Thymidylate synthase catalyzed H-transfers: two chapters in one tale

Area of Science:

• Biochemistry and Enzymology
• Chemical Kinetics
• Bioorganic Chemistry

Background:

• Enzymatic catalysis involves complex reaction pathways with multiple bond activations.
• Thymidylate synthase (TSase) is crucial for DNA synthesis, catalyzing a reaction with sequential hydride and proton transfers.
• Understanding the physical nature of these transfers is key to enzyme mechanism elucidation.

Purpose of the Study:

• To compare the physical nature of two sequential hydrogen transfers within the same enzymatic reaction catalyzed by TSase.
• To experimentally determine the intrinsic kinetic isotope effects (KIEs) for both hydride and proton transfer steps.
• To interpret the observed KIEs using a Marcus-like model to understand enzyme optimization.

Main Methods:

• Experimental determination of intrinsic kinetic isotope effects (KIEs) for sequential hydrogen transfers.
• Kinetic analysis of the enzymatic reaction catalyzed by thymidylate synthase.
• Application of the Marcus-like model for interpreting experimental data.

Main Results:

• Two distinct C-H bond activations, a hydride transfer and a proton transfer, were investigated in TSase catalysis.
• Intrinsic KIEs were extracted for both the rate-limiting hydride transfer and the non-rate-limiting proton transfer.
• Unlike the hydride transfer, the intrinsic KIEs for the proton transfer exhibited temperature dependence.

Conclusions:

• TSase optimizes donor-acceptor geometries for the rate-limiting hydride transfer step.
• Enzyme optimization is less pronounced for the faster proton transfer step.
• The findings provide insights into the differential control of sequential bond activations in enzymatic catalysis.