I Four electrophoretic variants of chloramphenicol acetyltransferase (E.C.2.3.1.99) have been purified to homogeneity from cell-free extracts of staphylococci by affinity chromatography. All four enzymes show similar Km values for the substrates, acetyl Coenzyme A and D, threo chloramphenicol. Amino acid analyses and tryptic peptide maps of the four enzymes are similar. II the N-terminal sequences of seven chloramphenicol acetyltransferase variants from both Gram positive and Gram negative bacteria have been determined by the method of Edman degradation of proteins covalently attached to solid phase supports. More than 90 percent of the primary sequence of one of the staphylococcal variants has been determined. III Kinetic studies and the results of chemical modification experiments have implicated the importance of a histidine residue in the mechanism of catalysis, and a unique histidine residue in the native enzyme has been found to react with iodoacetamide with consequent formation of 3-amidocarboxymethylhistidine. There is no evidence of a covalent acyl-enzyme intermediate in the catalytic process. IV The use of secondary structure prediction methods has allowed the comparison of both primary and predicted secondary structures of the N-termini of ten chloramphenicol acetyltransferase variants. The results of this study are consistent with the view that the chloramphenicol acetyltransferase enzymes have evolved from a common ancestral protein and, although their primary sequences differ considerably in some cases, their secondary structures and catalytic mechanism are likely to be similar.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:455557 |
| Date | January 1977 |
| Creators | Fitton, John Edward |
| Publisher | University of Leicester |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://hdl.handle.net/2381/35222 |
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