Phosphorus-31 Nuclear Magnetic Resonance Studies on Apoaspartate Aminotransferase Reconstituted with Cofactor Analogues

Summary

The ³¹P chemical shift of pyridoxal phosphate (PLP) in native cytosolic aspartate aminotransferase (cAAT) has been reported to be pH dependent with a pK of 6.2 (Schnackerz, 1984) even though X-ray data suggest that the cofactor phosphate group remains dianionic throughout. For further information on the pH dependence of ³¹P NMR spectra apo-cAAT reconstituted with phosphopyridoxyl aspartate and pyridoxal 5′deoxymethylene-phosphonate was measured. The chemical shifts for the two cofactor analogues when bound to apo-cAAT were found to be pH independent and should correspond to the phosph(on)ate dianion. Thus, the 31P NMR data on native cAAT can only be interpreted as the protonation/deprotonation equilibrium of the PLP-Lys 258 “internal aldimine” (pK=6.2). It is proposed that protonation of the aldimine exerts strain in the 5′-phosphate ester linkage, resulting in modified O-P-O bond angles which in turn cause changes in the ³¹P chemical shift.