Related Experiment Videos
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.
Insights
D-Alanine carboxypeptidase, a penicillin-sensitive enzyme, has a membrane-anchoring segment within its COOH-terminal 24 residues. This structure differs from typical transmembrane proteins, suggesting a unique membrane integration mechanism.
Area of Science:
- Biochemistry
- Molecular Biology
- Membrane Protein Structure
Background:
- D-Alanine carboxypeptidase is an intrinsic membrane enzyme sensitive to penicillin.
- It comprises a hydrophilic catalytic domain and a COOH-terminal membrane-bound segment.
Purpose of the Study:
- To determine the primary structure of the COOH-terminal amino acids of D-alanine carboxypeptidase.
- To elucidate the membrane-anchoring region and its integration mechanism.
Main Methods:
- Purification of D-alanine carboxypeptidase from bacterial membranes.
- Determination of the primary structure of COOH-terminal residues.
- Analysis of water-soluble fragments generated by enzymatic cleavage (carboxypeptidase A, B, and chymotrypsin).
Main Results:
- The membranous segment is localized within the COOH-terminal 24 amino acids.
- Cleavage occurred after Phe27 or Leu25, releasing specific chymotryptic peptides.
- The enzyme lacks a significant cytoplasmic domain typical of transmembrane proteins.
- Absence of a long uncharged residue stretch suggests an atypical membrane anchoring.
Conclusions:
- The membrane-anchoring segment of D-alanine carboxypeptidase is confined to its COOH-terminal 24 residues.
- Its membrane integration likely differs from that of simple transmembrane proteins.
- The study discusses potential structural models for the enzyme's membrane association.