STUDIES ON THE UREA CYCLE ENZYME ORNITHINE TRANSCARBAMYLASE USING HEAVY-ATOM ISOTOPE EFFECTS

Ornithine transcarbamylase (OTCase) catalyzes the reaction between L-o rnithine and carbamyl phosphate in the first step of the urea cycle. C -13 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 C-13 kinetic isotope effect was 1.0095, at high levels of L-orni thine (86 mM) the C-13 kinetic isotope effect was unity, and 0.83 mM o rnithine was found to eliminate half the isotope effect. Th-se results are indicative of an ordered kinetic mechanism in which carbamyl phos phate 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 C-13 kinetic isotope effect was large, 1.076. Th is value is most likely the intrinsic kinetic isotope effect with this substrate, and the chemistry of the enzyme catalyzed reaction has bec ome rate limiting. (C) 1998 Elsevier Science B.V.