Strain and catalysis in aspartate aminotransferase

Area of Science:

• Enzymology
• Biochemistry
• Structural Biology

Background:

• Ground-state destabilization is a known factor in enzyme catalysis, involving unfavorable interactions in enzyme-substrate complexes.
• Aspartate aminotransferase (AAT) is a key enzyme in amino acid metabolism, and its reaction mechanism has been extensively studied.

Purpose of the Study:

• To investigate a novel type of strain in the unliganded enzyme's internal aldimine and its role in AAT catalysis.
• To differentiate between classical strain and the strain of the distorted internal aldimine in AAT.
• To identify the true driving forces of catalysis in AAT, moving beyond pKa shifts.

Main Methods:

• Computational analysis of enzyme-substrate interactions.
• Investigation of reaction intermediates in AAT.
• Analysis of the internal aldimine's pKa values in relation to catalytic efficiency.

Main Results:

• A previously unrecognized strain in the internal aldimine of unliganded AAT was identified, which increases the k(cat)/K(m) value.
• The Michaelis complex of AAT with aspartate exhibits classical strain due to unfavorable interactions.
• Strain energy, rather than pKa shifts, was identified as the primary driver for proton transfer and catalysis in AAT.

Conclusions:

• Strain energy is a critical determinant of catalytic efficiency in enzymes like AAT.
• The observed pKa variations during catalysis are consequences of energy level adjustments driven by strain.
• This work reframes the understanding of catalytic mechanisms by highlighting the role of inherent strain energy.