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

The enzymatic process involving the incubation of a library of substrates with an enzyme is the key step for a few important experiments for bioanalytical chemistry including proteomics analysis, enzymatic labeling, substrate screening, etc. The relationship between the substrates' consumption and their abundances in a complex enzymatic system with a huge number of coexisting substrates of different abundances was not well-known. In this study, we have demonstrated theoretically and experimentally that the priority of substrate consumption depended on their specificity constants but not abundances. We derived the expression between the fractions of the substrates consumed (p i ) and their specificity constants. Using the enzymatic system of five synthetic peptide substrates of trypsin, we validated through 24 experiments that the ln(1 - p i ) values of competing substrates have linear correlation with their specificity constants, and thus, the priority of substrate depletion has no relation with their abundances. Using a state of the art quantitative proteomics approach, we found that the ln(1 - p i ) values of 144 competing substrates between any two of four experiments have a linear relationship and the prioritization of substrates can be achieved by sorting their consumption rates in the experiment. This study will improve our understanding of the enzymatic kinetics in the complex system and will benefit the design of enzymatic analytical approaches.