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Quantifying the Modulation of Elastase Enzyme Activity Through Colorimetric Analysis

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Carbon Acidity in Enzyme Active Sites.

Michael D Toney¹

¹Department of Chemistry, University of California, Davis, Davis, CA, United States.

Frontiers in Bioengineering and Biotechnology

|March 7, 2019

Summary

Enzymes significantly increase carbon acidity, lowering substrate C-H pK_a values by up to 23 units. This stabilization of carbanions is key to the enhanced reaction rates observed in carbon deprotonation enzymes.

Keywords:

PKA carbanion stability carbon acid enzyme general acid/base catalysis marcus theory pyridoxal phosphate

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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.