X-ray crystallography, we have assigned principal molecular directions of the transition dipole moment within the plane of the co-enzyme ring. Of two possible orientations, only one predicts the correct crystal extinction coefficients for the 436 nm band. In this orientation, when viewed from the B face of the ring (i.e. looking into the active site of the enzyme), the transition moment is related to the N-1-C-4 axis of the ring by counterclockwise rotation by 27 °. A tentative assignment of the principal molecular directions of the transition moment has also been made for the 368 nm band of the high pH form of the enzyme. In each case, the plane of the co-enzyme ring was located from the atomic co-ordinates of the ring atoms and of those atoms attached directly to the ring. The projection of the N-1 to C-4 axis on to this plane was used to evaluate the orientation of the transition moment, which was presumed to lie precisely within the plane of the ring. We have tilted this plane systematically to evaluate the error in transition moment direction resulting from uncertainties in the atomic co-ordinates. When 2-methylaspartate is diffused into the crystals it forms a Schiff base with the coenzyme in which the ring has tilted about 32 ° from its original position and the polarization ratio of the 436 nm band drops from 1·6 in the free enzyme to about 0·38. On the assumption that the orientation of the transition moment within the co-enzyme does not change during this rotation, this value of the polarization ratio is within experimental error of that predicted from X-ray structures on the two forms. The 2-methylaspartate binds only to subunit 1. X-ray studies show that the active site in subunit 2 is devoid of ligand, and spectroscopic studies indicate that its co-enzyme remains in the “high pH form” at pH 5·8. Spectra of crystals soaked with the non-productive dicarboxylate ligands 2-oxoglutarate, maleate and succinate and also with l-glutamate have also been recorded and analyzed, as have those of the pyridoxamine phosphate form of the enzyme obtained by soaking with a solution of l-cysteine sulfinate. We have assigned the principal molecular directions of the transition moment for the unoccupied subunit of the 2-methylaspartate complex and for the 334 nm band of the ketimine form as well as tentative values for the 335 nm band of the pyridoxamine 5-phosphate form.