The V108M mutation decreases the structural stability of catechol O-methyltransferase

Area of Science:

• Biochemistry
• Molecular Biology
• Genetics

Background:

• The catechol O-methyltransferase (COMT) gene has a common single-nucleotide polymorphism (108V/M) affecting enzyme activity and stability.
• The 108M variant of soluble COMT (s-COMT) shows reduced stability at physiological temperatures and is linked to increased risks of breast cancer and neuropsychiatric disorders.

Purpose of the Study:

• To investigate the impact of the 108V/M polymorphism on the stability of purified, recombinant s-COMT.
• To analyze the contributions of individual tryptophan residues (W143 and W38) to protein stability and fluorescence properties.

Main Methods:

• Circular dichroism (CD) spectroscopy to assess global secondary structure.
• Dynamic light scattering to detect aggregation and changes in hydration.
• Fluorescence spectroscopy to probe tertiary structure and tryptophan environments.
• Thermal and guanidine hydrochloride (GuHCl) denaturation studies.

Main Results:

• The 108M s-COMT variant demonstrated significantly lower thermal stability (5-7°C lower transition midpoint) and reduced unfolding free energy compared to 108V s-COMT.
• 108M s-COMT exhibited increased susceptibility to aggregation and partial unfolding at 37°C.
• W143 was identified as the primary contributor to tryptophan fluorescence in the folded protein.
• The co-substrate S-adenosylmethionine (SAM) stabilized the secondary structure of both variants.

Conclusions:

• The 108V/M polymorphism in s-COMT directly impacts protein stability, with the 108M variant being less stable.
• The observed differences in stability, particularly at physiological temperatures, may underlie the association of the 108M allele with various diseases.
• Understanding COMT protein stability is crucial for interpreting its role in health and disease.