Novel mechanism for carbamoyl-phosphate synthetase: A nucleotide switch for functionally equivalent domains

Abstract

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...

Mechanism of carbamoyl phosphate synthetase from Escherichia coli

“…for acetylglutamate to bring about all of the conformational Kothe et al [25] have recently put forward a mechanism in changes associated with activation. Thus, activation of the enzyme may have been incomplete before the addition of ATP.…”

“…have led to the proposal [25] of an ingenious, novel mechanism The amino acid sequence of the large subunit of the CPS from (Scheme 1) in which the ATP bound at the C-terminal half of Escherichia coli [10] (a heterodimeric enzyme in which the large the large subunit acts as a switch that triggers catalysis in the subunit catalyzes the reaction starting from ammonia and the N-terminal half, where the second ATP molecule phosphorylates small subunit cleaves glutamine [11]) or the equivalent region bicarbonate. The resulting carboxyphosphate is attacked by amin other CPS [12,13] exhibits internal homology between the monia, yielding a tetrahedral intermediate that collapses to car-N-terminal and C-terminal halves.…”

Aslfm, the D-Aspartate Ligase Responsible for the Addition of D-Aspartic Acid onto the Peptidoglycan Precursor of Enterococcus faecium

“…3A), indicating that the gene encoding the D-aspartate ligase (asl fm ) had been successfully identified. Asl fm belonged to the ATP-grasp protein superfamily composed of highly diverse enzymes that catalyze ATPdependent carboxylate-amine ligation reactions (22) and form acylphosphate intermediates (23,24).…”

3-Isobutylmethylxanthine Inhibits Hepatic Urea Synthesis

“…Precisely how AGM prevents the IBMX-induced inhibition of CPS-I is not obvious and deserves additional studies. One future study may involve examination of structure-function using crystallography (37)(38)(39), with the presence or absence of IBMX or IBMX ϩ AGM.…”

“…CPS-I has multiple active sites as well as "molecular tunnels" that facilitate the passage of reaction intermediates, thereby catalyzing the synthesis of carbamoyl phosphate by four independent chemical reactions (37)(38)(39). IBMX-mediated inhibition of CPS-I could reflect one of the following: (i) inhibition of a substrate binding to the enzyme; (ii) disruption of the binding of NAG to its activating site; (iii) the CPS-I reaction requires Mg 2ϩ , and IBMX may disrupt the Mg 2ϩ -ATP complex, and/or (iv) IBMX may obstruct one or more of the four active sites of CPS-I.…”