A mutation interfering with 5-lipoxygenase domain interaction leads to increased enzyme activity

Area of Science:

• Biochemistry
• Enzymology
• Molecular Biology

Background:

• Human 5-lipoxygenase (5-LOX) is crucial for synthesizing proinflammatory leukotrienes from arachidonic acid.
• The 5-LOX enzyme comprises an N-terminal C2-like β-sandwich domain and a catalytic domain.

Purpose of the Study:

• To investigate the interaction between the β-sandwich and catalytic domains of human 5-LOX.
• To elucidate the role of the Arg(101)-Asp(166) salt bridge in 5-LOX structure and function.
• To explore the impact of disrupting this interaction on enzyme activity.

Main Methods:

• Yeast two-hybrid system to assess domain interactions.
• Site-directed mutagenesis to alter specific amino acid residues (Arg(101) and Asp(166)).
• Enzyme kinetics assays to measure catalytic activity (vinit) and product formation.

Main Results:

• The β-sandwich and catalytic domains of 5-LOX were found to interact.
• Mutagenic disruption of the Arg(101)-Asp(166) salt bridge abolished domain interaction.
• Mutation of Arg(101) to Asp (5-LOX-R101D) significantly increased enzyme activity, showing higher initial velocity and product yield.
• These findings support a model where a flexible lid, controlled by domain interactions, regulates the 5-LOX active site.

Conclusions:

• The interaction between the 5-LOX β-sandwich and catalytic domains is mediated by the Arg(101)-Asp(166) salt bridge.
• Disruption of this salt bridge enhances 5-LOX catalytic efficiency.
• This study provides evidence for a flexible lid mechanism controlling substrate access to the active site in human 5-LOX.