Purification and characterization of ornithine acetyltransferase from Saccharomyces cerevisiae.
Ornithine acetyltransferase has been purified 4000-fold to homogeneity from Saccharomyces cerevisiae. The enzyme catalyses the freely reversible interchange of an acetyl group between N-acetylornithine and glutamate and has a specific activity of 22 mumol.min-1.mg-1 at 37 degrees C and pH 7.5. The Km values were determined for the substrates of the forward and reverse directions to be 1.0 mM for N-acetylornithine, 7.2 mM for glutamate, 1.5 mM for ornithine and 17.1 mM for N-acetylglutamate. The enzyme was localised to the mitochondrial matrix and was found to be a 57-kDa heterodimer consisting of subunits of 31 kDa and 26 kDa. Antibodies raised against the small subunit immunoprecipitated a single in vitro translation product of approximately 57 kDa suggesting that the subunits are processed from a single precursor protein. This is supported by N-terminal sequence analysis which shows that the 26-kDa subunit exhibits 40% sequence identity (8 out of 20) with the N-terminus of ornithine acetyltransferase from Neisseria gonorrhoeae whereas the N-terminus of the 31-kDa subunit exhibits 45% identity (9 out of 20) with a sequence located in the middle of the 60-kDa N. gonorrhoeae enzyme. The N-terminal sequence of the small subunit has the potential to form an amphiphilic helix, further suggesting that the precursor protein with the small subunit at its N-terminus could be targeted to mitochondria and processed into two subunits.