Hydrogen tunneling in enzyme reactions.

Area of Science:

• Biochemistry
• Enzymology
• Chemical Kinetics

Background:

• Yeast alcohol dehydrogenase (YADH) is crucial for alcohol metabolism.
• The hydrogen-transfer step in YADH-catalyzed reactions is often rate-limiting.
• Understanding kinetic isotope effects (KIEs) provides insights into reaction mechanisms.

Purpose of the Study:

• To determine primary and secondary protium-to-tritium (H/T) and deuterium-to-tritium (D/T) KIEs for YADH-catalyzed benzyl alcohol oxidation.
• To investigate the contribution of hydrogen tunneling to the reaction mechanism.
• To compare experimental KIEs with predictions from semiclassical mass considerations.

Main Methods:

• Enzymatic assays using benzyl alcohol as substrate.
• Measurement of reaction rates at 25°C.
• Determination of H/T and D/T kinetic isotope effects.
• Analysis of deviations from semiclassical predictions.

Main Results:

• Significant deviations were observed between experimental and predicted H/T and D/T KIEs.
• Experimental H/T ratios were substantially greater than those predicted without tunneling.
• These deviations persisted across a temperature range of 0–40°C.

Conclusions:

• The results strongly support the occurrence of significant hydrogen tunneling in the YADH-catalyzed reaction.
• The reaction coordinate involves a substantial quantum mechanical tunneling contribution.
• Deviations from classical predictions highlight the importance of quantum effects in enzyme catalysis.