Transition-state structures for enzymic and alkaline phosphotriester hydrolysis

Caldwell, Steven R.; Raushel, Frank M.; Weiss, Paul M.; Cleland, W. W.

doi:10.1021/bi00244a011

Cited by 114 publications

(155 citation statements)

References 15 publications

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“…Protio and deuterio GDP-fucose solutions were subjected to the coupled assay solution in the absence of FucT V to eliminate the possibility that the change in emission at 460 nm was due to either a GDP contamination or a compound that absorbs at either 340 or 460 nm. The observed isotope effects were corrected for protio contamination in deuterated GDP-[1-2 H]Fuc as described in the literature (Caldwell et al, 1991, Hengge & Hess, 1994. -1,4-Galactosyltransferase has been shown to have a secondary isotope effect (D V ) 1.21, D V/K ) 1.05) (Kim et al, 1988) Studies of glycosidases have used the R-hydrogen isotope effect to show sp 2 hybidization in the reaction center (Dahlquist et al, 1969;Kempton & Withers, 1992;Rosenberg & Kirsch, 1981;Smith et al, 1973).…”

Section: Resultsmentioning

confidence: 99%

“…The background, nonenzymatic contribution of the GDP-Fuc solution was accounted for by determining the change in emission at 460 nm as a function of GDP-Fuc concentration. The observed isotope effects were corrected for protio contamination in deuterated GDP-[1-2 H]Fuc as described in the literature (Caldwell et al, 1991;Hengge & Hess, 1994). Radiolabel R-1,3-Fucosyltransferase V ActiVity Assay.…”

Section: Guanosine 5′-diphospho-2-deoxy-2-fluoro--l-fucose Monoammonmentioning

confidence: 99%

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Mechanism of Human α-1,3-Fucosyltransferase V: Glycosidic Cleavage Occurs Prior to Nucleophilic Attack

et al. 1997

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R-1,3-Fucosyltransferase V (FucT V) catalyzes the transfer of l-fucose from the donor sugar guanosine 5′-diphospho--l-fucose (GDP-Fuc) to an acceptor sugar. A secondary isotope effect on the fucosyltransfer reaction with guanosine 5′-diphospho-[1-2 H]--l-fucose (GDP-[1-2 H]-Fuc) as the substrate was observed and determined to be D V ) 1.32 ( 0.13 and D V/K ) 1.27 ( 0.07. Competitive inhibition of FucT V by guanosine 5′-diphospho-2-deoxy-2-fluoro--l-fucose (GDP-2F-Fuc) was observed with an inhibition constant of 4.2 µM which represents the most potent inhibitor of this enzyme to date. Incubation of GDP-2F-Fuc with FucT V and an acceptor molecule prior to the addition of GDP-Fuc had no effect on the potency of inhibition, indicating that GDP-2F-Fuc is neither an inactivator nor a slow substrate. Both the observed secondary isotope effect and the inhibition by GDP-2F-Fuc are consistent with a charged, sp 2 -hybridized, transition-state structure. A convenient and efficient synthesis of GDP-[1-2 H]-Fuc and GDP-2F-Fuc and a nonradioactive, fluorescence assay for fucosyltransferase activity have been developed.The R-1,3-fucosylated oligosaccharide structures are central to numerous cell-cell interactions (Ichikawa et al., 1994) such as inflammation, tumor development, and blood clotting (Foxall et al., 1992;Parekh & Edge, 1994). Five distinct human R-1,3-fucosyltransferases have been cloned (KukowskaLatallo et al., 1990;Lowe et al., 1991;McCurley et al., 1995;Reguigne-Arnould et al., 1995;Sasaki et al., 1994; Weston et al., 1992a,b) and shown to have different acceptor sugar specificity. R-1,3-Fucosyltransferase V (FucT V) 1 is responsible for the terminal step in the biosynthesis of Lewis x (Le x ) and sialyl Lewis x (sLe x ) (Figure 1), a tetrasaccharide ligand involved in inflammatory cell adhesion and metastasis (Lasky, 1992;Muramatsu, 1993). Thus inhibition of this enzyme may impede inflammation or cancer progression, and understanding the mechanism of FucT V may lead to the development of effective inhibitors.The R-1,3-fucosyltransferase V-catalyzed reaction proceeds with inversion of configuration at the anomeric center of l-fucose (Weston et al., 1992a). Product inhibition studies have been used to establish that FucT V has an ordered, sequential, bi-bi mechanism with guanosine 5′-diphospho--l-fucose (GDP-Fuc) binding first and the product GDP releasing last (Qiao et al., 1996). FucT V has been shown to have a catalytic residue with pK a ) 4.1, presumably an active-site carboxylate residue . A solvent isotope effect was observed (D V ) 2.9, D V/K ) 2.1) and exploited in a proton inventory study to show that there is a one-proton transfer in the transition state . The transition-state structure of glycosyltransferasecatalyzed reactions has been proposed to have a flattened half-chair conformation with substantial oxocarbenium ion character at the anomeric position (Kim et al., 1988;Murray et al., 1996), analogous to that of the glycosidase reactions (Look et al., 1993;Sinnott, 1990). Consistent with...

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Section: Resultsmentioning

confidence: 99%

Section: Guanosine 5′-diphospho-2-deoxy-2-fluoro--l-fucose Monoammonmentioning

confidence: 99%

Mechanism of Human α-1,3-Fucosyltransferase V: Glycosidic Cleavage Occurs Prior to Nucleophilic Attack

et al. 1997

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“…PTEs have a number of attributes that make them interesting in this context. First, the enzymes house a highly reactive binuclear metal ion center that is involved in a relatively simple reaction: namely, a one-step S N 2 displacement reaction for the hydrolysis of the P-O bond of the phosphotriester paraoxon that does not involve any covalent enzyme-substrate intermediates (16)(17)(18). This makes PTE a less complicated system than other enzymes in which conformational changes are involved in multistep reactions and/or reactions involving cofactors.…”

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confidence: 99%

“…A second, sometimes overlooked, requirement of a catalyst is that it is not consumed during the reaction, i.e., it must turn over multiple reactions. With reported rate enhancements of up to 10 17 (1) and turnover rates reaching 10 4 s Ϫ1 (2), enzymes are truly extraordinary catalysts. In addition to efficient rate enhancement of the chemical reaction, enzymes are required to maintain fast rates for formation of the Michaelis complex and product release.…”

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confidence: 99%

Conformational sampling, catalysis, and evolution of the bacterial phosphotriesterase

Jackson

Foo

Tokuriki

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

114

134

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To efficiently catalyze a chemical reaction, enzymes are required to maintain fast rates for formation of the Michaelis complex, the chemical reaction and product release. These distinct demands could be satisfied via fluctuation between different conformational substates (CSs) with unique configurations and catalytic properties. However, there is debate as to how these rapid conformational changes, or dynamics, exactly affect catalysis. As a model system, we have studied bacterial phosphotriesterase (PTE), which catalyzes the hydrolysis of the pesticide paraoxon at rates limited by a physical barrier-either substrate diffusion or conformational change. The mechanism of paraoxon hydrolysis is understood in detail and is based on a single, dominant, enzyme conformation. However, the other aspects of substrate turnover (substrate binding and product release), although possibly rate-limiting, have received relatively little attention. This work identifies ''open'' and ''closed'' CSs in PTE and dominant structural transition in the enzyme that links them. The closed state is optimally preorganized for paraoxon hydrolysis, but seems to block access to/from the active site. In contrast, the open CS enables access to the active site but is poorly organized for hydrolysis. Analysis of the structural and kinetic effects of mutations distant from the active site suggests that remote mutations affect the turnover rate by altering the conformational landscape.dynamics ͉ enzyme catalysis ͉ evolution ͉ conformational fluctuation A catalyst is defined as a molecule that increases the rate of a chemical reaction by providing an alternative pathway of lower activation energy. A second, sometimes overlooked, requirement of a catalyst is that it is not consumed during the reaction, i.e., it must turn over multiple reactions. With reported rate enhancements of up to 10 17 (1) and turnover rates reaching 10 4 s Ϫ1 (2), enzymes are truly extraordinary catalysts. In addition to efficient rate enhancement of the chemical reaction, enzymes are required to maintain fast rates for formation of the Michaelis complex and product release. So, how does a single protein sequence satisfy these different demands?Our understanding of the mechanisms by which the active sites of enzymes lower the activation energy for various chemical reactions has developed steadily in the past decades and it is clear that the specific organization of chemical groups within the active sites of enzymes provides an electrostatic environment that is markedly different to the conditions in which the uncatalyzed reaction occurs, and results in remarkable rate enhancements (3). There have also been a number of studies showing that transitions between conformational substates (CSs) (4, 5) on a variable energy landscape (6, 7) are an integral part of the full catalytic cycles, or substrate turnover, in many enzymes (7-12). Despite much progress, there is some debate surrounding the exact role that such transitions, or dynamics, play in enzymatic catalysis (7, 13). The deb...

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“…The native enzyme contains two Zn 2ϩ ions, but these metal ions can be replaced with Co 2ϩ , Ni 2ϩ, Mn 2ϩ , or Cd 2ϩ with retention of full catalytic activity. From chemical, kinetic, and genetic studies, it has been demonstrated that the reaction proceeds via an S n 2-like associative mechanism in which a metal-bound hydroxide ion attacks the electrophilic phosphorus center of the substrate (2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16). The role of one of the two metal ions within the active site is thought to involve the activation of the hydrolytic water molecule, whereas the companion metal ion is most likely involved in the polarization of the phosphoryl oxygen bond of the substrate to increase the electrophilicity of the substrate for nucleophilic attack (16).…”

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confidence: 99%

Hydrolysis of Phosphodiesters through Transformation of the Bacterial Phosphotriesterase

Shim

Hong

Raushel

1998

Journal of Biological Chemistry

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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 >10 8 times faster than the uncatalyzed reaction (k cat ‫؍‬ 0.06 s ؊1 and K m ‫؍‬ 38 mM). Upon the addition of various alkylamines to the reaction mixture, the k 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 wildtype 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.The bacterial phosphotriesterase from Pseudomonas diminuta catalyzes the hydrolysis of a wide range of organophosphate nerve agents with high efficiency (1, 2). Paraoxon, the best characterized substrate for the zinc-substituted phosphotriesterase, is hydrolyzed with a k cat /K m of 4 ϫ 10and a k cat of 2100 s Ϫ1 . The active site of this enzyme contains a coupled binuclear metal center, which is absolutely essential for catalytic activity (1). The native enzyme contains two Zn 2ϩ ions, but these metal ions can be replaced with Co 2ϩ , Ni 2ϩ, Mn 2ϩ , or Cd 2ϩ with retention of full catalytic activity. From chemical, kinetic, and genetic studies, it has been demonstrated that the reaction proceeds via an S n 2-like associative mechanism in which a metal-bound hydroxide ion attacks the electrophilic phosphorus center of the substrate (2-16). The role of one of the two metal ions within the active site is thought to involve the activation of the hydrolytic water molecule, whereas the companion metal ion is most likely involved in the polarization of the phosphoryl oxygen bond of the substrate to increase the electrophilicity of the substrate for nucleophilic attack (16). Not surprisingly, the substrate-binding site pocket consists predominantly of ...

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Transition-state structures for enzymic and alkaline phosphotriester hydrolysis

Cited by 114 publications

References 15 publications

Mechanism of Human α-1,3-Fucosyltransferase V: Glycosidic Cleavage Occurs Prior to Nucleophilic Attack

Mechanism of Human α-1,3-Fucosyltransferase V: Glycosidic Cleavage Occurs Prior to Nucleophilic Attack

Conformational sampling, catalysis, and evolution of the bacterial phosphotriesterase

Hydrolysis of Phosphodiesters through Transformation of the Bacterial Phosphotriesterase

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