The imine-pyridine torsion of the pyridoxal 5'-phosphate Schiff base of aspartate aminotransferase lowers its pKa in the unliganded enzyme and is crucial for the successive increase in the pKa during catalysis.

In aspartate aminotransferase, pyridoxal 5'-phosphate (PLP) forms a Schiff base with the epsilon-amino group of Lys258 (internal aldimine). The internal aldimine has a pKa value of 6.8. Binding of a substrate amino acid to the enzyme yields the Michaelis complex, in which PLP still forms the internal aldimine with Lys258. This is followed by a transaldimination process to form a Schiff base of PLP with the alpha-amino group of substrates (external aldimine). Kinetic analysis of the spectral changes during the reaction of the enzyme with a substrate analogue 2-methylaspartate showed that the aldimine is 6.4-8.6% protonated in the Michaelis complex and 32-43% in the external aldimine. The bases that accept protons from the aldimines are considered to be the substrate alpha-amino group in the Michaelis complex and the epsilon-amino group of Lys258 in the external aldimine. Therefore, the intrinsic pKa value of the aldimine is expected to increase over a range of 3 during transformation from the unliganded enzyme (pKa = 6.8) to the Michaelis complex (pKa = 8.8) and the external aldimine (pKa > 10). When the Lys258 side chain of the internal aldimine was "cleaved" by the construction of an enzyme in which Lys258 was replaced by Ala and the aldimine was reconstituted with methylamine, the pKa of the internal aldimine was increased to 9.6. This indicates that the low pKa value of the internal aldimine of the unliganded enzyme is provided by the side chain of Lys258 which destabilizes the planar conformation of the aldimine suitable for protonation. This strained conformation is partially relaxed in the Michaelis complex, and the pKa is moderately increased. On formation of the external aldimine, Lys258 is released and the aldimine is fixed to a near planar conformation and has a high pKa value. Thus, the aldimine pKa is modulated by a mechanism that exploits the conformational differences between the intermediate structures. The strain of the protonated internal aldimine is interpreted to enhance the catalytic ability of the enzyme by increasing the energy level of the free enzyme plus substrate at neutral pH relative to the transition state.