Substrate-Based Allosteric Regulation of a Homodimeric Enzyme

Area of Science:

• Biochemistry
• Enzymology
• Structural Biology

Background:

• Many enzymes exhibit half-of-the-sites reactivity, with only one active site functional at a time.
• Fluoroacetate dehalogenase, a homodimeric enzyme, utilizes substrate binding to regulate protomer activity and dynamics.

Purpose of the Study:

• To investigate the role of interprotomer dynamics in fluoroacetate dehalogenase catalysis.
• To elucidate the mechanism of inhibition at high substrate concentrations and the function of an allosteric pocket.

Main Methods:

• Rigidity-based allosteric transmission theory
• Nuclear magnetic resonance (NMR) spectroscopy
• Functional assays

Main Results:

• High substrate concentrations lead to second substrate binding, dampening interprotomer dynamics and inhibiting catalysis.
• A mutation (K152I) abolishes second site binding and inhibition but reduces maximum catalytic rate, indicating the allosteric pocket's role in catalysis.
• The outer pocket desolvates substrate and facilitates its transfer to the active site, acting as an allosteric conduit.

Conclusions:

• Enzyme dynamics, regulated by allosteric networks, are crucial for fluoroacetate dehalogenase function.
• Second substrate binding at high concentrations inhibits catalysis by disrupting these essential dynamics.