Purification and Characterization of the Thermophilic d-Alanine Carboxypeptidase from Membranes of Bacillus stearothermophilus
R. Rogers Yocum 1, Peter M. Blumberg 1, and Jack L. Strominger 1
From the
1 From the Biological Laboratories, Harvard University, Cambridge, Massachusetts 02138
The d-alanine carboxypeptidase, the major penicillinbinding component in membranes of Bacillus stearothermophilus, was purified by covalent affinity chromatography. The enzyme was solubilized in the nonionic detergent Triton X-100, bound to a column of a 6-aminopenicillanic acid-Sepharose derivative, and eluted upon treatment of the column with neutral hydroxylamine. The enzyme was thermophilic, possessing a temperature optimum of 55°. Its molecular weight, 46,500, resembled that of the d-alanine carboxypeptidase from Bacillus subtilis. In addition to its carboxypeptidase activity, the enzyme was also capable of carrying out a simple transpeptidation reaction. This reaction, which was penicillin sensitive, involved UDP-N-acetylmuramyl -l - alanyl - d - glutamyl - mesodiaminopimelyl - d - alanyl-d-alanine as transpeptidation donor and either glycine or d-alanine as transpeptidation acceptor. The enzyme, however, did not appear to be the lethal target for ß-lactam antibiotics in this organism since the enzyme was 27,000-fold more resistant to cephalothin than was the organism.
Submitted on January 7, 1974
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