Phenylalanine ammonia-lyase (EC 4.3.1.5) was purified 40-fold from a cell homogenate of Streptomyces verticillatus. In many respects the enzyme was similar to phenylalanine ammonia-lyases isolated from plants and fungi. It was most active at pH 9.0 and the Km for L-phenylalanine was 1.6 × 10−4 M. It showed no requirement for metal ions but was inhibited by heavy metals, some sulfhydryl reagents, and carbonyl reagents. The Stokes' radius was estimated by gel filtration to be 5.45 nm. Sucrose gradient centrifugation gave an s20,w of 10.0, leading to calculated values of 226 000 and 1.61 for the molecular weight and frictional ratio, respectively, if a partial specific volume of 0.725 ml/g is assumed. The enzyme deaminated o-, m-, and p-fluoro-, p-chloro-, and p-methyl-phenylalanine but was without action on L-tyrosine. It was inhibited by trans-cinnamic acid and certain phenylalanine derivatives, as well as by some less closely related aromatic compounds, but not by trans-cinnamamide.

Radioactivity from L-phenylalanine-carboxyl-14C was incorporated specifically into the carboxyl group of cinnamamide by cultures of Streptomyces verticillatus. L-Phenylalanine ammonia-lyase (EC. 4.3.1.5) could be extracted from the mycelium. Cultures grown in defined medium produced little cinnamamide unless supplemented with L-phenylalanine; phenylalanine ammonia-lyase activity in the mycelium was directly related to the yield of cinnamamide.

1. Soluble and mitochondrial forms of histidine-pyruvate aminotransferase were separated from rat liver preparations by chromatography on DEAE-cellulose. 2. These enzymes were characterized with respect to substrate specificity, substrate affinity, pH optimum, stability and molecular weight by chromatography on Sephadex G-200. 3. Each enzyme has a relatively broad specificity showing significant activity towards l-phenylalanine and l-tyrosine and catalysing transamination with a number of monocarboxylic 2-oxo acids. 2-Oxoglutarate is not a substrate for either enzyme. 4. The molecular weights of the two enzymes, by chromatography on Sephadex G-200, are in the range 130000-150000. 5. The formation in vitro of imidazolyl-lactate from imidazolylpyruvate and NADH was demonstrated by using liver preparations. 6. From a study of imidazolyl-lactate-NAD(+) oxidoreductase activity after electrophoresis of liver preparations on polyacrylamide gel, and from an examination of the activity of l-lactate-NAD(+) oxidoreductase (EC 1.1.1.27) towards imidazolylpyruvate, it is concluded that this latter enzyme is responsible for the formation of imidazolyl-lactate in the liver. 7. Preparations of bacteria obtained from rat faeces form imidazolylpropionate from l-histidine and urocanate without further subculture. The amount of imidazolylpropionate formed is increased under anaerobic conditions and more so in an atmosphere of H(2). It is suggested that the gut flora of the rat contribute largely, if not exclusively, to the formation of imidazolylpropionate normally found in the urine.

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When chloramphenicol-producing cultures of Streptomyces species 3022a were administered [G-14C] chorismic, [G-14C] prephenic, and [G-14C, 6-3H] shikimic acids, radiochemical yields in the antibiotic were very low (0.01–0.1%). [G-14C] chorismic and prephenic acids labeled chloramphenicol to about the same specific activity as tyrosine, aspartic acid, and glutamic acid from mycelial protein, whereas protein phenylalanine was 100 times more active. We conclude that these two substrates are unable to penetrate the cell membrane and the radioactivity incorporated enters as [G-14C] phenylpyruvic acid. Labeled shikimic acid, on the other hand, was directly incorporated into the cells. The 14C: 3H ratio in chloramphenicol isolated after feeding the [G-14C, 6R-3H] and [G-14C, 6S-3H] labeled forms indicated that the pro-6R-hydrogen was eliminated during ring aromatization. Antibiotic labeled from [G-14C, 6S-3H] shikimic acid was chemically degraded to 2,4,6-tribromoaniline without change in the 14C: 3H ratio thus establishing the specific incorporation of shikimic acid into the phenyl ring, and locating the tritium at positions ortho to the propanoid substituent.