Primary Deuterium Kinetic Isotope Effects From Product Yields: Rationale, Implementation, and Interpretation

Area of Science:

• Biochemistry
• Chemical Kinetics
• Spectroscopy

Background:

• Determining primary deuterium kinetic isotope effects (1°DKIEs) is crucial for understanding reaction mechanisms.
• Isotope effects are sensitive probes of the transition state structure and bond-breaking/bond-forming events.
• Existing methods for measuring 1°DKIEs can be complex or limited in scope.

Purpose of the Study:

• To present a simple and convenient method for determining 1°DKIEs using 1H NMR.
• To apply this method to enzymatic decarboxylation reactions.
• To provide a framework for understanding product deuterium isotope effects (PDIEs) in relation to solvent and enzyme active site fractionation.

Main Methods:

• Utilized 1H NMR spectroscopy to analyze the ratio of H- and D-labeled products.
• Conducted reactions in a 50:50 (v/v) HOH/DOD solvent mixture.
• Defined and applied the concept of product deuterium isotope effect (PDIE) as 1/ϕ_EL, incorporating corrections for hydron partitioning when necessary.

Main Results:

• Successfully determined 1°DKIEs for decarboxylation reactions catalyzed by orotidine 5'-monophosphate decarboxylase (OMPDC) and its mutants.
• Extended the application to determine the 1°DKIE for the decarboxylation reaction catalyzed by 5-carboxyvanillate decarboxylase.
• Demonstrated the utility of 1H NMR for quantifying deuterium incorporation and elucidating reaction mechanisms.

Conclusions:

• The described 1H NMR method offers a straightforward approach to measure 1°DKIEs in reactions utilizing solvent water.
• The study provides valuable kinetic isotope effect data for key decarboxylase enzymes.
• This methodology facilitates mechanistic investigations of enzymatic reactions involving hydron transfer.