Differences in short peptide-substrate cleavage by two cell-envelope-located serine proteinases of Lactococcus lactis subsp. cremoris are related to secondary binding specificity.

Various chromophoric peptides have been tested as substates for two genetically related types (PI and PIII) of cell-envelope proteinases of Lactococcus lactis subsp. cremoris. The positively charged peptide methoxy-succinyl-arginyl-prolyl-tyrosyl-p-nitroanilide appeared to be cleaved with the highest catalytic efficiency by both enzymes, although in the case of PIII only at high ionic strength. A cation binding site in the PI-type proteinase that is not present in the related PIII-type appears to be mainly responsible for the difference between these enzymes with respect to the rate of conversion of this chromophoric substrate at relatively low ionic strength. This cation binding site most probably resides in the aspartic acid residue 166, which in PIII is substituted by asparagine. Substitution of the threonine residue 138 by lysine in PIII may also play a role. The binding step in the reaction pathway catalysed by PI at low ionic strength is governed mainly by an ionic interaction involving the cation binding site. In addition, hydrophobic interactions contribute to the binding process. Masking of the cation binding site only increases the Michaelis constant Km; the catalytic constant kcat is not affected. In the absence of the cation binding site (viz. in PIII) the free energy derived from the hydrophobic interactions only is too small to promote binding of the substrate effectively. High activities are measured only if a high ionic strength is introduced. Removal of electrostatic repulsion between the substrate and positively charged residues of the enzyme, among which is lysine 138, may contribute to this activation.(ABSTRACT TRUNCATED AT 250 WORDS)