Allosteric control of the oligomerization of carbamoyl phosphate synthetase from Escherichia coli.

Carbamoyl phosphate synthetase (CPS) from Escherichia coli is allosterically regulated by the metabolites ornithine, IMP, and UMP. Ornithine and IMP function as activators, whereas UMP is an inhibitor. CPS undergoes changes in the state of oligomerization that are dependent on the protein concentration and the binding of allosteric effectors. Ornithine and IMP promote the formation of an (alphabeta)4 tetramer while UMP favors the formation of an (alphabeta)2 dimer. The three-dimensional structure of the (alphabeta)4 tetramer has unveiled two regions of molecular contact between symmetry-related monomeric units. Identical residues within two pairs of allosteric domains interact with one another as do twin pairs of oligomerization domains. There are thus two possible structures for an (alphabeta)2 dimer: an elongated dimer formed at the interface of two allosteric domains and a more compact dimer formed at the interface between two oligomerization domains. Mutations at the two interfacial sites of oligomerization were constructed in an attempt to elucidate the mechanism for assembly of the (alphabeta)4 tetramer through disruption of the molecular binding interactions between monomeric units. When Leu-421 (located in the oligomerization domain) was mutated to a glutamate residue, CPS formed an (alphabeta)2 dimer in the presence of ornithine, UMP, or IMP. In contrast, when Asn-987 (located in the allosteric binding domain) was mutated to an aspartate, an (alphabeta) monomer was formed regardless of the presence of any allosteric effectors. These results are consistent with a model for the structure of the (alphabeta)2 dimer that is formed through molecular contact between two pairs of allosteric domains. Apparently, the second interaction, between pairs of oligomerization domains, does not form until after the interaction between pairs of allosteric domains is formed. The binding of UMP to the allosteric domain inhibits the dimerization of the (alphabeta)2 dimer, whereas the binding of either IMP or ornithine to this same domain promotes the dimerization of the (alphabeta)2 dimer. In the oligomerization process, ornithine and IMP must exert a conformational alteration on the oligomerization domain, which is approximately 45 A away from their site of binding within the allosteric domain. No significant dependence of the specific catalytic activity on the protein concentration could be detected, and thus the effects induced by the allosteric ligands on the catalytic activity and the state of oligomerization are unlinked from one another.

Hydrolysis of phosphotriesters: determination of transition states in parallel reactions by heavy-atom isotope effects.

The remote label method was used to measure primary and secondary (18)O isotope effects in the alkaline hydrolysis of O,O-diethylphosphorylcholine iodide (DEPC) and the primary (18)O effect in the alkaline hydrolysis of O,O-diethyl-m-nitrobenzyl phosphate (DEmNBP). Both the leaving group of interest (choline or m-nitrobenzyl alcohol) and ethanol can be ejected during hydrolysis due to the similarity of their pK values. The heavy-atom isotope effects were measured by isotope ratio mass spectrometry. Parallel reaction and incomplete labeling corrections were made for both systems. DEPC has a primary (18)O isotope effect of 1.041 +/- 0.003 and a secondary (18)O isotope effect of 1.033 +/- 0.002. The primary (18)O isotope effect for DEmNBP was 1.052 +/- 0.003. These large effects suggest a highly associative transition state in which the nucleophile approaches very close to the phosphorus atom to eject the leaving group. The large values are also indicative of a large compression, or general movement, on the reaction coordinate.

Stereoselective detoxification of chiral sarin and soman analogues by phosphotriesterase.

The catalytic activity of the bacterial phosphotriesterase (PTE) toward a series of chiral analogues of the chemical warfare agents sarin and soman was measured. Chemical procedures were developed for the chiral syntheses of the S(P)- and R(P)-enantiomers of O-isopropyl p-nitrophenyl methylphosphonate (sarin analogue) in high enantiomeric excess. The R(P)-enantiomer of the sarin analogue (k(cat)=2600 s(-1)) was the preferred substrate for the wild-type PTE relative to the corresponding S(P)-enantiomer (k(cat)=290 s(-1)). The observed stereoselectivity was reversed using the PTE mutant, I106A/F132A/H254Y where the k(cat) values for the R(P)- and S(P)-enantiomers were 410 and 4200 s(-1), respectively. A chemo-enzymatic procedure was developed for the chiral synthesis of the four stereoisomers of O-pinacolyl p-nitrophenyl methylphosphonate (soman analogue) with high diastereomeric excess. The R(P)R(C)-stereoisomer of the soman analogue was the preferred substrate for PTE. The k(cat) values for the soman analogues were measured as follows: R(P)R(C,) 48 s(-1); R(P)S(C), 4.8 s(-1); S(P)R(C), 0.3 s(-1), and S(P)S(C), 0.04 s(-1). With the I106A/F132A/H254Y mutant of PTE the stereoselectivity toward the chiral phosphorus center was reversed. With the triple mutant the k(cat) values for the soman analogues were found to be as follows: R(P)R(C,) 0.3 s(-1); R(P)S(C), 0.3 s(-1); S(P)R(C), 11s(-1), and S(P)S(C), 2.1 s(-1). Prior investigations have demonstrated that the S(P)-enantiomers of sarin and soman are significantly more toxic than the R(P)-enantiomers. This investigation has demonstrated that mutants of the wild-type PTE can be readily constructed with enhanced catalytic activities toward the most toxic stereoisomers of sarin and soman.

Channeling of substrates and intermediates in enzyme-catalyzed reactions.

The three-dimensional structures of tryptophan synthase, carbamoyl phosphate synthetase, glutamine phosphoribosylpyrophosphate amidotransferase, and asparagine synthetase have revealed the relative locations of multiple active sites within these proteins. In all of these polyfunctional enzymes, a product formed from the catalytic reaction at one active site is a substrate for an enzymatic reaction at a distal active site. Reaction intermediates are translocated from one active site to the next through the participation of an intermolecular tunnel. The tunnel in tryptophan synthase is approximately 25 A in length, whereas the tunnel in carbamoyl phosphate synthetase is nearly 100 A long. Kinetic studies have demonstrated that the individual reactions are coordinated through allosteric coupling of one active site with another. The participation of these molecular tunnels is thought to protect reactive intermediates from coming in contact with the external medium.

Molecular structure of dihydroorotase: a paradigm for catalysis through the use of a binuclear metal center.

Dihydroorotase plays a key role in pyrimidine biosynthesis by catalyzing the reversible interconversion of carbamoyl aspartate to dihydroorotate. Here we describe the three-dimensional structure of dihydroorotase from Escherichia coli determined and refined to 1.7 A resolution. Each subunit of the homodimeric enzyme folds into a "TIM" barrel motif with eight strands of parallel beta-sheet flanked on the outer surface by alpha-helices. Unexpectedly, each subunit contains a binuclear zinc center with the metal ions separated by approximately 3.6 A. Lys 102, which is carboxylated, serves as a bridging ligand between the two cations. The more buried or alpha-metal ion in subunit I is surrounded by His 16, His 18, Lys 102, Asp 250, and a solvent molecule (most likely a hydroxide ion) in a trigonal bipyramidal arrangement. The beta-metal ion, which is closer to the solvent, is tetrahedrally ligated by Lys 102, His 139, His 177, and the bridging hydroxide. L-Dihydroorotate is observed bound to subunit I, with its carbonyl oxygen, O4, lying 2.9 A from the beta-metal ion. Important interactions for positioning dihydroorotate into the active site include a salt bridge with the guanidinium group of Arg 20 and various additional electrostatic interactions with both protein backbone and side chain atoms. Strikingly, in subunit II, carbamoyl L-aspartate is observed binding near the binuclear metal center with its carboxylate side chain ligating the two metals and thus displacing the bridging hydroxide ion. From the three-dimensional structures of the enzyme-bound substrate and product, it has been possible to propose a unique catalytic mechanism for dihydroorotase. In the direction of dihydroorotate hydrolysis, the bridging hydroxide attacks the re-face of dihydroorotate with general base assistance by Asp 250. The carbonyl group is polarized for nucleophilic attack by the bridging hydroxide through a direct interaction with the beta-metal ion. During the cyclization of carbamoyl aspartate, Asp 250 initiates the reaction by abstracting a proton from N3 of the substrate. The side chain carboxylate of carbamoyl aspartate is polarized through a direct electrostatic interaction with the binuclear metal center. The ensuing tetrahedral intermediate collapses with C-O bond cleavage and expulsion of the hydroxide which then bridges the binuclear metal center.

Stereochemical specificity of organophosphorus acid anhydrolase toward p-nitrophenyl analogs of soman and sarin.

Organophosphorus acid anhydrolase (OPAA) catalyzes the hydrolysis of p-nitrophenyl analogs of the organophosphonate nerve agents, sarin and soman. The enzyme is stereoselective toward the chiral phosphorus center by displaying a preference for the R(P)-configuration of these analogs. OPAA also exhibits an additional preference for the stereochemical configuration at the chiral carbon center of the soman analog. The preferred configuration of the chiral carbon center is dependent upon the configuration at the phosphorus center. The enzyme displays a two- to four-fold preference for the R(P)-enantiomer of the sarin analog. The k(cat)/K(m) of the R(P)-enantiomer is 250 M(-1) s(-1), while that of the S(P)-enantiomer is 110 M(-1) s(-1). The order of preference for the stereoisomers of the soman analog is R(P)S(C) > R(P)R(C) > S(P)R(C) > S(P)S(C). The k(cat)/K(m) values are 36,300 M(-1)s(-1), 1250 M(-1) s(-1), 80 M(-1) s(-1) and 5 M(-1) s(-1), respectively. The R(P)S(C)-isomer of the soman analog is therefore preferred by a factor of 7000 over the S(P)S(C)-isomer.

High resolution X-ray structures of different metal-substituted forms of phosphotriesterase from Pseudomonas diminuta.

Phosphotriesterase, isolated from the soil-dwelling bacterium Pseudomonas diminuta, catalyzes the detoxification of organophosphate-based insecticides and chemical warfare agents. The enzyme has attracted significant research attention in light of its possible employment as a bioremediation tool. As naturally isolated, the enzyme is dimeric. Each subunit contains a binuclear zinc center that is situated at the C-terminal portion of a "TIM" barrel motif. The two zincs are separated by approximately 3.4 A and coordinated to the protein via the side chains of His 55, His 57, His 201, His 230, Asp 301, and a carboxylated Lys 169. Both Lys 169 and a water molecule (or hydroxide ion) serve to bridge the two zinc ions together. Interestingly, these metals can be replaced with cadmium or manganese ions without loss of enzymatic activity. Here we describe the three-dimensional structures of the Zn(2+)/Zn(2+)-, Zn(2+)/Cd(2+)-, Cd(2+)/Cd(2+)-, and Mn(2+)/Mn(2+)-substituted forms of phosphotriesterase determined and refined to a nominal resolution of 1.3 A. In each case, the more buried metal ion, referred to as the alpha-metal, is surrounded by ligands in a trigonal bipyramidal ligation sphere. For the more solvent-exposed or beta-metal ion, however, the observed coordination spheres are either octahedral (in the Cd(2+)/Cd(2+)-, Mn(2+)/Mn(2+)-, and the mixed Zn(2+)/Cd(2+)-species) or trigonal bipyramidal (in the Zn(2+)/Zn(2+)-protein). By measuring the anomalous X-ray data from crystals of the Zn(2+)/Cd(2+)-species, it has been possible to determine that the alpha-metal ion is zinc and the beta-site is occupied by cadmium.

Enhancement, relaxation, and reversal of the stereoselectivity for phosphotriesterase by rational evolution of active site residues.

The factors that govern the substrate reactivity and stereoselectivity of phosphotriesterase (PTE) toward organophosphotriesters containing various combinations of methyl, ethyl, isopropyl, and phenyl substituents at the phosphorus center were determined by systematic alterations in the dimensions of the active site. The wild type PTE prefers the S(P)-enantiomers over the corresponding R(P)-enantiomers by factors ranging from 10 to 90. Enlargement of the small subsite of PTE with the substitution of glycine and alanine residues for Ile-106, Phe-132, and/or Ser-308 resulted in significant improvements in k(cat)/K(a) for the R(P)-enantiomers of up to 2700-fold but had little effect on k(cat)/K(a) for the corresponding S(P)-enantiomers. The kinetic preferences for the S(P)-enantiomers were thus relaxed without sacrificing the inherent catalytic activity of the wild type enzyme. A reduction in the size of the large subsite with the mutant H257Y resulted in a reduction in k(cat)/K(a) for the S(P)-enantiomers, while the values of k(cat)/K(a) for the R(P)-enantiomers were essentially unchanged. The initial stereoselectivity observed with the wild type enzyme toward the chiral substrate library was significantly reduced with the H257Y mutant. Simultaneous alternations in the sizes of the large and small subsites resulted in the complete reversal of the chiral specificity. With this series of mutants, the R(P)-enantiomers were preferred as substrates over the corresponding S(P)-enantiomers by up to 500-fold. These results have demonstrated that the stereochemical determinants for substrate hydrolysis by PTE can be systematically altered through a rational reconstruction of the dimensions of the active site.

Structural determinants of the substrate and stereochemical specificity of phosphotriesterase.

Bacterial phosphotriesterase (PTE) catalyzes the hydrolysis of a wide variety of organophosphate nerve agents and insecticides. Previous kinetic studies with a series of enantiomeric organophosphate triesters have shown that the wild type PTE generally prefers the S(P)-enantiomer over the corresponding R(P)-enantiomers by factors ranging from 1 to 90. The three-dimensional crystal structure of PTE with a bound substrate analogue has led to the identification of three hydrophobic binding pockets. To delineate the factors that govern the reactivity and stereoselectivity of PTE, the dimensions of these three subsites have been systematically altered by site-directed mutagenesis of Cys-59, Gly-60, Ser-61, Ile-106, Trp-131, Phe-132, His-254, His-257, Leu-271, Leu-303, Phe-306, Ser-308, Tyr-309, and Met-317. These studies have shown that substitution of Gly-60 with an alanine within the small subsite dramatically decreased k(cat) and k(cat)/K(a) for the R(P)-enantiomers, but had little influence on the kinetic constants for the S(P)-enantiomers of the chiral substrates. As a result, the chiral preference for the S(P)-enantiomers was greatly enhanced. For example, the value of k(cat)/K(a) with the mutant G60A for the S(P)-enantiomer of methyl phenyl p-nitrophenyl phosphate was 13000-fold greater than that for the corresponding R(P)-enantiomer. The mutation of I106, F132, or S308 to an alanine residue, which enlarges the small or leaving group subsites, caused a significant reduction in the enantiomeric preference for the S(P)-enantiomers, due to selective increases in the reaction rates for the R(P)-enantiomers. Enlargement of the large subsite by the construction of an H254A, H257A, L271A, or M317A mutant had a relatively small effect on k(cat)/K(a) for either the R(P)- or S(P)-enantiomers and thus had little effect on the overall stereoselectivity. These studies demonstrate that by modifying specific residues located within the active site of PTE, it is possible to dramatically alter the stereoselectivity and overall reactivity of the native enzyme toward chiral substrates.

Capillary electrophoretic separation of the enantiomers of organophosphates with a phosphorus stereogenic center using the sodium salt of octakis(2,3-diacetyl-6-sulfo)-gamma-cyclodextrin as resolving agent.

The sodium salt of the single-isomer, chiral resolving agent, octakis(2,3-diacetyl-6-sulfo)-gamma-cyclodextrin (ODAS-gammaCD) has been used for the capillary electrophoretic separation of the enantiomers of alkylarylphosphates which carry a phosphorus-based stereogenic center. The effective mobilities and separation selectivities were measured at different ODAS-gammaCD and methanol concentrations to find the conditions under which the minor enantiomers could be adequately quantitated in samples obtained by chemical resolution of the racemic mixtures. This work extends the utility of ODAS-gammaCD to a hitherto unexplored field, the capillary electrophoretic separation of the enantiomers of organophosphorus compounds.

Experimental verification of a predicted, previously unseen separation selectivity pattern in the capillary electrophoretic separation of noncharged enantiomers by octakis(2,3-diacetyl-6-sulfato)-gamma-cyclodextrin.

The capillary electrophoretic separation of noncharged enantiomers with single-isomer anionic resolving agents is reexamined here with the help of the charged resolving agent migration model. Two general model parameters have been identified that influence the effective mobility, separation selectivity and mobility difference curves of the enantiomers: parameter b, called binding selectivity (K(RCD)/K(SCD)), and parameter s, called size selectivity (mu(o)RCD/mu(o)SCD). Analysis of the model in terms of these parameters indicates that in addition to the known, previously observed separation selectivity vs. resolving agent concentration patterns, a new pattern, increasing separation selectivity with increasing resolving agent concentration, is also possible provided that (i) K(RCD)/K(SCD)<1 and mu(o)RCD/mu(o)SCD>1 and (K(RCD)mu(o)RCD)/(K(SCD)mu(o)SCD)>1, or (ii) K(RCD)/ K(SCD)>1 and mu(o)SCD/mu(o)SCD<1 and (K(RCD)mu(o)RCD)/(K(SCD)mu(o)SCD)<1. This hitherto unseen separation selectivity pattern was experimentally verified during the capillary electrophoretic separation of the enantiomers of O-isopropyl p-nitrophenyl methylphosphonate with the single-isomer octakis(2,3-diacetyl-6-sulfato)-gamma-cyclodextrin as resolving agent.

Restricted passage of reaction intermediates through the ammonia tunnel of carbamoyl phosphate synthetase.

The x-ray crystal structure of the heterodimeric carbamoyl phosphate synthetase from Escherichia coli has identified an intermolecular tunnel that connects the glutamine binding site within the small amidotransferase subunit to the two phosphorylation sites within the large synthetase subunit. The tunneling of the ammonia intermediate through the interior of the protein has been proposed as a mechanism for the delivery of the ammonia from the small subunit to the large subunit. A series of mutants created within the ammonia tunnel were prepared by the placement of a constriction via site-directed mutagenesis. The degree of constriction within the ammonia tunnel of these enzymes was found to correlate to the extent of the uncoupling of the partial reactions, the diminution of carbamoyl phosphate formation, and the percentage of the internally derived ammonia that is channeled through the ammonia tunnel. NMR spectroscopy and a radiolabeled probe were used to detect and identify the enzymatic synthesis of N-amino carbamoyl phosphate and N-hydroxy carbamoyl phosphate from hydroxylamine and hydrazine. The kinetic results indicate that hydroxylamine, derived from the hydrolysis of gamma-glutamyl hydroxamate, is channeled through the ammonia tunnel to the large subunit. Discrimination between the passage of ammonia and hydroxylamine was observed among some of these tunnel-impaired enzymes. The overall results provide biochemical evidence for the tunneling of ammonia within the native carbamoyl phosphate synthetase.

Role of the hinge loop linking the N- and C-terminal domains of the amidotransferase subunit of carbamoyl phosphate synthetase.

Carbamoyl phosphate synthetase from Escherichia coli catalyzes the formation of carbamoyl phosphate from bicarbonate, glutamine, and two molecules of ATP. The enzyme consists of a large synthetase subunit and a small amidotransferase subunit. The small subunit is structurally bilobal. The N-terminal domain is unique compared to the sequences of other known proteins. The C-terminal domain, which contains the direct catalytic residues for the amidotransferase activity of CPS, is homologous to other members of the Triad glutamine amidotransferases. The two domains are linked by a hinge-like loop, which contains a type II beta turn. The role of this loop in the hydrolysis of glutamine and the formation of carbamoyl phosphate was probed by site-directed mutagenesis. Based upon the observed kinetic properties of the mutants, the modifications to the small subunit can be separated into two groups. The first group consists of G152I, G155I, and Delta155. Attempts to disrupt the turn conformation were made by the deletion of Gly-155 or substitution of the two glycine residues with isoleucine. However, these mutations only have minor effects on the kinetic properties of the enzyme. The second group includes L153W, L153G/N154G, and a ternary complex consisting of the intact large subunit plus the separate N- and C-terminal domains of the small subunit. Although the ability to synthesize carbamoyl phosphate is retained in these enzymes, the hydrolysis of glutamine is partially uncoupled from the synthetase reaction. It is concluded that the hinge loop, but not the type-II turn structure of the loop per se, is important for maintaining the proper interface interactions between the two subunits and the catalytic coupling of the partial reactions occurring within the separate subunits of CPS.

D-Ala-D-X ligases: evaluation of D-alanyl phosphate intermediate by MIX, PIX and rapid quench studies.

BACKGROUND: The D-alanyl-D-lactate (D-Ala-D-Lac) ligase is required for synthesis of altered peptidoglycan (PG) termini in the VanA phenotype of vancomycin-resistant enterococci (VRE), and the D-alanyl-D-serine (D-Ala-D-Ser) ligase is required for the VanC phenotype of VRE. Here we have compared these with the Escherichia coli D-Ala-D-Ala ligase DdlB for formation of the enzyme-bound D-alanyl phosphate, D-Ala(1)-PO(3)(2-) (D-Ala(1)-P), intermediate. RESULTS: The VanC2 ligase catalyzes a molecular isotope exchange (MIX) partial reaction, incorporating radioactivity from (14)C-D-Ser into D-Ala-(14)C-D-Ser at a rate of 0.7 min(-1), which approaches kinetic competence for the reversible D-Ala(1)-P formation from the back direction. A positional isotope exchange (PIX) study with the VanC2 and VanA ligases displayed a D-Ala(1)-dependent bridge to nonbridge exchange of the oxygen-18 label of [gamma-(18)O(4)]-ATP at rates of up to 0.6 min(-1); this exchange was completely suppressed by the addition of the second substrate D-Ser or D-Lac, respectively, as the D-Ala(1)-P intermediate was swept in the forward direction. As a third criterion for formation of bound D-Ala(1)-P, we conducted rapid quench studies to detect bursts of ADP formation in the first turnover of DdlB and VanA. With E. coli DdlB, there was a burst amplitude of ADP corresponding to 26-30% of the DdlB active sites, followed by the expected steady-state rate of 620-650 min(-1). For D-Ala-D-Lac and D-Ala-D-Ala synthesis by VanA, we measured a burst of 25-30% or 51% of active enzyme, respectively. CONCLUSIONS: These three approaches support the rapid (more than 1000 min(-1)), reversible formation of the enzyme intermediate D-Ala(1)-P by members of the D-Ala-D-X (where X is Ala, Ser or Lac) ligase superfamily.

Substrate and stereochemical specificity of the organophosphorus acid anhydrolase from Alteromonas sp. JD6.5 toward p-nitrophenyl phosphotriesters.

The enzyme OPAA hydrolyzes p-nitrophenyl phosphotriesters bearing substituents at the phosphorus center ranging in size from methyl to phenyl. The enzyme exhibits stereoselectivity toward the hydrolysis of chiral substrates with a preference for the Sp enantiomer.

Self-assembly of the binuclear metal center of phosphotriesterase.

The active site of the bacterial phosphotriesterase (PTE) from Pseudomonas diminuta contains two divalent metal ions and a carboxylated lysine residue. The native enzyme contains two Zn(2+) ions, which can be replaced with Co(2+), Cd(2+), Ni(2+), or Mn(2+) without loss of catalytic activity. Carbon dioxide reacts with the side chain of lysine-169 to form a carbamate functional group within the active site, which then serves as a bridging ligand to the two metal ions. The activation of apo-PTE using variable concentrations of divalent metal ions and bicarbonate was measured in order to establish the mechanism by which the active site of PTE is self-assembled. The time courses for the activation of apo-PTE are pseudo-first-order, and the observed rate constants are directly proportional to the concentration of bicarbonate. In contrast, the apparent rate constants for the activation of apo-PTE decrease as the concentrations of the divalent cations are increased and then become constant at higher concentrations of the divalent metal ions. These results are consistent with a largely ordered kinetic mechanism for the assembly of the binuclear metal center where CO(2)/bicarbonate reacts with the apo-PTE prior to the binding of the two metal ions. When apo-PTE is titrated with 0-8 equiv of Co(2+), Cd(2+), or Zn(2+), the concentration of activated enzyme increases linearly until 2 equiv of metal ion is added and then remains constant at elevated levels of the divalent cations. These results are consistent with the synergistic binding of the two metal ions to the active site, and thus the second metal ion binds more tightly to the protein than does the first metal ion. Measurement of the mean dissociation constant indicates that metal binding to the binuclear metal center is strong [(K(alpha)K(beta))(1/2) = 6.0 x 10(-)(11) M and k(off) = 1.5 x 10(-)(3) min(-)(1) for Zn(2+)]. The removal of the carbamate bridge through the mutagenesis of Lys-169 demonstrates that the carbamate bridge is required for both efficient catalysis and overall stability of the metal center.

The binding of substrate analogs to phosphotriesterase.

Phosphotriesterase (PTE) from Pseudomonas diminuta catalyzes the detoxification of organophosphates such as the widely utilized insecticide paraoxon and the chemical warfare agent sarin. The three-dimensional structure of the enzyme is known from high resolution x-ray crystallographic analyses. Each subunit of the homodimer folds into a so-called TIM barrel, with eight strands of parallel beta-sheet. The two zinc ions required for activity are positioned at the C-terminal portion of the beta-barrel. Here, we describe the three-dimensional structure of PTE complexed with the inhibitor diisopropyl methyl phosphonate, which serves as a mimic for sarin. Additionally, the structure of the enzyme complexed with triethyl phosphate is also presented. In the case of the PTE-diisopropyl methyl phosphonate complex, the phosphoryl oxygen of the inhibitor coordinates to the more solvent-exposed zinc ion (2.5 A), thereby lending support to the presumed catalytic mechanism involving metal coordination of the substrate. In the PTE-triethyl phosphate complex, the phosphoryl oxygen of the inhibitor is positioned at 3.4 A from the more solvent-exposed zinc ion. The two structures described in this report provide additional molecular understanding for the ability of this remarkable enzyme to hydrolyze such a wide range of organophosphorus substrates.

Synchronization of the three reaction centers within carbamoyl phosphate synthetase.

Carbamoyl phosphate synthetase from E. coli catalyzes the synthesis of carbamoyl phosphate through a series of four reactions occurring at three active sites connected by a molecular tunnel of 100 A. To understand the mechanism for coordination and synchronization among the active sites, the pre-steady-state time courses for the formation of phosphate, ADP, glutamate, and carbamoyl phosphate were determined. When bicarbonate and ATP were rapidly mixed with CPS, a stoichiometric burst of acid-labile phosphate and ADP was observed with a formation rate constant of 1100 min(-)(1). The burst phase was followed by a linear steady-state phase with a rate constant of 12 min(-)(1). When glutamine or ammonia was added to the initial reaction mixture, the magnitude and the rate of formation of the burst phase for either phosphate or ADP were unchanged, but the rate constant for the linear steady-state phase increased to an average value of 78 min(-)(1). These results demonstrate that the initial phosphorylation of bicarbonate is independent of the binding or hydrolysis of glutamine. The pre-steady-state time course for the hydrolysis of glutamine in the absence of ATP exhibited a burst of glutamate formation with a rate constant of 4 min(-)(1) when the reaction was quenched with base. In the presence of ATP and bicarbonate, the rate constant for the formation of the burst of glutamate was 1100 min(-)(1). The hydrolysis of ATP thus enhanced the hydrolysis of glutamine by a factor of 275, but there was no effect by glutamine on the initial phosphorylation of bicarbonate. The pre-steady-state time course for the formation of carbamoyl phosphate was linear with an overall rate constant of 72 min(-)(1). The absence of an initial burst of carbamoyl phosphate formation eliminates product release as a rate-determining step for CPS. Overall, these results have been interpreted to be consistent with a mechanism whereby the phosphorylation of bicarbonate serves as the initial trigger for the rest of the reaction cascade. The formation of the carboxy phosphate intermediate within the large subunit must induce a conformational change to the active site of the small subunit that enhances the hydrolysis of glutamine. Thus, ammonia is not released into the molecular tunnel until the activated bicarbonate is ready to form carbamate. The rate-limiting step for the steady-state assembly of carbamoyl phosphate is either the formation, migration, or phosphorylation of the carbamate intermediate.

Phosphotriesterase: an enzyme in search of its natural substrate.

The bacterial PTE is able to catalyze the hydrolysis of a wide range of organophosphate nerve agents. The active site has been shown to consist of a unique binuclear metal center that has evolved to deliver hydroxide to the site of bond cleavage. The reaction rate for the hydrolysis of activated substrates such as paraoxon is limited by product release or an associated protein conformational change.

An engineered blockage within the ammonia tunnel of carbamoyl phosphate synthetase prevents the use of glutamine as a substrate but not ammonia.

The heterodimeric carbamoyl phosphate synthetase (CPS) from Escherichia coli catalyzes the formation of carbamoyl phosphate from bicarbonate, glutamine, and two molecules of ATP. The enzyme catalyzes the hydrolysis of glutamine within the small amidotransferase subunit and then transfers ammonia to the two active sites within the large subunit. These three active sites are connected via an intermolecular tunnel, which has been located within the X-ray crystal structure of CPS from E. coli. It has been proposed that the ammonia intermediate diffuses through this molecular tunnel from the binding site for glutamine within the small subunit to the phosphorylation site for bicarbonate within the large subunit. To provide experimental support for the functional significance of this molecular tunnel, residues that define the interior walls of the "ammonia tunnel" within the small subunit were targeted for site-directed mutagenesis. These structural modifications were intended to either block or impede the passage of ammonia toward the large subunit. Two mutant proteins (G359Y and G359F) display kinetic properties consistent with a constriction or blockage of the ammonia tunnel. With both mutants, the glutaminase and bicarbonate-dependent ATPase reactions have become uncoupled from one another. However, these mutant enzymes are fully functional when external ammonia is utilized as the nitrogen source but are unable to use glutamine for the synthesis of carbamoyl-P. These results suggest the existence of an alternate route to the bicarbonate phosphorylation site when ammonia is provided as an external nitrogen source.