Introducing enzyme selectivity as a quantitative parameter to describe the effects of substrate concentration on protein hydrolysis

To understand the differences in peptide composition that result from variations in the conditions of enzymatic hydrolysis of proteins (e.g. substrate concentration) the mechanism of hydrolysis needs to be understood in detail. Therefore, methods and tools were developed to characterize and quantify the peptides formed during enzymatic protein hydrolysis. The information obtained was used to introduce a novel quantitative parameter: the selectivity of the enzyme towards the individual cleavage sites in the substrate, within the given specificity of the enzyme applied. The selectivity describes the rate of hydrolysis of a cleavage site compared to the rate of hydrolysis of all cleavage sites in the parental protein. Large differences in the selectivity of the enzyme towards the cleavage sites after the same type of amino acid residues in a protein were found. For β-lactoglobulin hydrolyzed by Bacillus licheniformis protease the selectivity was found to vary between 0.003 % and 17 % or even 0 for some cleavage sites. The effects of increasing substrate concentration and pH on the hydrolysis were studied. An increase in substrate concentration results in lower kinetics of hydrolysis, related to the available amount of water. This also resulted in significant changes in the enzyme selectivity towards the cleavage sites for which the enzyme has a high selectivity. Changing the pH of hydrolysis resulted in large changes in the kinetics of hydrolysis as well as in the enzyme selectivity. Due to the detailed analysis of the peptide composition, certain a-specific peptides were identified. It was shown that these originate from spontaneous cleavage of formed peptides. The changes in the mechanism of hydrolysis were compared to simulation data. The simulation data were obtained from a stochastic model based on random selection of the substrate and the cleavage site, given the specificity of the enzyme. A quite good agreement was obtained between simulated and experimental data. The parameters and methods developed in this study to describe the mechanism of hydrolysis can potentially be used for more complex systems.