Ground-state destabilization in orotate phosphoribosyltransferases by binding isotope effects

Area of Science:

• Biochemistry
• Enzymology
• Chemical Kinetics

Background:

• Orotate phosphoribosyltransferases (OPRTs) catalyze N-ribosidic bond formation and breakage in pyrimidines.
• These enzymes proceed through ribocation-like transition states (TSs), exhibiting significant kinetic isotope effects (KIEs).
• Substrate binding isotope effects (BIEs) can resolve ground-state destabilization from transition-state effects.

Purpose of the Study:

• To quantify the contribution of substrate binding to the catalytic mechanism of OPRTs.
• To differentiate between ground-state destabilization and transition-state effects in OPRT catalysis.
• To investigate the role of the 5'-phosphate group in substrate distortion and enzyme catalysis.

Main Methods:

• Measurement of primary α-secondary 1'-(3)H kinetic isotope effects (KIEs) for k(cat)/K(m).
• Measurement of primary α-secondary 1'-(3)H substrate binding isotope effects (BIEs).
• Comparison of BIEs and KIEs for substrates with and without the 5'-phosphate group.

Main Results:

• Large BIEs (1.104-1.108) were observed for [1'-(3)H]orotidine 5'-monophosphate (OMP) with Plasmodium falciparum and human OPRTs.
• These BIEs indicate significant ground-state destabilization of OMP upon binding, with C1' hybridization shifting towards the TS.
• Half of the catalytic k(cat)/K(m) KIE for OMP originates from this binding-induced distortion.
• Orotidine, lacking the 5'-phosphate, showed no significant BIEs, indicating no ground-state destabilization.
• Despite no ground-state destabilization, the k(cat)/K(m) KIE for orotidine was similar to OMP, suggesting TS distortion occurs post-binding.

Conclusions:

• OPRTs actively distort substrates, initiating transition-state geometry upon binding, particularly when the 5'-phosphate is present.
• The catalytic mechanism involves a two-step C1' distortion: half upon binding and half during transition state formation.
• The absence of the 5'-phosphate prevents initial ground-state destabilization, but the enzyme still achieves significant transition-state distortion, albeit through a single, rate-limiting barrier.