Frank M. Raushel

The Binding of Substrate Analogs to Phosphotriesterase

Phosphotriesterase (PTE) fromPseudomonas 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 β-sheet. The two zinc ions required for activity are positioned at the C-terminal portion of the β-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 Å), 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 Å 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.

The structure of carbamoyl phosphate synthetase determined to 2.1 Å resolution

Carbamoyl phosphate synthetase catalyzes the formation of carbamoyl phosphate from one molecule of bicarbonate, two molecules of Mg2+ATP and one molecule of glutamine or ammonia depending upon the particular form of the enzyme under investigation. As isolated from Escherichia coli, the enzyme is an alpha,beta-heterodimer consisting of a small subunit that hydrolyzes glutamine and a large subunit that catalyzes the two required phosphorylation events. Here the three-dimensional structure of carbamoyl phosphate synthetase from E. coli refined to 2.1 A resolution with an R factor of 17.9% is described. The small subunit is distinctly bilobal with a catalytic triad (Cys269, His353 and Glu355) situated between the two structural domains. As observed in those enzymes belonging to the alpha/beta-hydrolase family, the active-site nucleophile, Cys269, is perched at the top of a tight turn. The large subunit consists of four structural units: the carboxyphosphate synthetic component, the oligomerization domain, the carbamoyl phosphate synthetic component and the allosteric domain. Both the carboxyphosphate and carbamoyl phosphate synthetic components bind Mn2+ADP. In the carboxyphosphate synthetic component, the two observed Mn2+ ions are both octahedrally coordinated by oxygen-containing ligands and are bridged by the carboxylate side chain of Glu299. Glu215 plays a key allosteric role by coordinating to the physiologically important potassium ion and hydrogen bonding to the ribose hydroxyl groups of ADP. In the carbamoyl phosphate synthetic component, the single observed Mn2+ ion is also octahedrally coordinated by oxygen-containing ligands and Glu761 plays a similar role to that of Glu215. The carboxyphosphate and carbamoyl phosphate synthetic components, while topologically equivalent, are structurally different, as would be expected in light of their separate biochemical functions.

Carbamoyl phosphate synthetase: an amazing biochemical odyssey from substrate to product

Abstract. Carbamoyl phosphate synthetase (CPS) catalyzes one of the most remarkable reactions ever described in biological chemistry, in which carbamoyl phosphate is produced from one molecule of bicarbonate, two molecules of Mg2+ATP, and one molecule of either glutamine or ammonia. The carbamoyl phosphate so produced is utilized in the synthesis of arginine and pyrimidine nucleotides. It is also employed in the urea cycle in most terrestrial vertebrates. Due to its large size, its important metabolic role, and the fact that it is highly regulated, CPS has been the focus of intensive investigation for nearly 40 years. Numerous enzymological, biochemical, and biophysical studies by a variety of investigators have led to a quite detailed understanding of CPS. Perhaps one of the most significant advances on this topic within the last 2 years has been the successful X-ray crystallographic analysis of CPS from Escherichia coli. Quite unexpectedly, this structural investigation revealed that the three active sites on the protein are widely separated from one another. Furthermore, these active sites are connected by a molecular tunnel with a total length of approximately 100 Å, suggesting that CPS utilizes this channel to facilitate the translocation of reaction intermediates from one site to another. In this review, we highlight the recent biochemical and X-ray crystallographic results that have led to a more complete understanding of this finely tuned instrument of catalysis.

Success of pyridostigmine, physostigmine, eptastigmine and phosphotriesterase treatments in acute sarin intoxication

The acute toxicity of organophosphorus (OP) compounds in mammals is due to their irreversible inhibition of acetylcholinesterase (AChE) in the nervous system, which leads to increased synaptic acetylcholine levels. The protective actions of intravenously (i.v.) administered pyridostigmine, physostigmine, eptastigmine, and an organophosphate hydrolase, phosphotriesterase, in acute sarin intoxication were studied in mice. The acute intragastric (i.g.) toxicity (LD50) of sarin with and without the pretreatments was tested by the up-and-down method. The mice received pyridostigmine (0.06 mg/kg body weight), physostigmine (0.09 mg/kg body weight), the physostigmine derivative eptastigmine (0.90 mg/kg body weight) or phosphotriesterase (104 U/g, 10.7 microg/g body weight) 10 min prior to the i.g. administration of sarin. Physostigmine was also administered with phosphotriesterase. Phosphotriesterase was the most effective antidote in sarin intoxication. The LD50 value for sarin increased 3.4-fold in mice receiving phosphotriesterase. Physostigmine was the most effective carbamate in sarin exposure. The protective ratios of physostigmine and pyridostigmine were 1.5- and 1.2-1.3-fold, respectively. Eptastigmine did not give any protection against sarin toxicity. Both the phosphotriesterase and physostigmine treatments protected the brain AChE activities measured 24 h after sarin exposure. In phosphotriesterase and physostigmine-treated mice, a 4- and 2-fold higher sarin dose, respectively, was needed to cause a 50% inhibition of brain AChE activity. Moreover, the combination of phosphotriesterase-physostigmine increased the LD50 value for sarin 4.3-fold. The animals pretreated with phosphotriesterase-ephysostigmine tolerated four times the lethal dose in control animals, furthermore their survival time was 2-3 h in comparison to 20 min in controls. In conclusion, phosphotriesterase and physostigmine were the most effective treatments against sarin intoxication. However, eptastigmine did not provide any protection against sarin toxicity.

CARBAMOYL PHOSPHATE SYNTHETASE : A TUNNEL RUNS THROUGH IT

The direct transfer of metabolites from one protein to another in a biochemical pathway or between one active site and another within a single enzyme has been described as substrate channeling. The first structural visualization of such a phenomenon was provided by the X-ray crystallographic analysis of tryptophan synthase, in which a tunnel of approximately 25 A in length was observed. The recently determined three-dimensional structure of carbamoyl phosphate synthetase sets a new long distance record in that the three active sites are separated by nearly 100 A.

Hydrolysis of Phosphodiesters through Transformation of the Bacterial Phosphotriesterase

The phosphotriesterase from Pseudomonas diminuta catalyzes the hydrolysis of a wide array of phosphotriesters and related phosphonates, including organophosphate pesticides and military nerve agents. It has now been shown that this enzyme can also catalyze the hydrolysis of phosphodiesters, albeit at a greatly reduced rate. However, the enzymatic hydrolysis of ethyl-4-nitrophenyl phosphate (compound I) by the wild-type enzyme was >108 times faster than the uncatalyzed reaction (k cat = 0.06 s−1 andK m = 38 mm). Upon the addition of various alkylamines to the reaction mixture, thek cat/K m for the phosphodiester (compound I) increased up to 200-fold. Four mutant enzymes of the phosphotriesterase were constructed in a preliminary attempt to improve phosphodiester hydrolysis activity of the native enzyme. Met-317, which is thought to reside in close proximity to the pro-S-ethoxy arm of the paraoxon substrate, was mutated to arginine, alanine, histidine, and lysine. These mutant enzymes showed slight improvements in the catalytic hydrolysis of organophosphate diesters. The M317K mutant enzyme displayed the most improvement in catalytic activity (k cat = 0.34 s−1 and K m = 30 mm). The M317A mutant enzyme catalyzed the hydrolysis of the phosphodiester (compound I) in the presence of alkylamines up to 200 times faster than the wild-type enzyme in the absence of added amines. The neutralization of the negative charge on the oxygen atom of the phosphodiester by the ammonium cation within the active site is thought to be responsible for the rate enhancement by these amines in the hydrolytic reaction. These results demonstrate that an active site optimized for the hydrolysis of organophosphate triesters can be made to catalyze the hydrolysis of organophosphate diesters.

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.

Carbamoyl phosphate synthetase: a crooked path from substrates to products.

The formation of carbamoyl phosphate is catalyzed by a single enzyme using glutamine, bicarbonate and two molecules of ATP via a reaction mechanism that requires a minimum of four consecutive reactions and three unstable intermediates. The recently determined X-ray crystal structure of carbamoyl phosphate synthetase has revealed the location of three separate active sites connected by two molecular tunnels that run through the interior of the protein. It has been demonstrated that the amidotransferase domain within the small subunit of the enzyme from Escherichia coli hydrolyzes glutamine to ammonia via a thioester intermediate with Cys269. The ammonia migrates through the interior of the protein, where it reacts with carboxy phosphate to produce the carbamate intermediate. The carboxy phosphate intermediate is formed by the phosphorylation of bicarbonate by ATP at a site contained within the amino-terminal half of the large subunit. The carbamate intermediate is transported through the interior of the protein to a second site within the carboxy-terminal half of the large subunit, where it is phosphorylated by another ATP to yield the final product, carbamoyl phosphate. The entire journey from substrate to product covers a distance of nearly 100 A.

A VERSATILE MECHANISM BASED REACTION PROBE FOR THE DIRECT SELECTION OF BIOCATALYSTS

Abstract A mechanism based reaction probe was synthesized and shown to modify a bacterial phosphotriesterase; this strategy for generating a probe is general and should allow the isolation of a host of unique catalysts.

A COMBINATORIAL LIBRARY FOR THE BINUCLEAR METAL CENTER OF BACTERIAL PHOSPHOTRIESTERASE

Phosphotriesterase (PTE) is a zinc metalloenzyme that catalyzes the hydrolysis of an extensive array of organophosphate pesticides and mammalian acetylcholinesterase nerve agents. Although the three‐dimensional crystal structure of PTE has been solved (M. M. Benning et al., Biochemistry 34:7973–7978, 1995), the precise functions of the individual amino acid residues that interact directly with the substrate at the active site are largely unknown. To construct mutants of PTE with altered specificities for particular target substrates, a simple methodology for generating a library of mutants at specific sites was developed. In this investigation, four of the six protein ligands to the binuclear metal site (His‐55, His‐57, His‐201, and His‐230) were targeted for further characterization and investigation. Using the polymerase chain reaction (PCR) protocols, a library of modified PTE genes was generated by simultaneously creating random combinations of histidine and cysteine codons at these four positions. The 16 possible DNA sequences were isolated and confirmed by dideoxy‐DNA sequencing. The 16 mutant proteins were expressed in Escherichia coli and grown with the presence or absence of 1 mM CoCl2, ZnSO4, or CdSO4in the growth medium. When grown in the presence of CoCl2, the H57C protein cell lysate showed greater activity for the hydrolysis of paraoxon than the wild type PTE cell lysate. H201C and H230C exhibited up to 15% of the wild‐type activity, while H55C, a green protein, was inactive under all assay conditions. All other mutants had <10−5 of wild‐type activity. None of the purified mutants that exhibited catalytic activity had a significantly altered Km for paraoxon. Proteins 29:553–561, 1997.