Ground-state electronic destabilization via hyperconjugation in aspartate aminotransferase

Abstract

Binding isotope effects for l-aspartate reacting with the inactive K258A mutant of PLP-dependent aspartate aminotransferase to give a stable external aldimine intermediate are reported. They provide direct evidence for electronic ground-state destabilization via hyperconjugation. The smaller equilibrium isotope effect with deazaPLP-reconstituted K258A indicates that the pyridine nitrogen plays an important role in labilizing the Cα-H bond.

Figure 1

Formation and stabilization of the carbanionic intermediate in transamination.

Figure 2

Alignment of the Cα-H σ bond with the p orbitals of the conjugated π system in PLP external aldimine intermediates, selectively weakening the Cα-H bond through hyperconjugation.

Figure 3

(A) Top. Active site electron density for the K258A/deazaPLP/L-aspartate external aldimine. (PDB ID 4DBC) (B) Bottom. Overlay of the active sites of K258A/deazaPLP/L-aspartate with AAT/PLP/α-methyl-L-aspartate (PDB ID 1ART).

Figure 4

Calculated IE vs. change in Cα-H Pauling bond order (c = 0.3) for alanine combining with 3-hydroxy-pyridine-4-aldehyde to form an aldimine.