Regulation of hamster carbamoyl-phosphate synthase II by 5-phospho-α-d-ribosyl 1-diphosphate and uridine 5′-triphosphate

In mammals, carbamoyl phosphate for utilization in pyrimidine biosynthesis is synthesized by a glutamine-dependent carbamoyl-phosphate synthase II which is subject to regulation by 5-phospho-alpha-D-ribosyl 1-diphosphate (PRib-PP), a positive effector, and MgUTP, a negative effector [Mori, M., Ishida, H. and Tatibana, M. (1975) Biochemistry 14, 2622-2630]. We have found that Lineweaver-Burk plots of carbamoyl phosphate synthase activity versus 1/[MgATP] are described by a velocity equation which is a ratio of quadratic polynomials, consistent with a positive homotropic interaction between two catalytic sites for the binding of MgATP (Ks = 16.6 +/- 3.1 mM, interaction factor a = 0.00538 +/- 0.00245). The activating effect of PRib-PP upon carbamoyl-phosphate synthase is consistent with PRib-PP binding at an allosteric site (Ka = 31.4 +/- 6.4 microM) and promoting the binding of a first molecule of MgATP as substrate (interaction factor l = 0.0437 +/- 0.0063). Thus MgATP and PRib-PP bind to the E X MgATP complex with respective dissociation constants of a X Ks = 0.089 mM and l X Ka = 1.4 microM while MgATP binds to the E X PRib-PP complex with a dissociation constant of l X Ks = 0.73 mM. Data for the inhibitory effect of MgUTP upon carbamoyl-phosphate synthase indicate that MgUTP competes with MgATP for binding at the catalytic site (Ki = 0.203 +/- 0.016 mM). A computer model has recently been developed which enables quantitative stimulation of the time-dependent effects of blockade of the pyrimidine pathway by a tight-binding enzyme inhibitor [Duggleby, R.G. and Christopherson, R.I. (1984) Eur. J. Biochem. 143, 221-226]. The velocity equation derived in the present paper provides a quantitative basis for predicting changes in the flux through the de novo pyrimidine pathway in growing cells.