PURIFICATION AND CHARACTERIZATION OF ORNITHINE ACETYLTRANSFERASE FROMSACCHAROMYCES-CEREVISIAE

Ornithine acetyltransferase has been purified 4000-fold to homogeneity from Saccharomyces cerevisiae. The enzyme, catalyses the freely rever sible interchange of an acetyl group between N-acetylornithine and glu tamate and has a specific activity of 22 mu mol . min(-1) . mg(-1) at 37 degrees C and pH 7.5. The K-m values were determined for the substr ates of the forward and reverse directions to be 1.0 mM for N-acetylor nithine, 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 immunoprec ipitated a single in vitro translation product of approximately 57 kDa suggesting chat the subunits are processed from a single precursor pr otein. This is supported by N-terminal sequence analysis which shows t hat the 26-kDa subunit exhibits 40% sequence identity (8 out of 20) wi th the N-terminus of ornithine acetyltransferase from Neisseria gonorr hoeae whereas the N-terminus of the 31-kDa subunit exhibits 45% identi ty (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 t he precursor protein with the small subunit at its N-terminus could be targeted to mitochondria and processed into two subunits.