Carbon Acidity in Enzyme Active Sites.

Area of Science:

• Biochemistry
• Enzyme kinetics
• Physical organic chemistry

Background:

• Enzyme active sites utilize acid-base catalysis for C-H deprotonation.
• Understanding substrate C-H pK_a values is crucial for enzyme mechanism elucidation.

Purpose of the Study:

• To determine the pK_a values of substrates acting as carbon acids within enzyme active sites.
• To quantify the extent of carbon acidity enhancement by enzymes compared to aqueous solutions.

Main Methods:

• Calculation of enzymatic free energy profiles (FEPs) to determine proton transfer equilibrium constants.
• Extraction of proton transfer equilibrium constants from existing literature data.
• Calculation of substrate C-H pK_a values using determined equilibrium constants and known catalyst pK_as.

Main Results:

• Substrate C-H pK_a values in active sites range from 5.6 to 16 across 11 different enzymes.
• Enzymes lower substrate C-H pK_as by up to 23 units, providing up to 31 kcal/mol of carbanion stabilization.
• Marcus intrinsic barriers are reduced for cofactor-independent enzymes but slightly increased for pyridoxal phosphate-dependent enzymes.

Conclusions:

• Enzymes dramatically increase carbon acidity, stabilizing carbanions effectively.
• This enhanced carbon acidity is a primary driver for the significant rate accelerations observed in carbon deprotonation catalysis.
• Enzyme catalytic strategies modulate intrinsic reaction barriers differently based on cofactor dependence.