Loop residues and catalysis in OMP synthase.

Area of Science:

• Enzymology
• Protein Engineering
• Biochemistry

Background:

• Salmonella typhimurium orotate phosphoribosyltransferase (OMP synthase) is crucial for nucleotide biosynthesis.
• The enzyme's catalytic loop (residues 100-109) is highly conserved and essential for activity.
• Understanding the catalytic loop's role is key to enzyme mechanism studies.

Purpose of the Study:

• To investigate the function of the catalytic loop in Salmonella typhimurium OMP synthase.
• To elucidate the roles of specific residues within the catalytic loop through site-directed mutagenesis.
• To correlate structural changes with kinetic parameters and catalytic efficiency.

Main Methods:

• Site-directed mutagenesis was used to create alanine substitutions and deletions within the catalytic loop.
• Enzyme kinetics, including k(cat)/K(M) and kinetic isotope effects (KIEs), were measured for wild-type and mutant enzymes.
• Equilibrium binding studies were performed to assess substrate interactions.

Main Results:

• Mutations K103A and H105A significantly decreased catalytic efficiency (k(cat)/K(M)) for PRPP.
• Equilibrium binding was largely unaffected by loop mutations, suggesting limited ground-state contributions.
• H105A and E107A mutants fully expressed intrinsic [1'-(3)H]OMP kinetic isotope effects, indicating altered catalytic loop closure.

Conclusions:

• The catalytic loop plays a critical role in the catalytic mechanism of OMP synthase.
• Specific residues like K103, H105, and E107 are vital for efficient catalysis.
• The degree of catalytic loop closure influences the expression of kinetic isotope effects and the rate-limiting step in catalysis.