Engineering homooligomeric proteins to detect weak intersite allosteric communication: aminotransferases, a case...

Area of Science:

• Biochemistry
• Enzyme kinetics
• Structural biology

Background:

• Homooligomeric enzymes possess identical active sites, making it challenging to study intersubunit communication.
• Allosteric communication has not been previously demonstrated in E. coli aspartate aminotransferase (AATase) and tyrosine aminotransferase (TATase).

Purpose of the Study:

• To investigate the existence of allosteric communication between the active sites of homodimeric aminotransferases.
• To develop a novel method for probing intersubunit interactions in enzymes.

Main Methods:

• Engineered a hybrid aminotransferase by grafting an engineered TATase active site into one of the AATase active sites.
• Utilized selective inhibitors to probe the activity of each distinct catalytic pocket.
• Measured the impact of inhibitor binding on the activity of the non-inhibited active site.

Main Results:

• The engineered TATase/AATase hybrid enzyme exhibited distinct catalytic and inhibitor-binding properties at each active site.
• Simultaneous catalysis of both aminotransferase reactions occurred at their respective sites.
• Inhibitor binding to one active site reduced the activity of the other active site by up to 25%, demonstrating allosteric communication.

Conclusions:

• Unequivocally demonstrated allosteric communication between active sites in a homodimeric enzyme.
• The enzyme engineering strategy is potentially applicable to studying other homologous enzyme sets.
• Provides a novel approach to dissecting intersubunit communication in oligomeric enzymes.