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Properties of a fused protein formed by genetic manipulation. Histidinol dehydrogenase-imidazolylacetol phosphate: L-glutamate aminotransferase.

M. Rechler,C. Bruni

Published 1971 in Journal of Biological Chemistry

ABSTRACT

Abstract The enzymes specified by the second and third structural genes of the histidine operon of Salmonella typhimurium, histidinol dehydrogenase and imidazolylacetol phosphate:l-glutamate aminotransferase, respectively, have been covalently linked by genetic manipulation. The molecular weight of the fused enzyme subunit is the sum of the weights of the wild type aminotransferase subunit and of the wild type dehydrogenase subunit as determined by sodium dodecyl sulfate-acrylamide gel electrophoresis. All three proteins are isolated primarily as dimers, with molecular weights of 59,000 (aminotransferase), 84,000 (dehydrogenase), and 140,000 (fused enzyme). With respect to its aminotransferase activity, the fused enzyme does not differ significantly from the wild type enzyme in stability to heat, Vmax, Km for substrates histidinol phosphate and α-ketoglutarate, and absorption spectrum. With respect to its dehydrogenase activity, the fused enzyme is more labile to heat than is the wild type enzyme, and has a 7-fold higher Km for l-histidinol. Its Km for NAD is not altered. The remarkable functional integrity of both dehydrogenase and aminotransferase catalytic activities in the fused enzyme and the retention of dimeric structure suggest that the information for normal folding of the two single proteins is preserved when the two polypeptide chains are covalently fused.

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