An invariant threonine is involved in self-catalyzed cleavage of the precursor protein for ornithine acetyltransferase

In Bacillus stearothermophilus ornithine acetyltransferase is a bifunctiona l enzyme, catalyzing the first and the fifth steps of arginine biosynthesis ; it follows a ping-pong kinetic mechanism. A single chain precursor protei n is cleaved between the alanine and threonine residues in a highly conserv ed ATML sequence leading to the formation of alpha and beta subunits that a ssemble into a heterotetrameric 2 alpha2 beta molecule. The beta subunit ha s been shown to form an acetylated intermediate in the course of the transa cetylation reaction. The present data show that the precursor protein synth esized in vitro or in vivo undergoes a self-catalyzed cleavage involving an invariant threonine (Thr-197). Using site-directed mutagenesis T197G, T197 S, and T197C derivatives have been generated. The T197G substitution abolis hes both precursor protein cleavage and catalytic activity, whereas T197S a nd T197C substitutions reduce precursor cleavage and catalytic activity in the order Thr-197 (wild type) --> Ser-197 --> Cys-197. A mechanism is propo sed in which Thr-197 plays a crucial role in the autoproteolytic cleavage o f ornithine acetyltransferase.