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Volume 271, Number 46, Issue of November 15, 1996 pp. 28953-28959
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.

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The Two Acetyl-coenzyme A Synthetases of Saccharomyces cerevisiae Differ with Respect to Kinetic Properties and Transcriptional Regulation

(Received for publication, May 1, 1996, and in revised form, August 22, 1996)

Marco A. van den Berg ^§ , Patricia de Jong-Gubbels , Christine J. Kortland ^§ , Johannes P. van Dijken , Jack T. Pronk and H. Yde Steensma ^§

From the  Kluyver Laboratory of Biotechnology, Delft University of Technology, Julianalaan 67, 2628 BC Delft and the ^§ Institute of Molecular Plant Sciences, Leiden University, Wassenaarseweg 64, 2333 AL Leiden, The Netherlands

Saccharomyces cerevisiae contains two structural genes, ACS1 and ACS2, each encoding an active acetyl-coenzyme A synthetase. Characterization of enzyme activities in cell-free extracts from strains expressing either of the two genes revealed differences in the catalytic properties of the two enzymes. The K_m for acetate of Acs1p was about 30-fold lower than that of Acs2p and Acs1p, but not Acs2p, could use propionate as a substrate. Enzyme activity measurements and mRNA analyses showed that ACS1 and ACS2 were both expressed during carbon-limited growth on glucose, ethanol, and acetate in aerobic chemostat cultures. In anaerobic glucose-limited cultures, only the ACS2 gene was expressed. Based on these facts, the products of the ACS1 and ACS2 genes were identified as the previously described "aerobic" and "non-aerobic" forms of acetyl-coenzyme A synthetase, respectively. Batch and glucose-pulse experiments revealed that transcription of ACS1 is subject to glucose repression. A mutant strain lacking Acs2p was unable to grow on glucose in batch cultures, but grew readily in aerobic glucose-limited chemostat cultures, in which the low residual glucose concentration alleviated glucose repression. Experiments in which ethanol was pulsed to aerobic ethanol-limited chemostat cultures indicated that, in addition to glucose, ethanol also repressed ACS1 transcription, although to a lesser extent. In contrast, transcription of ACS2 was slightly induced by ethanol and glucose. Absence of ACS2 prevented complete glucose repression of ACS1, indicating that ACS2 (in)directly is involved in the transcriptional regulation of ACS1.

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