203 research outputs found
Modeling the Alkaline Hydrolysis of Diaryl Sulfate Diesters: A Mechanistic Study
Phosphate and sulfate esters have important roles in regulating cellular processes. However, while there has been substantial experimental and computational investigation of the mechanisms and the transition states involved in phosphate ester hydrolysis, there is far less work on sulfate ester hydrolysis. Here, we report a detailed computational study of the alkaline hydrolysis of diaryl sulfate diesters, using different DFT functionals as well as mixed implicit/explicit solvation with varying numbers of explicit water molecules. We consider the impact of the computational model on computed linear free-energy relationships (LFER) and the nature of the transition states (TS) involved. We obtain good qualitative agreement with experimental LFER data when using a pure implicit solvent model and excellent agreement with experimental kinetic isotope effects for all models used. Our calculations suggest that sulfate diester hydrolysis proceeds through loose transition states, with minimal bond formation to the nucleophile and bond cleavage to the leaving group already initiated. Comparison to prior work indicates that these TS are similar in nature to those for the alkaline hydrolysis of neutral arylsulfonate monoesters or charged phosphate diesters and fluorophosphates. Obtaining more detailed insights into the transition states involved assists in understanding the selectivity of enzymes that hydrolyze these reactions
Transition-State Interactions in a Promiscuous Enzyme: Sulfate and Phosphate Monoester Hydrolysis by Pseudomonas aeruginosa Arylsulfatase.
Pseudomonas aeruginosa arylsulfatase (PAS) hydrolyzes sulfate and, promiscuously, phosphate monoesters. Enzyme-catalyzed sulfate transfer is crucial to a wide variety of biological processes, but detailed studies of the mechanistic contributions to its catalysis are lacking. We present linear free energy relationships (LFERs) and kinetic isotope effects (KIEs) of PAS and analyses of active site mutants that suggest a key role for leaving group (LG) stabilization. In LFERs PASWT has a much less negative Brønsted coefficient (βleaving groupobs-Enz = -0.33) than the uncatalyzed reaction (βleaving groupobs = -1.81). This situation is diminished when cationic active site groups are exchanged for alanine. The considerable degree of bond breaking during the transition state (TS) is evidenced by an 18Obridge KIE of 1.0088. LFER and KIE data for several active site mutants point to leaving group stabilization by active site K375, in cooperation with H211. 15N KIEs and the increased sensitivity to leaving group ability of the sulfatase activity in neat D2O (Δβleaving groupH-D = +0.06) suggest that the mechanism for S-Obridge bond fission shifts, with decreasing leaving group ability, from charge compensation via Lewis acid interactions toward direct proton donation. 18Ononbridge KIEs indicate that the TS for PAS-catalyzed sulfate monoester hydrolysis has a significantly more associative character compared to the uncatalyzed reaction, while PAS-catalyzed phosphate monoester hydrolysis does not show this shift. This difference in enzyme-catalyzed TSs appears to be the major factor favoring specificity toward sulfate over phosphate esters by this promiscuous hydrolase, since other features are either too similar (uncatalyzed TS) or inherently favor phosphate (charge).BBSRC BB/I004327/1
EPSRC EP/E019390/1
Transition State Analysis of the Reaction Catalyzed by the Phosphotriesterase from Sphingiobium sp. TCM1
Organophosphorus flame retardants are stable toxic compounds used in nearly all durable plastic products and are considered major emerging pollutants. The phosphotriesterase from Sphingobium sp. TCM1 (Sb-PTE) is one of the few enzymes known to be able to hydrolyze organophosphorus flame retardants such as triphenyl phosphate and tris(2-chloroethyl) phosphate. The effectiveness of Sb-PTE for the hydrolysis of these organophosphates appears to arise from its ability to hydrolyze unactivated alkyl and phenolic esters from the central phosphorus core. How Sb-PTE is able to catalyze the hydrolysis of the unactivated substituents is not known. To interrogate the catalytic hydrolysis mechanism of Sb-PTE, the pH dependence of the reaction and the effects of changing the solvent viscosity were determined. These experiments were complemented by measurement of the primary and secondary 18-oxygen isotope effects on substrate hydrolysis and a determination of the effects of changing the pKa of the leaving group on the magnitude of the rate constants for hydrolysis. Collectively, the results indicated that a single group must be ionized for nucleophilic attack and that a separate general acid is not involved in protonation of the leaving group. The Brønsted analysis and the heavy atom kinetic isotope effects are consistent with an early associative transition state with subsequent proton transfers not being rate limiting. A novel binding mode of the substrate to the binuclear metal center and a catalytic mechanism are proposed to explain the unusual ability of Sb-PTE to hydrolyze unactivated esters from a wide range of organophosphate substrates
Catalytic Mechanism for the Conversion of Salicylate Into Catechol by the Flavin-Dependent Monooxygenase Salicylate Hydroxylase
Salicylate hydroxylase (NahG) is a flavin-dependent monooxygenase that catalyzes the decarboxylative hydroxylation of salicylate into catechol in the naphthalene degradation pathway in Pseudomonas putida G7. We explored the mechanism of action of this enzyme in detail using a combination of structural and biophysical methods. NahG shares many structural and mechanistic features with other versatile flavin-dependent monooxygenases, with potential biocatalytic applications. The crystal structure at 2.0 Å resolution for the apo form of NahG adds a new snapshot preceding the FAD binding in flavin-dependent monooxygenases. The kcat/Km for the salicylate reaction catalyzed by the holo form is \u3e105 M−1 s−1 at pH 8.5 and 25 °C. Hammett plots for Km and kcat using substituted salicylates indicate change in rate-limiting step. Electron-donating groups favor the hydroxylation of salicylate by a peroxyflavin to yield a Wheland-like intermediate, whereas the decarboxylation of this intermediate is faster for electron-withdrawing groups. The mechanism is supported by structural data and kinetic studies at different pHs. The salicylate carboxyl group lies near a hydrophobic region that aids decarboxylation. A conserved histidine residue is proposed to assist the reaction by general base/general acid catalysis
Phosphoryl Transfer Reactions
Phosphoryl transfer is the name given to the chemical process of the transfer of the phosphoryl group (PO3) from a phosphate ester or anhydride to a nucleophile. Nucleophilic attack by water on a phosphate monoester gives the hydrolysis product inorganic phosphate. This net dephosphorylation reaction is the process catalysed by phosphatases. The formation of phosphate esters is termed phosphorylation, and is accomplished in biological systems by kinases
Mechanistic Studies on Enzyme-Catalyzed Phosphoryl Transfer
Phosphoryl transfer reactions have essential roles throughout biochemistry. The enzymes that catalyze these reactions result in tremendous rate enhancements for their normally unreactive substrates. This fact has led to great interest in the enzymatic mechanisms, and debate as to whether the mechanisms for enzyme-catalyzed hydrolysis of phosphate esters differ from those of uncatalyzed reactions. This review summarizes the uncatalyzed reactions of monoesters, diesters and triesters. A selection of enzymatic phosphoryl transfer reactions that have been the most studied and are the best understood are discussed, with examples of phosphatases, diesterases, and triesterases
The Transition State of the Phosphoryl Transfer Reaction Catalyzed by the Lambda Ser/Thr Protein Phosphatase
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