Nucleophilic Attack Of Water Molecule Research Articles

Direct Synthesis of Artificial Esterase through Molecular Imprinting Using a Substrate-Mimicking Acylthiourea Template.

Most reported artificial esterases hydrolyze only activated esters. We here report a one-pot synthesis of artificial esterases via molecular imprinting. An acylthiourea template hydrogen bonds with 4-vinylbenzoic acid and coordinates to a polymerizable zinc complex inside a cross-linkable surfactant micelle. Double cross-linking of the micelle yields a polymeric nanoparticle catalyst that mimics a metalloenzyme to activate a water molecule for nucleophilic attack on the bound ester. The catalyst hydrolyzes both activated and unactivated esters under mild conditions with selectivity.

Regulating Ru−O Bonding Interactions by Ir Doping Boosts the Acid Oxygen Evolution Performance

AbstractHighly active and stable oxygen evolution reaction (OER) catalysts are crucial for the large‐scale application of proton exchange membrane water electrolyzers. However, the dynamic reconfiguration of the catalyst surface structure and active centers is still undefined, which greatly hinders the development and application of efficient OER catalysts. Herein, we report an Ir0.3Ru0.7Ox/C catalyst with a facile low‐temperature synthesis route, which can reach 10 mA cm−2 at an overpotential of 217 mV with a Tafel slope as low as 39.4 mV dec−1, and yields a mass activity 61 times that of commercial IrO2/C at an overpotential of 300 mV. The lattice oxygen structure of RuOx is stabilized by the introduction of Ir species, thus greatly promoting the OER activity and durability. Further in situ Raman reveals that RuOx emerges as the active species at high potentials, and Ru−O bonding interactions are enhanced with Ir regulation, stabilizing the solvation of Ru at high potentials and accelerating the nucleophilic attack of water molecules, leading to the improved OER performance. This work deepens the fundamental understanding of OER and offers an effective way to advance the utilization of Ru‐based OER catalysts.

High-valence metal-doped amorphous IrOx as active and stable electrocatalyst for acidic oxygen evolution reaction

Proton-exchange membrane water electrolysis, particularly driven by renewable electricity, is a sustainable strategy for green hydrogen production. However, developing highly active and stable electrocatalysts to accelerate the oxygen evolution reaction (OER) remains challenging. Herein, we introduce an effective strategy for constructing high-valence metal-doped IrOx with abundant oxygen vacancies, while simultaneously enhancing the catalytic activity and stability of the acidic OER. The synthesized Ta-doped IrOx (350-Ta@IrOx) exhibits ultramicroscopic nanoparticle morphology and abundant surface oxygen vacancies</a>, enabling a rapid OER process with a low overpotential of 223 mV at 10 mA cm-2 and 147.7 times higher mass activity (1207.4 A gIr−1) than that of commercial IrO2 at 1.55 V</a> versus the reversible hydrogen electrode. More importantly, 350-Ta@IrOx affords excellent stability with insignificant potential degradation after 500 h of electrolysis at 10 mA cm2, originating from the low operating potential and suppressed dissolution and oxidation of oxygen vacancy active sites via Ta doping. Density functional theory calculations suggest that Ta doping and oxygen defect engineering are effective in facilitating the nucleophilic attack of water molecules, thereby accelerating the rate-determining step toward high catalytic OER activity on Ta-doped IrOx. We anticipate that this study will provide an effective method to obtain active and stable electrocatalysts via high-valence metal doping.

Expanding the β-substitution reactions of serine synthase through mutagenesis of aromatic active site residues

The Gram-negative bacterium, Fusobacterium nucleatum, possesses a fold II type pyridoxal 5′-phosphate-dependent enzyme that catalyzes the reversible β-replacement of l-cysteine and l-serine, generating H2S and H2O, respectively. This enzyme, termed serine synthase (FN1055), contains an active site Asp232 that serves as a general base in the activation of a water molecule for nucleophilic attack of the ⍺-aminoacrylate intermediate. A network of hydrophobic residues surrounding Asp232 are key to catalysis as they increase the basicity of the side chain. However, these residues severely restrict the range of nucleophilic substrates that can react with the ⍺-aminoacrylate, making the enzyme an ineffective biocatalyst for noncanonical amino acid biosynthesis. Herein, we systematically substituted four aromatic active residues (Trp99, Phe125, Phe148 and Phe234) to an alanine to determine their catalytic importance in serine/cysteine synthase reactions and if their substitution could broaden the scope of nucleophiles that could react with the ⍺-aminoacrylate intermediate. All four single site mutants W99A, F125A, F148A, and F234A could form the ⍺-aminoacrylate intermediate upon reaction with either l-cysteine or l-serine; however, the rate constant associated with the elimination of the β-hydroxyl group from l-serine was 150 to 200-fold lower in the F125A and F148A variants. Substitution of Phe125 and Phe148, situated ∼3–4 Å from the general base, also abolished the serine synthase reaction due to their inability to activate a water molecule for nucleophilic attack of the ⍺-aminoacrylate. Overall, the mutational studies indicate that the clustering of aromatic residues disproportionately benefits the serine synthase reaction as they increase the binding affinity for l-cysteine, decrease the binding of the product, l-serine, and promote the activation of a water molecule. Notably, the aminoacrylate species present in F125A and F148A was able to react with thiophenol, signifying that serine synthase has biocatalytic potential in the synthesis of noncanonical amino acids.

Catalytic deAMPylation in AMPylation-inhibitory/assistant forms of FICD protein.

DeAMPylation, as a reversible reaction of AMPylation and mediated by the endoplasmic reticulum-localized enzyme FICD (filamentation induced by cAMP domain protein, also known as HYPE), is an important process in protein posttranslational modifications (PTMs). Elucidating the function and catalytic details of FICD is of vital importance to provide a comprehensive understanding of protein folding homeostasis. However, the detailed deAMPylation mechanism is still unclear. Furthermore, the role of a conserved glutamine (Glu234), that plays an inhibitory role in the AMPylation response, is still an open question in the deAMPylation process. In the present work, the elaborated deAMPylation mechanisms with AMPylation-inhibitory/assistant forms of FICD (wild type and Glu234Ala mutant) were investigated based on the QM(DFT)/MM MD approach. The results revealed that deAMPylation was triggered by proton transfer from protonated histidine (His363) to AMPylated threonine, instead of a nucleophilic attack of water molecules adding to the phosphorus of AMP. The free energy barrier of deAMPylation in the wild type (∼17.3kcal/mol) is consistent with that in the Glu234Ala mutant of FICD (∼17.1kcal/mol), suggesting that the alteration of the Glu234 residue does not affect the deAMPylation reaction and indirectly verifying the inducement of deAMPylation in FICD. In the wild type, the proton in the nucleophilic water molecule is transferred to Glu234, whereas it is delivered to Asp367 through the hydrogen-bond network of coordinated water molecules in the Glu234Ala mutant. The present findings were inspirational for understanding the catalytic and inhibitory mechanisms of FICD-mediated AMP transfer, paving the way for further studies on the physiological role of FICD protein.

Theoretical Analysis of the Catalytic Hydrolysis Mechanism of HCN over Cu-ZSM-5

HCN catalytic hydrolysis mechanism over Cu-ZSM-5 was investigated based on the density functional theory (DFT) with 6-31++g (d, p) basis set. Five paths (A, B, C, D, and E) were designed. For path A and path B, the first step is the nucleophilic attack of water molecule. Next, the hydrogen atom of H2O is transferred to the nitrogen atom first for path A, while in path B, the hydrogen atom of the HCN is first transferred to the nitrogen atom. In path C, HCN isomerizes to HNC initially, and the remaining steps are similar to that of path A. The H atom of HCN shifts to Cu-ZSM-5 initially in path D, and the H atom is transferred to N atom subsequently. The last step is the attack on water molecule. The first step for path E is similar to that of path D. The next step is the attack on water molecule, in which the H atom of water molecule shifts to N atom, and the H on Cu-ZSM-5 shifts to the N atom. Meanwhile, the H atom of oxygen atom is transferred to the N atom. The results show that path C is the most favorable path, with the lowest free energy barrier (35.45 kcal/mol). The results indicate that the Cu-ZSM-5 strongly reduces the energy barrier of HCN and isomerizes to HNC, making it an effective catalyst for HCN hydrolysis.

Activation by oxidation and ligand exchange in a molecular manganese vanadium oxide water oxidation catalyst.

Despite their technological importance for water splitting, the reaction mechanisms of most water oxidation catalysts (WOCs) are poorly understood. This paper combines theoretical and experimental methods to reveal mechanistic insights into the reactivity of the highly active molecular manganese vanadium oxide WOC [Mn4V4O17(OAc)3]3− in aqueous acetonitrile solutions. Using density functional theory together with electrochemistry and IR-spectroscopy, we propose a sequential three-step activation mechanism including a one-electron oxidation of the catalyst from [Mn23+Mn24+] to [Mn3+Mn34+], acetate-to-water ligand exchange, and a second one-electron oxidation from [Mn3+Mn34+] to [Mn44+]. Analysis of several plausible ligand exchange pathways shows that nucleophilic attack of water molecules along the Jahn–Teller axis of the Mn3+ centers leads to significantly lower activation barriers compared with attack at Mn4+ centers. Deprotonation of one water ligand by the leaving acetate group leads to the formation of the activated species [Mn4V4O17(OAc)2(H2O)(OH)]− featuring one H2O and one OH ligand. Redox potentials based on the computed intermediates are in excellent agreement with electrochemical measurements at various solvent compositions. This intricate interplay between redox chemistry and ligand exchange controls the formation of the catalytically active species. These results provide key reactivity information essential to further study bio-inspired molecular WOCs and solid-state manganese oxide catalysts.

GTP Hydrolysis Without an Active Site Base: A Unifying Mechanism for Ras and Related GTPases.

GTP hydrolysis is a biologically crucial reaction, being involved in regulating almost all cellular processes. As a result, the enzymes that catalyze this reaction are among the most important drug targets. Despite their vital importance and decades of substantial research effort, the fundamental mechanism of enzyme-catalyzed GTP hydrolysis by GTPases remains highly controversial. Specifically, how do these regulatory proteins hydrolyze GTP without an obvious general base in the active site to activate the water molecule for nucleophilic attack? To answer this question, we perform empirical valence bond simulations of GTPase-catalyzed GTP hydrolysis, comparing solvent- and substrate-assisted pathways in three distinct GTPases, Ras, Rab, and the Gαi subunit of a heterotrimeric G-protein, both in the presence and in the absence of the corresponding GTPase activating proteins. Our results demonstrate that a general base is not needed in the active site, as the preferred mechanism for GTP hydrolysis is a conserved solvent-assisted pathway. This pathway involves the rate-limiting nucleophilic attack of a water molecule, leading to a short-lived intermediate that tautomerizes to form H2PO4- and GDP as the final products. Our fundamental biochemical insight into the enzymatic regulation of GTP hydrolysis not only resolves a decades-old mechanistic controversy but also has high relevance for drug discovery efforts. That is, revisiting the role of oncogenic mutants with respect to our mechanistic findings would pave the way for a new starting point to discover drugs for (so far) "undruggable" GTPases like Ras.

Structure and Reaction Mechanism of the LigJ Hydratase: An Enzyme Critical for the Bacterial Degradation of Lignin in the Protocatechuate 4,5-Cleavage Pathway.

LigJ from the soil bacterium Sphingobium sp. SYK-6 catalyzes the reversible hydration of (3 Z)-2-keto-4-carboxy-3-hexenedioate (KCH) to 4-carboxy-4-hydroxy-2-oxoadipate (CHA) in the degradation of lignin in the protocatechuate 4,5-cleavage pathway. LigJ is a member of the amidohydrolase superfamily and an enzyme in cog2159. The three-dimensional crystal structure of wild-type LigJ was determined in the presence [Protein Data Bank (PDB) entry 6DXQ ] and absence of the product CHA (PDB entry 6DWV ). The protein folds as a distorted (β/α)8-barrel, and a single zinc ion is bound in the active site at the C-terminal end of the central β-barrel. The product CHA is ligated to the zinc ion in the active site via the displacement of a single water molecule from the coordination shell of the metal center in LigJ. The product-bound structure reveals that the enzyme catalyzes the hydration of KCH with the formation of a chiral center at C4 with S stereochemistry. The E284Q mutant was unable to catalyze the hydration of KCH to CHA, and the structure of this mutant was determined in the presence of the substrate KCH (PDB entry 6DXS ). On the basis of the structure of LigJ in the presence of KCH and CHA, it is proposed that the side chain carboxylate of Glu-284 functions as a general base in the abstraction of a proton from a bound water molecule for nucleophilic attack at C4 of the substrate. The reaction is facilitated by the delocalization of the negative charge to the metal center via the carbonyl group at C2 of the substrate. C3 of the substrate is subsequently protonated by Glu-284 functioning as a general acid. The overall reaction occurs by the syn addition of water to the double bond between C4 and C3 of the substrate KCH. The kinetic constants for the hydration of KCH to CHA by LigJ at pH 8.0 are 25 s-1 ( kcat) and 2.6 × 106 M-1 s-1 ( kcat/ Km).

A Rational Active-Site Redesign Converts a Decarboxylase into a C=C Hydratase: "Tethered Acetate" Supports Enantioselective Hydration of 4-Hydroxystyrenes.

The promiscuous regio- and stereoselective hydration of 4-hydroxystyrenes catalyzed by ferulic acid decarboxylase from Enterobacter sp. (FDC_Es) depends on bicarbonate bound in the active site, which serves as a proton relay activating a water molecule for nucleophilic attack on a quinone methide electrophile. This “cofactor” is crucial for achieving improved conversions and high stereoselectivities for (S)-configured benzylic alcohol products. Similar effects were observed with simple aliphatic carboxylic acids as additives. A rational redesign of the active site by replacing the bicarbonate or acetate “cofactor” with a newly introduced side-chain carboxylate from an adjacent amino acid yielded mutants that efficiently acted as C=C hydratases. A single-point mutation of valine 46 to glutamate or aspartate improved the hydration activity by 40% and boosted the stereoselectivity 39-fold in the absence of bicarbonate or acetate.

Human acid sphingomyelinase structures provide insight to molecular basis of Niemann-Pick disease.

Acid sphingomyelinase (ASM) hydrolyzes sphingomyelin to ceramide and phosphocholine, essential components of myelin in neurons. Genetic alterations in ASM lead to ASM deficiency (ASMD) and have been linked to Niemann–Pick disease types A and B. Olipudase alfa, a recombinant form of human ASM, is being developed as enzyme replacement therapy to treat the non-neurological manifestations of ASMD. Here we present the human ASM holoenzyme and product bound structures encompassing all of the functional domains. The catalytic domain has a metallophosphatase fold, and two zinc ions and one reaction product phosphocholine are identified in a histidine-rich active site. The structures reveal the underlying catalytic mechanism, in which two zinc ions activate a water molecule for nucleophilic attack of the phosphodiester bond. Docking of sphingomyelin provides a model that allows insight into the selectivity of the enzyme and how the ASM domains collaborate to complete hydrolysis. Mapping of known mutations provides a basic understanding on correlations between enzyme dysfunction and phenotypes observed in ASMD patients.

Density functional theory calculations on oxidative C-C bond cleavage and N-O bond formation of [Ru(II)(bpy)2(diamine)](2+) via reactive ruthenium imide intermediates.

DFT calculations are performed on [Ru(II)(bpy)2(tmen)](2+) (M1, tmen = 2,3-dimethyl-2,3-butanediamine) and [Ru(II)(bpy)2(heda)](2+) (M2, head = 2,5-dimethyl-2,5-hexanediamine), and on the oxidation reactions of M1 to give the C-C bond cleavage product [Ru(II)(bpy)2(NH=CMe2)2](2+) (M3) and the N-O bond formation product [Ru(II)(bpy)2(ONCMe2CMe2NO)](2+) (M4). The calculated geometrical parameters and oxidation potentials are in good agreement with the experimental data. As revealed by the DFT calculations, [Ru(II)(bpy)2(tmen)](2+) (M1) can undergo oxidative deprotonation to generate Ru-bis(imide) [Ru(bpy)2(tmen-4 H)](+) (A) or Ru-imide/amide [Ru(bpy)2(tmen-3 H)](2+) (A') intermediates. Both A and A' are prone to C-C bond cleavage, with low reaction barriers (ΔG(≠)) of 6.8 and 2.9 kcal mol(-1) for their doublet spin states (2)A and (2)A', respectively. The calculated reaction barrier for the nucleophilic attack of water molecules on (2)A' is relatively high (14.2 kcal mol(-1)). These calculation results are in agreement with the formation of the Ru(II)-bis(imine) complex M3 from the electrochemical oxidation of M1 in aqueous solution. The oxidation of M1 with Ce(IV) in aqueous solution to afford the Ru(II)-dinitrosoalkane complex M4 is proposed to proceed by attack of the cerium oxidant on the ruthenium imide intermediate. The findings of ESI-MS experiments are consistent with the generation of a ruthenium imide intermediate in the course of the oxidation.

Hydrolysis of DFP and the Nerve Agent (S)-Sarin by DFPase Proceeds along Two Different Reaction Pathways: Implications for Engineering Bioscavengers

Organophosphorus (OP) nerve agents such as (S)-sarin are among the most highly toxic compounds that have been synthesized. Engineering enzymes that catalyze the hydrolysis of nerve agents (“bioscavengers”) is an emerging prophylactic approach to diminish their toxic effects. Although its native function is not known, diisopropyl fluorophosphatase (DFPase) from Loligo vulgaris catalyzes the hydrolysis of OP compounds. Here, we investigate the mechanisms of diisopropylfluorophosphate (DFP) and (S)-sarin hydrolysis by DFPase with quantum mechanical/molecular mechanical umbrella sampling simulations. We find that the mechanism for hydrolysis of DFP involves nucleophilic attack by Asp229 on phosphorus to form a pentavalent intermediate. P–F bond dissociation then yields a phosphoacyl enzyme intermediate in the rate-limiting step. The simulations suggest that a water molecule, coordinated to the catalytic Ca2+, donates a proton to Asp121 and then attacks the tetrahedral phosphoacyl intermediate to liberate the diisopropylphosphate product. In contrast, the calculated free energy barrier for hydrolysis of (S)-sarin by the same mechanism is highly unfavorable, primarily because of the instability of the pentavalent phosphoenzyme species. Instead, simulations suggest that hydrolysis of (S)-sarin proceeds by a mechanism in which Asp229 could activate an intervening water molecule for nucleophilic attack on the substrate. These findings may lead to improved strategies for engineering DFPase and related six-bladed β-propeller folds for more efficient degradation of OP compounds.

The Mechanism of Cellulose Hydrolysis by a Two-Step, Retaining Cellobiohydrolase Elucidated by Structural and Transition Path Sampling Studies

Glycoside hydrolases (GHs) cleave glycosidic linkages in carbohydrates, typically via inverting or retaining mechanisms, the latter of which proceeds via a two-step mechanism that includes formation of a glycosyl-enzyme intermediate. We present two new structures of the catalytic domain of Hypocrea jecorina GH Family 7 cellobiohydrolase Cel7A, namely a Michaelis complex with a full cellononaose ligand and a glycosyl-enzyme intermediate, that reveal details of the 'static' reaction coordinate. We also employ transition path sampling to determine the 'dynamic' reaction coordinate for the catalytic cycle. The glycosylation reaction coordinate contains components of forming and breaking bonds and a conformational change in the nucleophile. Deglycosylation proceeds via a product-assisted mechanism wherein the glycosylation product, cellobiose, positions a water molecule for nucleophilic attack on the anomeric carbon of the glycosyl-enzyme intermediate. In concert with previous structures, the present results reveal the complete hydrolytic reaction coordinate for this naturally and industrially important enzyme family.

Reaction Pathways for Oxygen Evolution Promoted by Cobalt Catalyst

The in-depth understanding of the molecular mechanisms regulating the water oxidation catalysis is of key relevance for the rationalization and the design of efficient oxygen evolution catalysts based on earth-abundant transition metals. Performing ab initio DFT+U molecular dynamics calculations of cluster models in explicit water solution, we provide insight into the pathways for oxygen evolution of a cobalt-based catalyst (CoCat). The fast motion of protons at the CoCat/water interface and the occurrence of cubane-like Co-oxo units at the catalyst boundaries are the keys to unlock the fast formation of O-O bonds. Along the resulting pathways, we identified the formation of Co(IV)-oxyl species as the driving ingredient for the activation of the catalytic mechanism, followed by their geminal coupling with O atoms coordinated by the same Co. Concurrent nucleophilic attack of water molecules coming directly from the water solution is discouraged by high activation barriers. The achieved results suggest also interesting similarities between the CoCat and the Mn4Ca-oxo oxygen evolving complex of photosystem II.

Directed Evolution of a Thermostable Quorum-quenching Lactonase from the Amidohydrolase Superfamily

A thermostable quorum-quenching lactonase from Geobacillus kaustophilus HTA426 (GI: 56420041) was used as an initial template for in vitro directed evolution experiments. This enzyme belongs to the phosphotriesterase-like lactonase (PLL) group of enzymes within the amidohydrolase superfamily that hydrolyze N-acylhomoserine lactones (AHLs) that are involved in virulence pathways of quorum-sensing pathogenic bacteria. Here we have determined the N-butyryl-L-homoserine lactone-liganded structure of the catalytically inactive D266N mutant of this enzyme to a resolution of 1.6 Å. Using a tunable, bioluminescence-based quorum-quenching molecular circuit, the catalytic efficiency was enhanced, and the AHL substrate range increased through two point mutations on the loops at the C-terminal ends of the third and seventh β-strands. This E101N/R230I mutant had an increased value of k(cat)/K(m) of 72-fold toward 3-oxo-N-dodecanoyl-L-homoserine lactone. The evolved mutant also exhibited lactonase activity toward N-butyryl-L-homoserine lactone, an AHL that was previously not hydrolyzed by the wild-type enzyme. Both the purified wild-type and mutant enzymes contain a mixture of zinc and iron and are colored purple and brown, respectively, at high concentrations. The origin of this coloration is suggested to be because of a charge transfer complex involving the β-cation and Tyr-99 within the enzyme active site. Modulation of the charge transfer complex alters the lactonase activity of the mutant enzymes and is reflected in enzyme coloration changes. We attribute the observed enhancement in catalytic reactivity of the evolved enzyme to favorable modulations of the active site architecture toward productive geometries required for chemical catalysis.

A facile and rapid synthesis of unsubstituted polythiophene with high electrical conductivity using binary organic solvents

Dichloromethane and acetonitrile containing thiophene and oxidant, respectively, were adopted as a binary organic solvent for oxidative synthesis of unsubstituted polythiophene (UPTh) under various conditions, i.e., oxidant/monomer molar ratio, polymerization temperature, and reaction time. We also investigated the effects of polymerization medium on the chemical and physical properties of the UPTh. The electrical conductivities of the UPThs polymerized in only acetonitrile or aqueous medium were 1.0 × 10−4 Scm−1 and zero, respectively. However, the UPTh obtained in binary organic solvent system exhibited the highest electrical conductivity of 20.1 Scm−1 without post-doping process under an oxidant/monomer molar ratio of 5:1 and a reaction time of 0.2 h at 0 °C. Furthermore, it showed an enhanced crystalline structure, thermal stability, and longer π-conjugation length than those in acetonitrile or aqueous medium. We also found that the presence of water as a polymerization medium inevitably gave rise to the incorporation of hydroxyl and carbonyl groups into the UPTh main chains by nucleophilic attack of water molecules. Therefore, these undesired structures broke the conjugated structures, resulting in UPTh with an electrically non-conducting property. The morphology of UPTh prepared in binary organic solvent appeared to be an aggregate of nanoparticles (50–200 nm). The combination of organic solvents should provide another systematic approach to the facile and rapid synthesis of UPThs with high conductivity.

Studies on the Catalytic Mechanism of a Glutamic Peptidase

Scytalidoglutamic peptidase (SGP) is the prototype of fungal glutamic peptidases that are characteristically pepstatin insensitive. These enzymes have a unique catalytic dyad comprised of Gln(53) and Glu(136) that activate a bound water molecule for nucleophilic attack on the carbonyl carbon atom of the scissile peptide bond. The hydrolysis by SGP at peptide bonds with proline in the P(1)' position is a rare event among peptidases that we investigated using the series of fluorescence resonance energy transfer peptides, Abz-KLXPSKQ-EDDnp, compared with the series Abz-KLXSSKQ-EDDnp. The preference observed in these two series for Phe and His over Leu, Ile, Val, Arg, and Lys, seems to be related to the structure of the S(1) subsite of SGP. These results and the pH profiles of SGP activity showed that its S(1) subsite can accommodate the benzyl group of Phe at pH 4 as well as the positively charged imidazolium group of His. In the pH range 2 to 7, SGP maintains its structure and activity, but at pH 8 or higher it is irreversibly denatured. The intrinsic fluorescence of the Trp residues of SGP were sensitive to the titration of carboxyl groups having low pK values; this can be attributed to the buried Asp(57) and/or Asp(43) as described in SGP three-dimensional structure. The solvent kinetic isotope effects and the proton inventory experiments support a mechanism for the glutamic peptidase SGP that involves the nucleophilic attack of the general base (Glu(136)) activated water, and establish a fundamental role of the S(1) subsite interactions in promoting catalysis.

Allosteric modulation of Ras positions Q61 for a direct role in catalysis

Ras and its effector Raf are key mediators of the Ras/Raf/MEK/ERK signal transduction pathway. Mutants of residue Q61 impair the GTPase activity of Ras and are found prominently in human cancers. Yet the mechanism through which Q61 contributes to catalysis has been elusive. It is thought to position the catalytic water molecule for nucleophilic attack on the gamma-phosphate of GTP. However, we previously solved the structure of Ras from crystals with symmetry of the space group R32 in which switch II is disordered and found that the catalytic water molecule is present. Here we present a structure of wild-type Ras with calcium acetate from the crystallization mother liquor bound at a site remote from the active site and likely near the membrane. This results in a shift in helix 3/loop 7 and a network of H-bonding interactions that propagates across the molecule, culminating in the ordering of switch II and placement of Q61 in the active site in a previously unobserved conformation. This structure suggests a direct catalytic role for Q61 where it interacts with a water molecule that bridges one of the gamma-phosphate oxygen atoms to the hydroxyl group of Y32 to stabilize the transition state of the hydrolysis reaction. We propose that Raf together with the binding of Ca(2+) and a negatively charged group mimicked in our structure by the acetate molecule induces the ordering of switch I and switch II to complete the active site of Ras.

The Crystal Structure of DehI Reveals a New α-Haloacid Dehalogenase Fold and Active-Site Mechanism

Haloacid dehalogenases catalyse the removal of halides from organic haloacids and are of interest for bioremediation and for their potential use in the synthesis of industrial chemicals. We present the crystal structure of the homodimer DehI from Pseudomonas putida strain PP3, the first structure of a group I α-haloacid dehalogenase that can process both l- and d-substrates. The structure shows that the DehI monomer consists of two domains of ∼130 amino acids that have ∼16% sequence identity yet adopt virtually identical and unique folds that form a pseudo-dimer. Analysis of the active site reveals the likely binding mode of both l- and d-substrates with respect to key catalytic residues. Asp189 is predicted to activate a water molecule for nucleophilic attack of the substrate chiral centre resulting in an inversion of configuration of either l- or d-substrates in contrast to d-only enzymes. These details will assist with future bioengineering of dehalogenases.