Primary structure of the COOH-terminal membranous segment of a penicillin-sensitive enzyme purified from two Bacilli.

Abstract

D-Alanine carboxypeptidase is a penicillin-sensitive intrinsic membrane enzyme which is composed of a hydrophilic NH2-terminal catalytic domain (Mr congruent to 45,000 to 47,000) and a COOH-terminal membranous segment (approximately 20 to 30 amino acids in length) (Waxman, D. J., and Strominger, J. L. (1979) J. Biol. Chem. 254, 4863-4875; Waxman, D. J., and Strominger, J. L. (1981) J. Biol. Chem. 256, 2059-2066). The primary structures of the COOH-terminal 30 amino acids of two D-alanine carboxypeptidase purified from bacterial membranes were determined (residues numbered from the COOH terminus): Bacillus stearothermophilus: (formula see text) Water-soluble fragments of the B. stearothermophilus D-alanine carboxypeptidase were shown to be formed by cleavage after Phe27 or after Leu25 as indicated by carboxypeptidase A and B analysis and by the release of the four COOH-terminal chymotryptic peptides (Val26-Leu25, Ser24-Phe16, Val15-Trp12, and Thr11-Leu1) upon formation of water-soluble chymotrypsin D-alanine carboxypeptidase. This indicates that the membranous fragment is largely contained within the COOH-terminal 24 residues. Thus, this bacterial membrane protein probably does not contain the significant cytoplasmic domain characteristic of transmembrane proteins such as glycophorin. The absence of an uninterrupted stretch of 20 to 25 uncharged residues suggests that the membrane anchoring of D-alanine carboxypeptidase may differ from that of simple transmembrane proteins. Possible structures for the membranous segment of D-alanine carboxypeptidase are discussed.