Motional dynamics of the catalytic loop in OMP synthase

Area of Science:

• Biochemistry
• Enzymology
• Structural Biology

Background:

• Orotate phosphoribosyltransferase (OPRTase) is key in de novo pyrimidine biosynthesis, catalyzing OMP formation from orotic acid and PRPP.
• The enzyme's dimeric structure features a solvent-exposed active site with a mobile loop containing essential catalytic residues.
• Previous studies indicated this loop can cover the active site of an adjacent subunit in enzyme-inhibitor complexes.

Purpose of the Study:

• To investigate the dynamic properties of the mobile loop in orotate phosphoribosyltransferase.
• To understand the role of loop flexibility in enzyme catalysis and substrate/product binding.
• To elucidate the mechanism of PRPP release and its impact on enzymatic activity.

Main Methods:

• Limited proteolysis with trypsin to assess loop accessibility.
• Two-dimensional 1H-15N NMR relaxation experiments (T1, T2, NOE) on histidine-labeled OPRTase.
• 1H and 31P NMR titrations to monitor ligand binding and protein motion.

Main Results:

• Limited proteolysis confirmed the loop's solvent-exposed nature.
• NMR data revealed high flexibility of the surface loop in the unliganded enzyme, with significant reduction in flexibility upon analogue binding.
• Quantification of loop movement rates during product release indicated a two-step process, partially rate-limiting for reverse pyrophosphorolysis.

Conclusions:

• The mobile surface loop of OPRTase exhibits significant flexibility in the absence of ligands.
• Ligand binding, particularly PRPP, induces a conformational change, reducing loop flexibility and impacting catalysis.
• Loop opening and PRPP release constitute a two-step mechanism, with protein motion being integral to the enzymatic cycle.