Role of calcium in activity and stability of the Lactococcus lactis cell envelope proteinase

The mature lactococcal cell envelope proteinase (CEP) consists of an N-term inal subtilisin-like proteinase domain and a large C-terminal extension of unknown function whose far end anchors the molecule in the cell envelope. D ifferent types of CEP can be distinguished on the basis of specificity and amino acid sequence. Removal of weakly bound Ca2+ from the native cell-boun d CEP of Lactococcus lactis SK11 (type III specificity) is coupled with a s ignificant reversible decrease in specific activity and a dramatic reversib le reduction in thermal stability, as a result of which no activity at 25 d egrees C (pH 6.5) can be measured. The consequences of Ca2+ removal are les s dramatic for the CEP of strain Wg2 (mixed type I-type III specificity). A utoproteolytic release of CEP from cells concerns this so-called "Ca-free" form only and occurs most efficiently in the case of the Wg2 CEP. The resul ts of a study of the relationship between the Ca2+ concentration and the st ability and activity of the cell-bound SK11 CEP at 25 degrees C suggested t hat binding of at least two Ca2+ ions occurred. Similar studies performed w ith hybrid CEPs constructed from SK11 and Wg2 wild-type CEPs revealed that the C-terminal extension plays a determinative role with respect to the ult imate distinct Ca2+ dependence of the cell-bound CEP, The results are discu ssed in terms of predicted Ca2+ binding sites in the subtilisin like protei nase domain and Ca-triggered structural rearrangements that influence both the conformational stability of the enzyme and the effectiveness of the cat alytic site. We argue that distinctive primary folding of the proteinase do main is guided and maintained by the large C-terminal extension.