INVOLVEMENT OF ENZYME-SUBSTRATE CHARGE INTERACTIONS IN THE CASEINOLYTIC SPECIFICITY OF LACTOCOCCAL CELL ENVELOPE-ASSOCIATED PROTEINASES

Three series of oligopeptides were synthesized to investigate the prop osal that a major factor in determining the differences in specificity of the lactococcal cell surface-associated proteinases against casein s is the interactions between charged amino acids in the substrate and in the enzyme. The sequences of the oligopeptides were based on two r egions of kappa-casein (residues 98 to 111 and 153 to 169) which show markedly different susceptibilities to P-I- and P-III-type lactococcal proteinases. In each series, one oligopeptide had an identical sequen ce to that of the kappa-casein region, while in the others, one or mor e charged residues were substituted by an amino acid of opposite charg e, i.e., His <-> Glu. Generally, substitution of His by Glu in the oli gopeptides corresponding to residues 98 to 111 of kappa-casein resulte d in reduced cleavage of susceptible bonds by the P-I-type proteinase and increased cleavage of susceptible bonds by the P-III-type proteina se. In the case of the oligopeptide corresponding to residues 153 to 1 69 of kappa-casein, one major cleavage site was evident, and the bond was hydrolyzed bg both types of proteinase (even though this sequence in kappa-casein itself is extremely resistant to the P-I-type enzyme), Substitution of Glu by His in this oligopeptide, even in the P-7 posi tion, resulted in increased cleavage of the bond by the P-I-type prote inase and reduced cleavage by the P-m-type proteinase. C-terminal trun cation of this oligopeptide resulted in a 100 fold decrease in the rat e of hydrolysis of the susceptible bond and a change in the pattern of cleavage. While the charge on residues in the immediate vicinity of p otentially susceptible bonds has a marked effect on their rate of clea vage by the proteinase, it is clear that other factors are important i n determining which bonds in the casein molecule are actually hydrolyz ed.