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

Apparent rate constants of tryptic hydrolysis of amide bonds containing Arg and Lys residues in beta-casein were determined by the analysis of kinetic s of accumulation of 17 major peptide components revealed by high performan ce liquid chromatography. When studying pH influence on Arg/Lys bond cleava ge preference, averaged rate constants over several Arg-X and Lys-X bonds w ere used for analysis of kinetics of wild-type trypsin, K188H, K188F, K188Y , K188W, and of K188D/D189K mutants. The pK(alpha 1) value of 6.5 was found for all studied trypsins, For wild-type trypsin and its K188D/D189K mutant , pK(alpha 2) was found to be 10. The lowest among studied engineered tryps ins pK(alpha 2) = 9.3 was determined for K188Y mutant. Considerable prefere nce for the cleavage of Arg over Lys containing peptide bonds was demonstra ted for all trypsins with engineered S2 site except for K188H and K188F. Th e comparison of individual rate constants for various bonds showed that dur ing the hydrolysis by wild-type trypsin, the probabilities of splitting dep end on secondary specificity and local hydrophobicity of amino acid residue s, which are nearest to the hydrolyzed peptide bond (P2 site). The improvem ent of prediction of hydrolysis rates performed by the used program was ach ieved after considering the presence of hydrophobic neighborhood of Lys48-I le49 and Arg202-Gly203 bonds. (C) 2000 John Wiley & Sons, Inc.