ABSTRACT
A spontaneous high-copy-number plasmid derivative of plasmid pBR322 was isolated. This plasmid bears two point mutations adjacent to the unpaired region in the stem of loop II of RNA I and expresses its high-copy-number phenotype only when the harboring cells grow on solid support (L-agar).
Subject(s)
DNA, Bacterial/analysis , Plasmids , Replicon , Base Sequence , Mutation , Nucleic Acid Conformation , Phenotype , RNA, BacterialABSTRACT
Transpeptidation reactions catalyzed by chymotrypsin, pepsin, leucine aminopeptidase and thermolysin have been studied in heavy oxygen water (H2 18O). The 18O incorporation into the peptide bond of transpeptidation products and into the non-hydrolyzed substrate has been measured. The rates of 18O exchange in the carboxylic groups of N-acetylphenylalanine and leucine, catalyzed by pepsin and leucine aminopeptidase, respectively, have also been determined. These rates have been compared with that of the exchange in the presence of amino compounds which reversibly form amide bonds with the above carboxyl-containing substances. The data obtained show that, in contrast to chymotrypsin, other enzymes studied do not form 'acyl-enzymes' but function by the mechanism of general-base catalysis. In other words, their catalytically active groups promote the abstraction of a proton from the water molecule, which attacks the susceptible bond of the substrate. The structure of intermediate compounds in this type of catalysis and the mechanism of the transpeptidation reaction are discussed.
