D-alanine carboxypeptidase activity of Micrococcus lysodeikticus released into the protoplasting medium.
Conversion of whole cells of Micrococcus lysodeikticus to protoplasts allowed the release of a soluble form of a D-alanine carboxypeptidase into the protoplasting medium. The enzyme cleaves the terminal D-alanine from the radioactively labelled UDP-N-acetylmuramyl-pentapeptide containing L-lysine as the diamino acid. However, the enzyme is only minimally active in this fraction so that it had to be enriched and partially purified before its properties could be studied. Chromatography on carboxymethyl-Sephadex removed the lysozyme used in the protoplasting of the cells. The material which was unadsorbed to the column was applied to an affinity chromatography column of Ampicillin-Sepharose. Most of the contaminating protein was washed from the column while the D-alanine carboxypeptidase adhered to the resin and could be eluted with 0.5 M Tris-HCl buffer pH 8.6. Some of the properties of the enzymic activity were studied using this preparation. The enzyme was activated by Mg2+ ions with a broad optimum from 15--35 mM. It was maximally active when NaCl at a concentrations of 0.06--0.08 M was added to the assay, and the pH curve was biphasic with an alkaline optimum. The Km for substrate was found to be 0.118 mM. Enzymic activity was completely inhibited by low concentrations of Ampicillin and penicillin G.
D000409:Alanine; D000667:Ampicillin / Q000494:pharmacology; D002268:Carboxypeptidases / Q000302:isolation & purification / Q000378:metabolism*; D002846:Chromatography, Affinity; D004789:Enzyme Activation / Q000187:drug effects; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D008837:Micrococcus / Q000201:enzymology*; D009112:Muramic Acids; D009842:Oligopeptides; D009994:Osmolar Concentration; D010400:Penicillin G / Q000494:pharmacology; D011523:Protoplasts / Q000201:enzymology*; D012965:Sodium Chloride / Q000494:pharmacology
D009112:Muramic Acids; D009842:Oligopeptides; D012965:Sodium Chloride; D000667:Ampicillin; D002268:Carboxypeptidases; D008274:Magnesium; D000409:Alanine; D010400:Penicillin G