Mechanism-Based Inhibitors for the Inactivation of the Bacterial Phosphotriesterase (original) (raw)

Mechanistic and Kinetic Studies of Inhibition of Enzymes

Cell Biochemistry and Biophysics, 2000

A graphical method for analyzing enzyme data to obtain kinetic parameters, and to identify the types of inhibition and the enzyme mechanisms, is described. The method consists of plotting experimental data as v/(V o -v) vs 1/(I) at different substrate concentrations. I is the inhibitor concentration; V o and v are the rates of enzyme reaction attained by the system in the presence of a fixed amount of substrate, and in the absence and presence of inhibitor, respectively. Complete inhibition gives straight lines that go through the origin; partial inhibition gives straight lines that converge on the 1-I axis, at a point away from the origin. For competitive inhibition, the slopes of the lines increase with increasing substrate concentration; with noncompetitive inhibition, the slopes are independent of substrate concentration; with uncompetitive inhibition, the slopes of the lines decrease with increasing substrate concentrations. The kinetic parameters, K m , K i , K i ′, and β (degree of partiality) can best be determined from respective secondary plots of slope and intercept vs substrate concentration, for competitive and noncompetitive inhibition mechanism or slope and intercept vs reciprocal substrate concentration for uncompetitive inhibition mechanism. Functional consequencs of these analyses are represented in terms of specific enzyme-inhibitor systems.

Mechanism-based enzyme inactivation using an allyl sulfoxide-allyl sulfenate ester rearrangement

Journal of the American Chemical Society, 1980

Imoto, T.; Shibata. S.; Hatano, H. J. Magn. Reson. 1975, 78, 328-343. (b) Zens, A. P.; Bryson, T. A,; Dunlap, R. B.; Fisher, R. R.; Ellis, P. D.

Stereoselective Hydrolysis of Organophosphate Nerve Agents by the Bacterial Phosphotriesterase

Biochemistry, 2010

Organophosphorus compounds include many synthetic, neurotoxic substances that are commonly used as insecticides. The toxicity of these compounds is due to their ability to inhibit the enzyme acetylcholine esterase. Some of the most toxic organophosphates have been adapted for use as chemical warfare agents; the most well-known are GA, GB, GD, GF, VX, and VR. All of these compounds contain a chiral phosphorus center, with the S P enantiomers being significantly more toxic than the R P enantiomers. Phosphotriesterase (PTE) is an enzyme capable of detoxifying these agents, but the stereochemical preference of the wild-type enzyme is for the R P enantiomers. A series of enantiomerically pure chiral nerve agent analogues containing the relevant phosphoryl centers found in GB, GD, GF, VX, and VR has been developed. Wild-type and mutant forms of PTE have been tested for their ability to hydrolyze this series of compounds. Mutant forms of PTE with significantly enhanced, as well as relaxed or reversed, stereoselectivity have been identified. A number of variants exhibited dramatically improved kinetic constants for the catalytic hydrolysis of the more toxic S P enantiomers. Improvements of up to 3 orders of magnitude relative to the value of the wild-type enzyme were observed. Some of these mutants were tested against racemic mixtures of GB and GD. The kinetic constants obtained with the chiral nerve agent analogues accurately predict the improved activity and stereoselectivity against the authentic nerve agents used in this study. (PTE). Human paraoxonase is capable of hydrolyzing GB and GD, but the overall catalytic activity is relatively low (4). The DFPase from Loligo vulgaris is able to hydrolyze GA, GB, GD, GF, and DFP (diisopropyl fluorophosphate). The value of k cat / K m for the hydrolysis of DFP is ∼1.3 Â 10 6 M -1 s -1 , but the catalytic activities for the hydrolysis of GB and GD are significantly lower (5, 6). Organophosphorus acid anhydrolase from Alteromonas sp. JD6.5 is capable of hydrolyzing a wide variety of organophosphorus compounds, including GB, GD, and GF but not VX . Phosphotriesterase was first isolated from soil microbes (8). The best substrate identified to date for this enzyme is the agricultural pesticide paraoxon, and the value of k cat /K m approaches the diffusion-controlled limit of ∼10 8 M -1 s -1 (9). The enzymatic reaction for the hydrolysis of paraoxon to p-nitrophenol and diethyl phosphate is shown in Scheme 2. The substrate specificity of PTE is quite broad, and this enzyme is capable of hydrolyzing GA, GB, GD, GF, VR, and VX (10). PTE is a homodimeric protein that contains a binuclear metal center embedded within a (β/R) 8 -barrel structure and is a member of the amidohydrolase superfamily (11). The native enzyme contains two Zn 2þ ions, and these metal ions can be substituted with Cd 2þ , Co 2þ , Ni 2þ , or Mn 2þ without a loss of catalytic activity (9). Previous investigations have identified three subsites within the active site of PTE that help to define the substrate specificity for this enzyme (12).

Purification and Properties of the Phosphotriesterase from Pseudomonas diminuta

Journal of Biological Chemistry, 1989

The phosphotriesterase produced from the opd cistron of Pseudomonas diminuta was purified 1500-fold to homogeneity using a combination of gel filtration, ion exchange, hydrophobic, and dye matrix chromatographic steps. This is the first organophosphate triesterase or organophosphofluoridate hydrolyzing enzyme to be purified to homogeneity. The enzyme is a monomeric, spherical protein having a molecular weight of 39,000. A single zinc atom is bound to the enzyme and is required for catalytic activity. Incubation with metal chelating compounds, o-phenanthroline, EDTA, or 2,6-pyridine dicarboxylate inactivate the enzyme. The kinetic rate constants, koa, and kcat/ K,, for the hydrolysis of paraoxon are 2100 s-' and 4 X 10' M" s-I , respectively. The enzyme is inhibited competitively by dithiothreitol, dithioerithritol, and 8mercaptoethanol. In addition to paraoxon the phosphotriesterase was found to hydrolyze the commonly used organophosphorus insecticides, dursban, parathion, coumaphos, diazinon, fensulfothion, methyl parathion, and cyanophos. An enzyme capable of hydrolyzing the acetylcholinesterase inhibitor diisopropylfluorophosphate (DFP)' was first reported by Mazur (1) in the same year the synthesis of DFP was published (2). DFPases have since been discovered in squid (3), bacteria (4), protozoa (5), fresh water clam (6), and many mammals (7). While none of these enzymes will catalyze the hydrolysis of the related acetylcholinesterase inhibitors parathion and paraoxon, several parathionases and paraoxonases have been described. Among other sources, these enzymes are found in mammals (8), insects (9), bacteria (lo), and fungi (11). Despite over 40 years of study, no organophosphate hydrolyzing enzyme2 has ever been purified to

The bioremediator glycerophosphodiesterase employs a non-processive mechanism for hydrolysis

Journal of Inorganic Biochemistry, 2010

Glycerophosphodiesterase (GpdQ) from Enterobacter aerogenes is a binuclear metallohydrolase that catalyzes the breakdown of a broad range of phosphate ester substrates, and it is of interest for its potential application in the destruction of organophosphate nerve agents and pesticides. The reaction mechanism of GpdQ has been proposed to involve a nucleophilic attack by a terminally bound hydroxide molecule. The hydroxide species bridging the two metal ions is suggested to activate the nucleophile, thus favoring a sequential rather than a processive mechanism of action. Here, the hydrolysis of the two ester bonds in the substrate bis(para-nitrophenyl) phosphate (bpNPP) is probed using 31 P NMR. The kinetic rates measured compare well with those determined spectrophotometrically. Furthermore, the data indicate that the diester bonds are cleaved in two separate (non-processive) reactions, indicating that only a single nucleophile (the terminal hydroxide molecule) is likely to be employed as a nucleophile for GpdQ.

Structure-Based Rational Design of a Phosphotriesterase

Applied and Environmental Microbiology, 2009

In silico substrate docking of both stereoisomers of the pesticide chlorfenvinphos (CVP) in the phosphotriesterase from Agrobacterium radiobacter identified two residues (F131 and W132) that prevent productive substrate binding and cause stereospecificity. A variant (W131H/F132A) was designed that exhibited ca. 480-fold and 8-fold increases in the rate of Z-CVP and E-CVP hydrolysis, respectively, eliminating stereospecificity.

Inhibition studies of purple acid phosphatases: implications for the catalytic mechanism

Journal of the Brazilian Chemical Society, 2006

Fosfatases ácidas púrpuras (PAP) pertencem à família das metalo-hidrolases binucleares que catalisam a hidrólise de um grande grupo de substratos fosfoésteres em pH ácido. Apesar dos seus sítios ativos serem estruturamente conservados as PAP apresentam versatilidade mecanística. Neste trabalho são investigados alguns aspectos do mecanismo catalítico de duas PAP, usando os inibidores vanadato e fluoreto como sondas. Enquanto as magnitudes das constantes de inibição das duas enzimas pelo vanadato são semelhantes, as enzimas diferem com relação ao modo de inibição; vanadato interage de forma não-competitiva com a PAP de porco (K i = 40 µmol L-1), porém inibe a PAP de feijão competitivamente (K i = 30 µmol L-1). De modo semelhante, o fluoreto também age como inibidor competitivo da PAP de feijão, independentemente do pH, enquanto que o fluoreto simplesmente interage com o complexo formado pela enzima de porco e seu substrato(PAPsubstrato) em pH baixo e inibe de forma não competitiva esta enzima em pH mais alto, independentemente da composição do íon metálico. Além disso, enquanto a inibição pelo fluoreto se dá através da interação lenta com a PAP de porco, ele se liga rapidamente ao sítio catalítico da enzima do feijão. Visto que se propõe que o vanadato e o fluoreto mimetizem o estado de transição e o nucleófilo, respectivamente, as diferenças observadas na cinética de inibição indicam sutis, porém distintas diferenças no mecanismo de reação destas enzimas. Purple acid phosphatases (PAPs) belong to the family of binuclear metallohydrolases and catalyse the hydrolysis of a large group of phosphoester substrates at acidic pH. Despite structural conservation in their active sites PAPs appear to display mechanistic versatility. Here, aspects of the catalytic mechanism of two PAPs are investigated using the inhibitors vanadate and fluoride as probes. While the magnitude of their vanadate inhibition constants are similar the two enzymes differ with respect to the mode of inhibition; vanadate interacts in a non-competitive fashion with pig PAP (K i = 40 μmol L-1) while it inhibits red kidney bean PAP competitively (K i = 30 μmol L-1). Similarly, fluoride also acts as a competitive inhibitor for red kidney bean PAP, independent of pH, while the inhibition of pig PAP by fluoride is uncompetitive at low pH and non-competitive at higher pH, independent of metal ion composition. Furthermore, while fluoride acts as a slow-binding inhibitor in pig PAP it binds rapidly to the catalytic site of the red kidney bean enzyme. Since vanadate and fluoride are proposed to act as transition state and nucleophile mimics, respectively, the observed differences in inhibition kinetics indicate subtle but distinct variations in the reaction mechanism of these enzymes.