Channeling of Ammonia through the Intermolecular Tunnel Contained within Carbamoyl Phosphate Synthetase

Ammonia can replace glutamine as the ultimate source for the nitrogen requirement in the reaction presented above.3 A chemical mechanism for the enzymatic transformation is presented below in Scheme 1. The first ATP phosphorylates bicarbonate to form the unstable intermediate, carboxyphosphate. Phosphate is displaced from this intermediate by ammonia to produce carbamate. This second intermediate is subsequently phosphorylated by an additional ATP to liberate the final product, carbamoyl phosphate. Indirect experimental evidence has been obtained for the participation of the three reaction intermediates in the overall chemical mechanism.4 The CPS from E. coli is a heterodimer.5 The larger subunit contains the binding sites for each of the two phosphorylation events, whereas the smaller subunit has been shown to catalyze the hydrolysis of glutamine through the intermediacy of a thiol ester with Cys-269.6 The recent crystal structure of CPS from the laboratories of Holden and of Rayment has clearly shown that the three active sites contained within the heterodimer are separated in three-dimensional space by nearly 100 A.7 This observation has prompted speculation about the precise mechanism for the translocation of two reaction intermediates, ammonia and carbamate, from their respective sites of formation to their specific sites of utilization. Facilitated diffusion through the interior of the protein is one of a number of possible scenarios for this migration. This latter proposal has been bolstered by the identification of an intermolecular tunnel in CPS, which leads from the active site within the small subunit toward the two active sites found within the large subunit of this protein. A ribbon representation of CPS with the relative location of the three active sites and the molecular tunnel is presented in Figure 1. The physical and chemical mechanism for the incorporation of the amide nitrogen from glutamine into carbamoyl phosphate is unknown for the reaction catalyzed by CPS. There are, however, a number of plausible mechanisms that can be postulated for this transformation. For example, the ammonia derived from the hydrolysis of glutamine could migrate directly from the small subunit through the interior of the protein to the large subunit. It would then react with the carboxyphosphate intermediate formed at the active site within the N-terminal domain of the large subunit of CPS. Alternatively, the ammonia could dissociate into solution and then rebind to the large subunit where it could complete the reaction cycle. Instead of forming ammonia as a discrete and independent intermediate, the initial nucleophile for carbamate formation could be the amino group of the tetrahedral adduct formed from the attack of Cys-269 on the amide carbonyl group of glutamine.8 However, the substantial physical separation of the active site for glutamine hydrolysis and the site for carboxyphosphate formation would appear to preclude any mechanism which requires the close association of the two active sites for glutamine and carboxyphosphate. The most viable mechanism for the amidotransferase reaction of CPS would thus entail NH3 as a true intermediate. The primary objective of the present investigation is a determination of whether the NH3 diffuses * To whom correspondence may be sent. FAX: 409-845-9452. E-mail: raushel@tamu.edu. (1) Raushel, F. M.; Thoden, J. B.; Reinhart, G. D.; Holden, H. M. Curr. Opin. Chem. Biol. 1998, 2, 624. (2) (a) Stapleton, M. A.; Javid-Majd, F.; Harmon, M. F.; Hanks, B. A.; Grahmann, J. L.; Mullins, L. S.; Raushel, F. M. Biochemistry 1996, 35, 14352. (b) Javid-Majd, F.; Stapleton, M. A.; Harmon, M. F.; Hanks, B. A.; Mullins, L. S.; Raushel, F. M. Biochemistry 1996, 35, 14362. (c) Rubino, S. D.; Nyumoya, H.; Lusty, C. J. J. Biol. Chem. 1986, 261, 11320. (3) Trotta, P. P.; Estis, L. F.; Meister, A.; Haschemeyer, R. H. J. Biol. Chem. 1974, 249, 482. (4) (a) Raushel, F. M.; Villafranca, J. J. Biochemistry 1980, 19, 3170. (b) Wimmer, M. J.; Rose I. A.; Powers, S. G.; Meister, A. J. Biol. Chem. 1979, 254, 1854. (5) Matthews, S. L.; Anderson, P. M. Biochemistry 1972, 11, 1176. (6) Thoden, J. B.; Miran, S. G.; Phillips, J. C.; Howard, A. J.; Raushel, F. M.; Holden, H. M. Biochemistry 1998, 37, 8825. (7) Thoden, J. B.; Holden, H. M.; Wesenberg, G.; Raushel, F. M.; Rayment, I. Biochemistry 1997, 36, 6305. (8) Boehlein, S. K.; Richards, N. G. J.; Walworth, E. S.; Schuster, S. M. J. Biol. Chem. 1994, 269, 26789. Glutamine + HCO3 + 2 MgATP + H2O f Glutamate + 2 MgADP + Pi + carbamoyl-P (1)