We have carried out a site-directed mutagenic analysis that is consistent with ATP binding to a palmate motif rather than to a Walker A͞B motif in domains B and C. To accommodate our present findings, as well as the other recent findings of structural and functional equivalence, we are proposing a novel mechanism for CPS. In this mechanism utilization of ATP bound to domain C is coupled to carbamoylphosphate synthesis at domain B via a nucleotide switch, with the energy of ATP hydrolysis at domain C allowing domain B to cycle between two alternative conformations.Carbamoyl-phosphate (CP) is a high-energy biological compound that plays a key role in the introduction of both ammonia and single carbon units into the metabolic pool. CP may serve either as the precursor for synthesis of pyrimidine nucleotides or, in an alternative pathway, as the precursor for arginine. In addition to serving as a required component of almost all proteins, arginine is used in the liver for ammonia detoxification via the urea cycle and, under appropriate physiological conditions, it is also used in a variety of tissues for nitric oxide formation. Depending on the physiological role, CP synthetases (CPSs) vary in mode of regulation and subunit composition (1). However, all of the CPSs thus far sequenced display marked primary sequence identity (2, 3), and all catalyze the formation of CP, P i , and two ADP from HCO 3 Ϫ , NH 3 , and two ATP. Sequence analysis demonstrated regions of internal duplication within CPS and suggested, on the basis of similarities to the ATP-binding sequence motifs of Walker A͞B proteins (i.e., proteins involving a ␤␣␤ nucleotide binding fold; ref. 4), that each region of internal duplication contains one ATP site (5, 6). Subsequent studies (7-10) demonstrated that the synthetase duplications correspond to independently folded domains (B and C) and that binding of one ATP molecule is localized to each of the two synthetase domains (6,(11)(12)(13). A great deal of experimental evidence (14-23) has established that the role of one ATP is to form the enzyme-bound intermediate carboxy-phosphate (CxP) and that domain B binds this ATP (ATP B ) whereas domain C binds the other ATP molecule (ATP C ).When we carried out the presently described studies, further definition of the utilization of ATP B and ATP C by CPS could not be based on a solved three-dimensional structure because there was none available for any CPS at that time. However, we found that domains B and C of CPS could each be fit to the structural coordinates for the biotin carboxylase (BN) component of Escherichia coli acetyl Co-A carboxylase (24). This BN homology modeling seemed appropriate for the following reasons. First, there is significant sequence identity shared by portions of BN (as well as other biotin-dependent enzymes) and each of the CPS domains B and C (2). Also, both CPS and BN couple cleavage of ATP to ADP with the activation of HCO 3 Ϫ to yield CxP, and both catalyze reaction of the activated carboxyl group with an amino group (NH 3 for...