Kinetics of β-casein hydrolysis by wild-type and engineered trypsin

Abstract

Apparent rate constants of tryptic hydrolysis of amide bonds containing Arg and Lys residues in β‐casein were determined by the analysis of kinetics of accumulation of 17 major peptide components revealed by high performance liquid chromatography. When studying pH influence on Arg/Lys bond cleavage preference, averaged rate constants over several ArgX and LysX bonds were used for analysis of kinetics of wild‐type trypsin, K188H, K188F, K188Y, K188W, and of K188D/D189K mutants. The pKa1 value of 6.5 was found for all studied trypsins. For wild‐type trypsin and its K188D/D189K mutant, pKa2 was found to be 10. The lowest among studied engineered trypsins pKa2 = 9.3 was determined for K188Y mutant. Considerable preference for the cleavage of Arg over Lys containing peptide bonds was demonstrated for all trypsins with engineered S2 site except for K188H and K188F. The comparison of individual rate constants for various bonds showed that during the hydrolysis by wild‐type trypsin, the probabilities of splitting depend on secondary specificity and local hydrophobicity of amino acid residues, which are nearest to the hydrolyzed peptide bond (P2 site). The improvement of prediction of hydrolysis rates performed by the used program was achieved after considering the presence of hydrophobic neighborhood of Lys48Ile49 and Arg202Gly203 bonds. © 2000 John Wiley & Sons, Inc. Biopoly 54: 355–364, 2000

Monitoring of Demasking of Peptide Bonds During Proteolysis by Analysis of the Apparent Spectral Shift of Intrinsic Protein Fluorescence

“…17 In the comparison with globular proteins, casein is known to be easily hydrolysed by proteases because of its elevated conformational flexibility and the abundance of the enzymeaccessible peptide bonds. 14 It was reported that clusters of hydrophobic amino acid residues in the polypeptide chain of casein substrate cause hydrophobic interaction to be responsible for the steric obstacles shielding peptide bonds against enzymatic attack. 3,14 For the proteolysis of β-CN, the fluorescence shift was also attributed to the increase of the water accessibility of Trp residue.…”

“…This effect known as masking of peptide bonds was pointed to be one of the main limiting factors of proteolysis. 3,14 Additionally, enzyme inhibition is a complex process, which includes interaction of the enzyme with the set of various proteolysis products. Consequently, simplifications are required for proteolysis models that can be used for quantitative estimations.…”

Conformational Equilibria and Rates of Localized Motion within Hepatitis B Virus Capsids

“…Because the initial proteolytic reaction is focused on a single site, the kinetic analysis is practical and unambiguous. Computational modeling of proteolytic reactions indicates that even a minor secondary cleavage pathway can have a significant impact on the calculated equilibrium and rates (63,64). These concerns make it vital that measurements of dynamic motion based on the disappearance of starting material be the result of a single cleavage pathway.…”

Determination of the influence of the pH of hydrolysis on enzyme selectivity of Bacillus licheniformis protease towards whey protein isolate

“…In that case, the pH (7e10) affects the charge state of the amino acids on the substrate (Lys, Arg). This also resulted in significant changes in the hydrolysis patterns (Vorob'ev et al, 2000). Different behaviour in the formation of peptides was also observed with optimum pH for the formation of some of the peptides.…”

“…In this way, the selectivity is more detailed than the enzyme specificity, which only describes the amino acids after which an enzyme will cleave a peptide bond. An indication that the pH of hydrolysis influences the enzyme selectivity was found in the tryptic hydrolysis of b-casein (Vorob'ev, Dalgalarrondo, Chobert, & Haertl e, 2000). It was shown that the kinetics of peptide formation and release of peptides with Arg residues were not affected by the changes in pH, while, for peptides after Lys, the pH of hydrolysis did influence the peptide kinetics.…”