Regulatory Control of the Amidotransferase Domain of Carbamoyl Phosphate Synthetase

337

331

Substrate channeling is the process of direct transfer of an intermediate between the active sites of two enzymes that catalyze sequential reactions in a biosynthetic pathway (for reviews see Refs. 1 and 2). The active sites can be located either on separate domains in a multifunctional enzyme or on separate subunits in a multienzyme complex.Substrate channeling has been proposed to decrease transit time of intermediates, prevent loss of intermediates by diffusion, protect labile intermediates from solvent, and forestall entrance of intermediates into competing metabolic pathways (2). Loss of an intermediate by diffusion may be especially important in the case of a neutral species, such as indole, which could escape from the cell by passive diffusion across cell membranes (2-5). Nevertheless, there has been considerable debate over whether channeling actually occurs and whether it is advantageous (5, 6).X-ray crystallographic studies on several enzyme complexes have revealed two molecular mechanisms for channeling. The discovery of an intramolecular tunnel in tryptophan synthase (7) provided the first molecular mechanism for channeling. For a long time this was a unique example, but recent structure determinations have revealed plausible evidence for tunneling of ammonia and carbamate in carbamoyl-phosphate synthase (CPS) 1 (8) and of ammonia in phosphoribosylpyrophosphate amidotransferase (GPATase) (9).The structure of dihydrofolate reductase-thymidylate synthase, however, showed no evidence for a tunnel between the two active sites (10). Neither are the two active sites adjacent to one another. Instead, there are positively charged residues along the surface between the active sites that form an electrostatic highway sufficient to channel the negatively charged dihydrofolate with high efficiency (10, 11).The focus of this minireview will be on the structural basis of channeling in the four enzyme structures cited above and on solution studies of these systems.2 Scheme I summarizes the reactions catalyzed by the four enzymes. A comparison of the structural and kinetic results reveals that these enzymes frequently exhibit allosteric interactions that synchronize the reactions to prevent the build-up of excess intermediate (12)(13)(14)(15)(16). TunnelingTryptophan Synthase-Tryptophan synthase catalyzes the last two reactions in the biosynthesis of L-tryptophan (Scheme I, A) (for reviews see . In bacteria, the two reactions are catalyzed by separate ␣ and ␤ subunits, which combine to form a stable multienzyme complex, (␣␤) 2 . In yeast and molds, the two reactions are catalyzed by separate ␣ and ␤ sites in a single bifunctional polypeptide chain, (␣-␤) 2 .3 Evidence that indole is not liberated as a free intermediate in the overall conversion of indole-3-glycerol phosphate and L-serine to L-tryptophan suggested that indole passes directly from the ␣ site to the ␤ site without release to the surrounding solvent (20 -23). The 2.8-Å resolution crystal structure of tryptophan synthase from Salmonella typhimurium showe...

Section: From the ‡Enzyme Structure And Function Section Laboratory mentioning

confidence: 99%

The Molecular Basis of Substrate Channeling

Miles¹,

Rhee²,

Davies³

1999

337

331

“…For example, binding of ATP and bicarbonate to the large subunit of E. coli carbamoyl phosphate synthetase large subunit stimulates the k cat for glutamine hydrolysis at a site about 45 Å removed in the small enzyme subunit (11). Likewise, binding of chorismate to the anthranilate synthase component I subunit stimulates glutamine hydrolysis by the component II subunit by more than 30-fold (12).…”

Section: Replacement Of Ilementioning

confidence: 99%

Interdomain Signaling in Glutamine Phosphoribosylpyrophosphate Amidotransferase

Bera¹,

Chen²,

Smith³

et al. 1999

The glutamine phosphoribosylpyrophosphate (PRPP) amidotransferase-catalyzed synthesis of phosphoribosylamine from PRPP and glutamine is the sum of two half-reactions at separated catalytic sites in different domains. Binding of PRPP to a C-terminal phosphoribosyltransferase domain is required to activate the reaction at the N-terminal glutaminase domain. Glutamine PRPP 1 amidotransferase, an N-terminal nucleophile type glutamine amidotransferase (1), catalyzes the first step in purine nucleotide synthesis, shown by Equation 1. This reaction takes place in two steps (Equations 2 and 3).X-ray structures have defined the structure-function relationship (2, 3). An N-terminal glutaminase domain catalyzes the first half-reaction (Equation 2) and a C-terminal PRTase domain the second step (Equation 3). These catalytic sites are separated by 16 Å. Binding of PRPP to the PRTase catalytic site activates the glutaminase step (4, 5), a feature that prevents the wasteful hydrolysis of glutamine independent of PRA synthesis. We have investigated two key questions regarding the enzyme mechanism. First, how is the PRPP-binding signal communicated to the glutamine site over a distance of 16 Å? Second, how is NH 3 produced at the glutamine site sequestered from solvent and delivered to the PRTase domain for nucleophilic attack on PRPP to produce PRA and PP i ? The substrate for the second half-reaction, shown by Equation 3, is NH 3 , not NH 4 ϩ (4). X-ray crystal structures of an Escherichia coli ligandfree enzyme (2) and an enzyme-substrate analog ternary complex (3) have provided the framework to investigate these questions. The ligand-free and ternary complex structures have been referred to as state I and state III conformers, respectively (6). The structure of the state I enzyme is incompatible with catalysis. The unfavorable properties include: (i) the PRPP site is exposed to solvent; (ii) an important arginine residue (Arg 73 ) needed for glutamine binding is unfavorably positioned; and (iii) sites for glutamine hydrolysis and reaction of NH 3 with PRPP are separated by a 16 Å solvent-exposed space. These barriers to catalysis are corrected in the structure of the state III enzyme-substrate ternary complex. A PRTase "flexible loop" (residues 326 -350) has closed over the bound PRPP, thus protecting it from hydrolysis. Arg 73 is optimally positioned for binding glutamine and the glutamine, and PRTase sites are connected by a 20 Å NH 3 channel. However, the x-ray structures of the two enzyme conformers do not indicate how glutamine binds because in each conformer the glutamine site is closed thus restricting entry to the site. Thus, glutamine must initially bind to a different enzyme conformer. Recently, we have engineered enzymes containing a single tryptophan fluorescence reporter in positions that change conformation upon formation of the enzyme-substrate ternary complex (6). Measurements of steady state and pre-steady state intrinsic tryptophan fluorescence have identified an intermediate state II enzyme⅐PRPP conformer. ...

“…molecular tunnel for transport of ammonia and carbamate. In the case of carbamoyl phosphate synthetase, glutaminase activity is accelerated 1000-fold by bicarbonate phosphorylation at a site 45 Å away (3,4,11).…”

mentioning

confidence: 99%

“…Much of the catalytic power of a GAT domain involves protection of ammonia gas as a reactant (2)(3)(4)11). In this study, we identified two classes (I and III, involving amino acid substitutions at amino acids 111 and 112) of mutants in the glutaminase domain that inhibit synthetase activity, using either glutamine or ammonia as the amide donor.…”

mentioning

confidence: 99%

Glutamine-dependent NAD+ Synthetase

Wójcik

Seidle

Bieganowski

et al. 2006

Glutamine-dependent NAD؉ synthetase, Qns1, utilizes a glutamine aminotransferase domain to supply ammonia for amidation of nicotinic acid adenine dinucleotide (NaAD ؉ ) to NAD ؉ . Earlier characterization of Qns1 suggested that glutamine consumption exceeds NAD ؉ production by 40%. To explore whether Qns1 is systematically wasteful or whether additional features account for this behavior, we performed a careful kinetic and molecular genetic analysis. In fact, Qns1 possesses remarkable properties to reduce waste. The glutaminase active site is stimulated by NaAD ؉ more than 50-fold such that glutamine is not appreciably consumed in the absence of NaAD ؉ . Glutamine consumption exceeds NAD؉ production over the whole range of glutamine and NaAD ؉ substrate concentrations with greatest efficiency occurring at saturation of both substrates. Kinetic data coupled with site-directed mutagenesis of amino acids in the predicted ammonia channel indicate that NaAD ؉ stimulates the glutaminase active site in the k cat term by a synergistic mechanism that does not require ammonia utilization by the NaAD ؉ substrate. Six distinct classes of Qns1 mutants that fall within the glutaminase domain and the synthetase domain selectively inhibit components of the coordinated reaction.Glutamine-dependent NAD ϩ synthetase Qns1 (1) is one of many enzymes that couples a glutamine amidotransferase (GAT) 2 domain to a second active site that requires ammonia gas as a reactant (2-4). The second active site of Qns1 is an NAD ϩ synthetase domain that reacts nicotinic acid adenine dinucleotide (NaAD ϩ ) with ATP to form an activated, adenylylated intermediate (NaAD-AMP), releasing pyrophosphate. In turn, NaAD-AMP is attacked by ammonia to produce NAD ϩ plus AMP. Ideally, the enzyme would produce one equivalent of NAD ϩ and AMP for every glutamine hydrolyzed to glutamate. This ideal stoichiometry, depicted in Fig. 1, has been attributed to glutamine-dependent NAD ϩ synthetase since the first description of the enzyme by Preiss and Handler (5). The simplicity of Fig. 1 notwithstanding, characterization of the native (6) and recombinant (1) purified enzyme has never been entirely consistent with ideality. Whether the enzyme is, in fact, capable of perfect stoichiometry, potentially at a "sweet spot" of substrate concentrations, has not been investigated.Though eukaryotic and some prokaryotic NAD ϩ synthetases have long been understood to be glutamine-dependent enzymes (5, 7), the GAT domain of these enzymes initially escaped recognition as a glutaminase because it is neither a triad-type GAT, an N-terminal nucleophile-type GAT (2), nor one related to aminoacyl-tRNA transamidases (8). In the course of characterizing the domain structure of members of the nitrilase superfamily, we observed that the glutamine dependence of all eukaryotic and particular prokaryotic NAD ϩ synthetases could be accounted for by the presence of a putative GAT domain related to nitrilase (9, 10). Site-directed mutagenesis of yeast Qns1 coupled to biochemical and physiologi...