Probing catalytic rate enhancement during intramembrane proteolysis

Area of Science:

• Biochemistry
• Molecular Biology
• Enzymology

Background:

• Rhomboid proteases are intramembrane serine proteases crucial for cellular signaling.
• Previous studies revealed the active site structure of E. coli rhomboid GlpG but lacked mechanistic details due to unknown substrates.
• The physiological substrate for the AarA rhomboid remains the only known substrate pair.

Purpose of the Study:

• To elucidate the catalytic roles of specific residues in the AarA rhomboid protease.
• To quantify the contribution of active site residues to reaction rate enhancement and transition state stabilization.
• To gain insight into the mechanism of nucleophile generation in rhomboid proteases.

Main Methods:

• Utilized a homology model of AarA refined with molecular dynamics (MD).
• Performed site-directed mutagenesis to substitute catalytically important residues (H83, N87, M154).
• Quantified changes in enzymatic activity and transition state stabilization energy (ΔΔG‡).

Main Results:

• AarA active site geometry was found to be strict and intolerant to alterations.
• Substitution of H83 abolished AarA activity or reduced ΔΔG‡ by 3.1 kcal/mol.
• Substitution of N87 decreased ΔΔG‡ by 1.6-3.9 kcal/mol, and M154 substitution provided insights into nucleophile generation.

Conclusions:

• Identified critical roles for H83 and N87 in transition state stabilization and catalytic efficiency.
• Demonstrated the importance of residue M154 in stabilizing the catalytic general base for nucleophile generation.
• Provided quantitative data on residue contributions to hydrolytic efficiency in intramembrane proteolysis.