Ground-state electronic destabilization via hyperconjugation in aspartate aminotransferase

Area of Science:

• Biochemistry
• Enzyme kinetics
• Structural biology

Background:

• Aspartate aminotransferase (AAT) is a pyridoxal 5'-phosphate (PLP)-dependent enzyme crucial for amino acid metabolism.
• Understanding the catalytic mechanism of AAT, particularly the role of key residues and the PLP cofactor, is vital for enzyme engineering and drug development.

Purpose of the Study:

• To investigate the electronic ground-state destabilization of the Cα-H bond in the reaction of l-aspartate with a mutant aspartate aminotransferase.
• To elucidate the role of the pyridine nitrogen in the PLP cofactor and the active site lysine in the enzyme's catalytic mechanism.

Main Methods:

• Utilizing binding isotope effect studies with l-aspartate and the K258A mutant of PLP-dependent aspartate aminotransferase.
• Employing deazaPLP-reconstituted K258A mutant to assess the specific contribution of the pyridine nitrogen.

Main Results:

• Direct evidence for electronic ground-state destabilization via hyperconjugation was observed.
• A smaller equilibrium isotope effect in the deazaPLP-reconstituted K258A mutant indicated a significant role for the pyridine nitrogen.
• The K258A mutation and cofactor modifications revealed the importance of the pyridine nitrogen in labilizing the Cα-H bond.

Conclusions:

• The study provides direct evidence for hyperconjugation contributing to Cα-H bond destabilization in the external aldimine intermediate.
• The pyridine nitrogen of the PLP cofactor is essential for weakening the Cα-H bond, highlighting its importance in the catalytic mechanism of aspartate aminotransferase.