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-terminal subtilisin-like proteinase domain and a large C-terminal extension of unknown function whose far end anchors the molecule in the cell envelope. Different types of CEP can be distinguished on the basis of specificity and amino acid sequence. Removal of weakly bound Ca2+ from the native cell-bound CEP of Lactococcus lactis SK11 (type III specificity) is coupled with a significant reversible decrease in specific activity and a dramatic reversible reduction in thermal stability, as a result of which no activity at 25 degrees C (pH 6.5) can be measured. The consequences of Ca2+ removal are less dramatic for the CEP of strain Wg2 (mixed type I-type III specificity). Autoproteolytic 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 results of a study of the relationship between the Ca2+ concentration and the stability and activity of the cell-bound SK11 CEP at 25 degrees C suggested that binding of at least two Ca2+ ions occurred. Similar studies performed with hybrid CEPs constructed from SK11 and Wg2 wild-type CEPs revealed that the C-terminal extension plays a determinative role with respect to the ultimate distinct Ca2+ dependence of the cell-bound CEP. The results are discussed in terms of predicted Ca2+ binding sites in the subtilisin-like proteinase domain and Ca-triggered structural rearrangements that influence both the conformational stability of the enzyme and the effectiveness of the catalytic site. We argue that distinctive primary folding of the proteinase domain is guided and maintained by the large C-terminal extension.
Exterkate
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FA
Department of Biophysical Chemistry, Netherlands Institute for Dairy Research (NIZO), 6710 BA Ede, The Netherlands. exterkate@nizo.nl
Alting
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AC
D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D002118:Calcium / Q000378:metabolism*; D002413:Cations, Divalent / Q000378:metabolism; D002462:Cell Membrane / Q000201:enzymology; D002851:Chromatography, High Pressure Liquid; D010450:Endopeptidases / Q000737:chemistry* / Q000378:metabolism*; D013294:Lactococcus lactis / Q000201:enzymology* / Q000254:growth & development; D008892:Milk / Q000382:microbiology; D008958:Models, Molecular
D002413:Cations, Divalent; D010450:Endopeptidases; D002118:Calcium