Investigating the Relationship between the Substrates' Consumption and Their Abundances in a Complex Enzymatic System

Area of Science:

• Biochemistry
• Analytical Chemistry
• Enzymology

Background:

• Enzymatic reactions are crucial for bioanalytical chemistry, including proteomics and substrate screening.
• The relationship between substrate consumption and abundance in complex enzymatic systems was previously unclear.

Purpose of the Study:

• To theoretically and experimentally investigate substrate consumption priority in complex enzymatic systems.
• To determine if substrate abundance or specificity constants dictate consumption order.

Main Methods:

• Derivation of mathematical expressions relating substrate consumption fractions to specificity constants.
• Experimental validation using a trypsin enzymatic system with five synthetic peptide substrates.
• Application of quantitative proteomics to analyze 144 competing substrates across four experiments.

Main Results:

• Substrate consumption priority is dictated by specificity constants, not initial abundances.
• A linear correlation was observed between ln(1 - pᵢ) and specificity constants for competing substrates.
• Prioritization of substrates can be achieved by analyzing their consumption rates.

Conclusions:

• Enzymatic substrate consumption follows specificity constants, independent of abundance.
• This study enhances understanding of enzymatic kinetics in complex biological systems.
• Findings will benefit the design and optimization of enzymatic analytical methods.