Physical Studies Comparing a Genetically Fused Enzyme of the Histidine Operon with Its Component Enzymes
Year: 1973
Type: article
Abstract: Physical studies have been performed on a genetically fused enzyme and its two component wild type enzymes, l-histidinol dehydrogenase and imidazolylacetol phosphate:l-glutamate aminotransferase. The circular dichroic activity of the peptide chromophore of the fused enzyme is approximately accounted for by a weighted average of the activities of the two wild type enzymes. All three proteins lose circular dichroic activity in dilute alkali. The fluorescence behavior of the wild type dehydrogenase is significantly modified in the fused enzyme. Tyrosyl fluorescence is quenched and tryptophanyl fluorescence is strongly red-shifted in the fused enzyme compared to their behavior in the wild type dehydrogenase. The absorption and emission properties of the cofactor (pyridoxal 5'-phosphate) are quite different in the two enzymes. At neutral pH, the wild type aminotransferase two exhibits absorption peaks at 330 and 430 nm whereas only the 330-nm band is evident with the fused enzyme. Titration of either enzyme between pH 10 and 12 results in the disappearance of the 330-nm peak with the parallel formation of a band centered at 385 nm. The 430-nm band of the aminotransferase is replaced by a band located at 355 nm when the pH is increased between 7 and 11. The wild type aminotransferase and the fused enzyme fluoresce with a peak near 400 nm when excited at 330 nm. Three pH-dependent fluorescence transitions are observed in the cofactor of the aminotransferase whereas only two are apparent in the fused enzyme. These occur in the former enzyme, between pH 4 and 6, pH 7.5 and 9.5, and pH 10 and 12.5. The most acidic transition in both enzymes probably represents the ionization of the pyridinium group of the Schiff base form of the pyridoxal 5'-phosphate cofactor. The most alkaline transition appears to result from the release of pyridoxal 5'-phosphate from the two enzymes and the ionization of its phenolic group.
Source: Journal of Biological Chemistry
Institution: National Institutes of Health
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: hybrid
Estimated APC paid: $3,430
OpenAlex ID: W1490690829