Carbamoyl phosphate synthethase I synthesizes carbamoyl phosphate from ammonia, HCO−3 and two molecules of ATP, one of which, ATPA, yields Pi while the other, ATPB, yields the phosphoryl group of carbamoyl phosphate. Pulse‐chase experiments with [γ‐32P]ATP without added HCO−3 demonstrate separate binding sites for ATPA and ATPB. Bound ATPA dissociates readily from its site (t1/2∼ 1–2 s) and the Kd is 0.2–0.7 mM. For the ATPB binding site the t1/2 for dissociation is 5–12 s and the Kd∼ 10 μM. Kd for ATPA seems to increase with enzyme concentration whereas Kd for ATPB does not change. HClO4 releases the ATP unchanged from the enzyme · ATPB and enzyme · ATPB· ATPA complexes. In the presence of HCO−3, ATP and N‐acetylglutamate, an enzyme · ATPB· HCO−3· ATPA complex is formed. Its formation by the addition of HCO−3 to the enzyme · ATPB· ATPA complex appears to involve an initial bimolecular addition reaction followed by an isomerization. Treatment with HClO4 releases Pi from ATPA but ATPH is released unchanged. Spontaneous hydrolysis of ATPA is responsible for the ATPase activity of the enzyme. Thus, a covalent bond may form between HCO−3 and ATPA. However, ATPA can dissociate rapidly (t1/2≪ 10 s). The Kd for ATPA is approximately 0.2 mM. ATPB appears unable to dissociate from the enzyme. ATPB· HCO−3· ATPA complex since the t1/2 for dissociation of ATPB from the enzyme is lengthened about five times in the presence of 19 mM HCO−3 and at 1 mM ATP. ATPA may also hydrolyse in this complex and be replaced by another molecule of ATP in the absence of exchange of ATPB. However, the ATPA binding site must be occupied to prevent ATPB release. ATPB may be bound in a pocket which becomes inaccessible to the solution when HCO−3 and ATPA also bind. In contrast, HCO−3 does not inhibit the binding of ATPB to the enzyme. Various intermediate steps in the formation of the enzyme · ATPB· HCO−3· ATPA complex are discussed. Additional evidence is presented that the ATPB binding site is only periodically accessible to ATP in solution and that ATPB in the steady‐state reaction binds when the products leave. Since ⋝ 1.3 mol ATPB and ⋝ 1.8 mol ATPA bind/mol enzyme dimer, the enzyme monomer may be an active species. Copyright © 1979, Wiley Blackwell. All rights reserved
