THE CONVERSION OF THE ALPHA-S1-CASEIN-(1-23)-FRAGMENT BY THE FREE ANDBOUND FORM OF THE CELL-ENVELOPE PROTEINASE OF LACTOCOCCUS-LACTIS SUBSP CREMORIS UNDER CONDITIONS PREVAILING IN CHEESE

The effect of pH, salt (NaCl, 4% w/v) and Ca++-ions (10 mM) on the spe cificity of two related cell-envelope proteinases (PI-type and PIII-ty pe) of Lactococcus lactis subsp. cremoris towards cleavage of the alph a(s1)-casein-(1-23)-fragment (cf. Exterkate et al., 1991, Biochem. J. 273: 135-139) and on the rate of conversion of this peptide was invest igated. In addition, the cell-bound (in situ) and the free forms of th ese enzymes were compared. At pH 6.5 and a relatively low ionic streng th the selection of peptide bonds to be cleaved (viz. bonds 8-9,9-10 a nd 13-14 by PI and bonds 16-17,17-18 and 21-22 by PIII) is determined in the first instance by long-range electrostatic attraction and repul sion. Substrate binding with respect to the cleavage of bond 13-14 by PI and of bonds 16-17 and 17-18 by PIII is not essentially electrostat ic but involves hydrophobic interactions together with other chemical forces. Binding is governed by electrostatic forces with respect to cl eavage of bonds 8-9 and 9-10 by PI and of bond 21-22 by PIII. The latt er cleavage is unique seeing that an electrostatic binding of the p'1 residue with the leaving group binding site of the enzyme is essential . In situ proteinase action largely reflects the specificity of the so luble enzymes. However, some impact of the micro-environment on specif icity could be established. Moreover, the pH dependence of the in situ PI-type proteinase deviates remarkably from that of the free enzyme. It is therefore suggested that specific catalytic properties of the en zyme are imposed by the in situ structural organisation. The initial a ction of the cell-envelope proteinase is essential for efficient conve rsion of the alpha(s1)-casein-(1-23)-fragment to amino acids by the co ncerted actions of other cell-bound peptidases.