Mitochondrial aspartate aminotransferase independent function of the catalytic binding sites

Yan-Hwa Wu Lee, J. E. Churchich

研究成果: Article同行評審

16 引文 斯高帕斯(Scopus)

摘要

The enzyme mitochondrial aspartate aminotransferase from beef liver is a dimer of identical subunits. The enzymatic activity of the resolved enzyme is restored upon addition of the cofactor pyridoxal 5 phosphate. The binding of 1 molecule of cofactor restores 50% of the original enzymatic activity, whereas the binding of a 2nd molecule of cofactor brings about more than 95% recovery of the catalytic activity. Following addition of 1 mol of pyridoxal 5 P per dimer, 3 forms of the enzyme may exist in solution: apoenzyme 2 pyridoxal 5' phosphate, apoenzyme 1 pyridoxal 5' phosphate, and apoenzyme. The enzyme species are separated by affinity chromatography and the following distribution was found: apoenzyme 2 pyridoxal 5' phosphate/apoenzyme 1 pyridoxal 5' phosphate/apoenzyme, 2/6/2. Similar distribution was observed after reduction with NaBH4 of the mixture containing apoenzyme and pyridoxal 5 P at a mixing ratio of 1:1. Fluorometric titrations conducted on samples of apoenzyme and apoenzyme 1 pyridoxal 5' phosphate reveal that the enzyme species display identical affinity towards the inhibitor 4 pyridoxic 5 P (K(D) = 1.1 x 10-6M). It is concluded that the binding of the cofactor to one of the catalytic sites does not affect the affinity of the second site for the inhibitor. These results, obtained by 2 independent methods, lend strong support to the hypothesis that the 2 subunits of the enzyme function independently.

原文English
頁(從 - 到)5604-5608
頁數5
期刊Journal of Biological Chemistry
250
發行號14
出版狀態Published - 25 七月 1975

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