Autoproteolysis of the Extracellular Serine Proteinase of Lactococcus lactis subsp. cremoris Wg2.

The molecular masses of purified extracellular serine proteinase of a number of Lactococcus lactis strains vary significantly, and these molecular mass values do not correspond to the values estimated on the basis of genetic data. The discrepancies can only partially be explained by N-terminal processing during maturation of the precursor enzyme and by C-terminal cleaving during the release from the cell envelope. With a monoclonal antibody that binds in the active site region of the L. lactis proteinase, the processing of the released proteinase was followed. At 30 degrees C the proteinase was degraded with a concomitant loss of beta-casein hydrolytic activity. In the presence of CaCl(2), proteinase degradation was inhibited, and new degradation products were detected. The specific serine proteinase inhibitors phenylmethylsulfonyl fluoride and diisopropylfluorophosphate also inhibited proteinase degradation. Two major high-molecular-mass proteinase fragments (165 and 90 kDa) were found to have the same N-terminal amino acid sequence as the mature proteinase, i.e., [Asp-1-Ala-2-Lys-3-Ala-4-Asn-5-Ser-6, indicating that both fragments were formed by cleavage at the C terminus. The N terminus of a proteinase fragment with low molecular mass (58 kDa) started with Gln-215. In this fragment part of the active site region was eliminated, suggesting that it is proteolytically inactive. Unlike larger fragments, this 58-kDa fragment remained intact after prolonged incubations. These results indicate that autoproteolysis of the L. lactis subsp. cremoris Wg2 proteinase ultimately leads to inactivation of the proteinase by deletion of the active site region.