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Kinetics of β‐casein hydrolysis by wild‐type and engineered trypsin
Author(s) -
Vorob'ev Mikhail M.,
Dalgalarrondo Michèle,
Chobert JeanMarc,
Haertlé Thomas
Publication year - 2000
Publication title -
biopolymers
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.556
H-Index - 125
eISSN - 1097-0282
pISSN - 0006-3525
DOI - 10.1002/1097-0282(20001015)54:5<355::aid-bip60>3.0.co;2-h
Subject(s) - chemistry , peptide bond , hydrolysis , trypsin , peptide , kinetics , casein , enzyme kinetics , cleavage (geology) , bond cleavage , hydrogen bond , stereochemistry , amide , active site , enzyme , organic chemistry , biochemistry , molecule , catalysis , physics , geotechnical engineering , quantum mechanics , fracture (geology) , engineering
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 ArgX and LysX bonds were used for analysis of kinetics of wild‐type trypsin, K188H, K188F, K188Y, K188W, and of K188D/D189K mutants. The pK a1 value of 6.5 was found for all studied trypsins. For wild‐type trypsin and its K188D/D189K mutant, pK a2 was found to be 10. The lowest among studied engineered trypsins pK a2 = 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 Lys48Ile49 and Arg202Gly203 bonds. © 2000 John Wiley & Sons, Inc. Biopoly 54: 355–364, 2000