The cell-free formation of tryptophan synthetase in Escherichia coli. Studies with crude extracts

Abstract

The cell-free formation of tryptophan synthetase (l-serine hydro-lyase (adding indole), EC 4.2.1.20) in Escherichia coli was studied in an effort to investigate the genetic control of specific enzyme formation at the subcellular level. Activities of A and B component proteins of this enzyme and that of indole-3-glycerol phosphate synthetase (indole-3-glycerol phosphate d-glyceraldehyde-3-phosphate lyase, EC 4.1.2.8) increased when crude extracts prepared from partially derepressed wild-type cells were incubated with the supplements. A detailed characterization of the activity increase with respect to the B protein of tryptophan synthetase showed that the increase in enzyme activity depended on the presence of ATP and its generating system and was stimulated by a mixture of other ribonucleoside triphosphates and by that of l-amino acids. The system was partially sensitive to inhibition by DNAase (deoxyribonucleate oligonucleotidohydrolase, EC 3.1.4.5), RNAase (polyribonucleotide 2-oligonucleotidotransferase (cyclizing), EC 2.7.7.16), chloramphenicol, puromycin and notably by l-tryptophan. In a parallel experiment, amino acid incorporation into protein in the present system was completely inhibited by RNAase. The newly developed enzyme activity was found entirely in the supernatant fraction, and the increase in enzyme activity seems to be accompanied by the corresponding increase in protein immunologically reactive with anti-enzyme serum. Crude extracts of various mutant strains have also been examined for their capacity to form the enzymes.