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.