Studies on the urea cycle enzyme ornithine transcarbamylase using heavy atom isotope effects.

Ornithine transcarbamylase (OTCase) catalyzes the reaction between L-ornithine and carbamyl phosphate in the first step of the urea cycle. 13C isotope effects were measured in carbamyl phosphate, using OTCase obtained from E. coli in a one-column purification which yielded 30 mg of very pure enzyme from 51 of cell culture. At near zero L-ornithine, the 13C kinetic isotope effect was 1.0095, at high levels of L-ornithine (86 mM) the 13C kinetic isotope effect was unity, and 0.83 mM ornithine was found to eliminate half the isotope effect. These results are indicative of an ordered kinetic mechanism in which carbamyl phosphate binds to the enzyme before L-ornithine. Similar experiments were performed using the slow substrate L-lysine in place of L-ornithine. At 90 mM L-lysine the 13C kinetic isotope effect was large, 1.076. This value is most likely the intrinsic kinetic isotope effect with this substrate, and the chemistry of the enzyme catalyzed reaction has become rate limiting.