Cleavage of a COOH-terminal hydrophobic region from D-alanine carboxypeptidase, a penicillin-sensitive bacterial membrane enzyme. Characterization of active, water-soluble fragments.
D-Alanine carboxypeptidase (CPase), a detergent-soluble penicillin-sensitive membrane enzyme of Bacillus stearothermophilus, Mr = 46,500, was digested with either trypsin or alpha-chymotrypsin to yield water-soluble fragments, designated T-CPase and Chy-CPase, respectively, each of Mr = approximately 45,000. These fragments were generated and purified in milligram quantities by digestion of CPase covalently immobilized on a penicillin affinity column. They retained full enzymatic activity, became significantly more resistant to thermal inactivation, and lost micellar detergent binding upon proteolysis. Each was derived from CPase by loss of a COOH-terminal hydrophobic peptide. CPase was reconstituted into bacterial lipid vesicles in an enzymatically active form. Penicillin-binding sites were equally distributed on both sides of the lipid bilayer, suggesting a random orientation of the CPase molecules. Neither T-CPase nor Chy-CPase reconstituted into lipid vesicles when treated in an identical manner. CPase was slowly cleaved from the surface of these vesicles by either trypsin or alpha-chymotrypsin, yielding T-CPase and Chy-CPase, respectively. These results demonstrate that CPase is comprised of a water-soluble catalytic domain and a COOH-terminal hydrophobic region which mediates the anchoring of this enzyme to the bacterial membrane.
D002268:Carboxypeptidases / Q000378:metabolism; D002462:Cell Membrane / Q000201:enzymology; D002918:Chymotrypsin; D001411:Geobacillus stearothermophilus / Q000201:enzymology*; D008970:Molecular Weight; D002267:Muramoylpentapeptide Carboxypeptidase / Q000378:metabolism; D010406:Penicillins / Q000494:pharmacology*; D010446:Peptide Fragments / Q000032:analysis; D014357:Trypsin
D010406:Penicillins; D010446:Peptide Fragments; D002268:Carboxypeptidases; D002267:Muramoylpentapeptide Carboxypeptidase; D002918:Chymotrypsin; D014357:Trypsin