Three series of oligopeptides were synthesized to investigate the proposal that a major factor in determining the differences in specificity of the lactococcal cell surface-associated proteinases against caseins is the interactions between charged amino acids in the substrate and in the enzyme. The sequences of the oligopeptides were based on two regions of -casein (residues 98 to 111 and 153 to 169) which show markedly different susceptibilities to P I -and P III -type lactococcal proteinases.

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... In each series, one oligopeptide had an identical sequence to that of the -casein region, while in the others, one or more charged residues were substituted by an amino acid of opposite charge, i.e., His7Glu. Generally, substitution of His by Glu in the oligopeptides corresponding to residues 98 to 111 of -casein resulted in reduced cleavage of susceptible bonds by the P I -type proteinase and increased cleavage of susceptible bonds by the P III -type proteinase. In the case of the oligopeptide corresponding to residues 153 to 169 of -casein, one major cleavage site was evident, and the bond was hydrolyzed by both types of proteinase (even though this sequence in -casein itself is extremely resistant to the P I -type enzyme). Substitution of Glu by His in this oligopeptide, even in the P 7 position, resulted in increased cleavage of the bond by the P I -type proteinase and reduced cleavage by the P III -type proteinase. C-terminal truncation of this oligopeptide resulted in a 100-fold decrease in the rate of hydrolysis of the susceptible bond and a change in the pattern of cleavage. While the charge on residues in the immediate vicinity of potentially susceptible bonds has a marked effect on their rate of cleavage by the proteinase, it is clear that other factors are important in determining which bonds in the casein molecule are actually hydrolyzed.