Arginine-178 is an essential residue for ITPA function.

Area of Science:

• Biochemistry
• Genetics
• Enzymology

Background:

• The inosine triphosphate pyrophosphatase (ITPA) enzyme is vital for maintaining cellular nucleotide pools by hydrolyzing noncanonical nucleoside triphosphates.
• A significant mutation, R178C, in the ITPA gene is linked to developmental and epileptic encephalopathy 35 (DEE 35), a fatal infantile disorder.
• Accumulation of inosine triphosphate (ITP) due to ITPA dysfunction is implicated in the pathogenesis of DEE 35.

Purpose of the Study:

• To investigate the biochemical consequences of the R178C ITPA mutation and other mutations at position 178.
• To elucidate the structural and functional impact of these mutations on ITPA enzyme activity.
• To explore potential therapeutic strategies for DEE 35 based on ITPA function.

Main Methods:

• Michaelis-Menten enzyme kinetics assays were performed on the R178C ITPA mutant and three other mutants at position 178.
• Molecular dynamics simulations were utilized to assess the structural impact of the R178C mutation.
• Biochemical data were analyzed to correlate structural changes with enzyme activity.

Main Results:

• The R178C ITPA mutant exhibited significantly reduced ITP hydrolyzing activity.
• Mutations at position 178, including the conservative R178K, substantially diminished ITPA enzyme activity.
• Structural analysis indicated that the R178C mutation disrupts active-site hydrogen bond networks, leading to increased flexibility and reduced function.

Conclusions:

• Position 178 of the ITPA enzyme is critical for its catalytic activity.
• Disruption of the active-site hydrogen bond network is a primary mechanism underlying the reduced ITPA activity observed in the R178C mutation.
• Understanding the molecular basis of ITPA dysfunction provides a foundation for developing targeted therapies for DEE 35.