Jian-Guo Yu

Mechanism of tungsten-dependent acetylene hydratase from quantum chemical calculations

Acetylene hydratase is a tungsten-dependent enzyme that catalyzes the nonredox hydration of acetylene to acetaldehyde. Density functional theory calculations are used to elucidate the reaction mechanism of this enzyme with a large model of the active site devised on the basis of the native X-ray crystal structure. Based on the calculations, we propose a new mechanism in which the acetylene substrate first displaces the W-coordinated water molecule, and then undergoes a nucleophilic attack by the water molecule assisted by an ionized Asp13 residue at the active site. This is followed by proton transfer from Asp13 to the newly formed vinyl anion intermediate. In the subsequent isomerization, Asp13 shuttles a proton from the hydroxyl group of the vinyl alcohol to the α-carbon. Asp13 is thus a key player in the mechanism, but also W is directly involved in the reaction by binding and activating acetylene and providing electrostatic stabilization to the transition states and intermediates. Several other mechanisms are also considered but the energetic barriers are found to be very high, ruling out these possibilities.

Quantum Chemical Modeling of Enzymatic Reactions: The Case of Decarboxylation

We present a systematic study of the decarboxylation step of the enzyme aspartate decarboxylase with the purpose of assessing the quantum chemical cluster approach for modeling this important class of decarboxylase enzymes. Active site models ranging in size from 27 to 220 atoms are designed, and the barrier and reaction energy of this step are evaluated. To model the enzyme surrounding, homogeneous polarizable medium techniques are used with several dielectric constants. The main conclusion is that when the active site model reaches a certain size, the solvation effects from the surroundings saturate. Similar results have previously been obtained from systematic studies of other classes of enzymes, suggesting that they are of a quite general nature.

Mechanistic Insight into the NN Bond‐Cleavage of Azo‐Compounds that was Induced by an AlAl‐bonded Compound [L2−AlIIAlIIL2−]

An α-diimine-stabilized Al-Al-bonded compound [L(2-)Al(II)-Al(II)L(2-)] (L = [{(2,6-iPr(2)C(6)H(3))NC(Me)}(2)]; 1) consists of dianionic α-diimine ligands and sub-valent Al(2+) ions and thus could potentially behave as a multielectron reductant. The reactions of compound 1 with azo-compounds afforded phenylimido-bridged products [L(-)Al(III)(μ(2)-NPh)(μ(2)-NAr)Al(III)L(-)] (2-4). During the reaction, the dianionic ligands and Al(2+) ions were oxidized into monoanions and Al(3+), respectively, whilst the [NAr](2-) imides were produced by the four-electron reductive cleavage of the N=N double bond. Upon further reduction by Na, the monoanionic ligands in compound 2 were reduced to the dianion to give [(L(2-))(2)Al(III)(2)(μ(2)-NPh)(2)Na(2)(thf)(4)] (5). Interestingly, when asymmetric azo-compounds were used, the asymmetric adducts were isolated as the only products (compounds 3 and 4). DFT calculations indicated that the reaction was quite feasible in the singlet electronic state, but the final product with the triplet-state monoanionic ligands could result from an exothermic singlet-to-triplet conversion during the reaction process.

Reaction Mechanism of the Trinuclear Zinc Enzyme Phospholipase C: A Density Functional Theory Study

Phosphatidylcholine-preferring phospholipase C is a trinuclear zinc-dependent phosphodiesterase, catalyzing the hydrolysis of choline phospholipids. In the present study, density functional theory is used to investigate the reaction mechanism of this enzyme. Two possible mechanistic scenarios were considered with a model of the active site designed on the basis of the high resolution X-ray crystal structure of the native enzyme. The calculations show that a Zn1 and Zn3 bridging hydroxide rather than a Zn1 coordinated water molecule performs the nucleophilic attack on the phosphorus center. Simultaneously, Zn2 activates a water molecule to protonate the leaving group. In the following step, the newly generated Zn2 bound hydroxide makes the reverse attack, resulting in the regeneration of the bridging hydroxide. The first step is calculated to be rate-limiting with a barrier of 17.3 kcal/mol, in good agreement with experimental kinetic studies. The zinc ions are suggested to orient the substrate for nucleophilic attack and provide electrostatic stabilization to the dianionic penta-coordinated trigonal bipyramidal transition states, thereby lowering the barrier.

Theoretical Investigation of the Reaction Mechanism of the Dinuclear Zinc Enzyme Dihydroorotase

The reaction mechanism of the dinuclear zinc enzyme dihydroorotase was investigated by using hybrid density functional theory. This enzyme catalyzes the reversible interconversion of dihydroorotate and carbamoyl aspartate. Two reaction mechanisms in which the important active site residue Asp250 was either protonated or unprotonated were considered. The calculations establish that Asp250 must be unprotonated for the reaction to take place. The bridging hydroxide is shown to be capable of performing nucleophilic attack on the substrate from its bridging position and the role of Zn(beta) is argued to be the stabilization of the tetrahedral intermediate and the transition state leading to it, thereby lowering the barrier for the nucleophilic attack. It is furthermore concluded that the rate-limiting step is the protonation of the amide nitrogen by Asp250 coupled with C-N bond cleavage, which is consistent with previous experimental findings from isotope labeling studies.

Phosphate Mono- and Diesterase Activities of the Trinuclear Zinc Enzyme Nuclease P1-Insights from Quantum Chemical Calculations

Nuclease P1 is a trinuclear zinc enzyme that catalyzes the hydrolysis of single-stranded DNA and RNA. Density functional calculations are used to elucidate the reaction mechanism of this enzyme with a model of the active site designed on the basis of the X-ray crystal structure. 2-Tetrahydrofuranyl phosphate and methyl 2-tetrahydrofuranyl phosphate substrates are used to explore the phosphomonoesterase and phosphodiesterase activities of this enzyme, respectively. The calculations reveal that for both activities, a bridging hydroxide performs an in-line attack on the phosphorus center, resulting in inversion of the configuration. Simultaneously, the P-O bond is cleaved, and Zn2 stabilizes the negative charge of the leaving alkoxide anion and assists its departure. All three zinc ions, together with Arg48, provide electrostatic stabilization to the penta-coordinated transition state, thereby lowering the reaction barrier.

Reaction mechanism of the dinuclear zinc enzyme N-acyl-L-homoserine lactone hydrolase: a quantum chemical study.

N-acyl-L-homoserine lactone hydrolase (AHL lactonase) is a dinuclear zinc enzyme responsible for the hydrolytic ring opening of AHLs, disrupting quorum sensing in bacteria. The reaction mechanism is investigated using hybrid density functional theory. A model of the active site is designed on the basis of the X-ray crystal structure, and stationary points along the reaction pathway are optimized and analyzed. Two possible mechanisms based on two different substrate orientations are considered. The calculations give support to a reaction mechanism that involves two major chemical steps: nucleophilic attack on the substrate carbonyl carbon by the bridging hydroxide and ring opening by direct ester C-O bond cleavage. The roles of the two zinc ions are analyzed. Zn1 is demonstrated to stabilize the charge of the tetrahedral intermediate, thereby facilitating the nucleophilic attack, while Zn2 stabilizes the charge of the alkoxide resulting from the ring opening, thereby lowering the barrier for the C-O bond cleavage.

Theoretical studies on pyridoxal 5′‐phosphate‐dependent transamination of α‐amino acids

Density functional methods have been applied to investigate the irreversible transamination between glyoxylic acid and pyridoxamine analog and the catalytic mechanism for the critical [1,3] proton transfer step in aspartate aminotransferase (AATase). The results indicate that the catalytic effect of pyridoxal 5′‐phosphate (PLP) may be attributed to its ability to stabilize related transition states through structural resonance. Additionally, the PLP hydroxyl group and the carboxylic group of the amino acid can shuttle proton, thereby lowering the barrier. The rate‐limiting step is the tautomeric conversion of the aldimine to ketimine by [1,3] proton transfer, with a barrier of 36.3 kcal/mol in water solvent. A quantum chemical model consisting 142 atoms was constructed based on the crystal structure of the native AATase complex with the product L‐glutamate. The electron‐withdrawing stabilization by various residues, involving Arg386, Tyr225, Asp222, Asn194, and peptide backbone, enhances the carbon acidity of 4′‐C of PLP and Cα of amino acid. The calculations support the proposed proton transfer mechanism in which Lys258 acts as a base to shuttle a proton from the 4′‐C of PLP to Cα of amino acid. The first step (proton transfer from 4′‐C to lysine) is shown to be the rate‐limiting step. Furthermore, we provided an explanation for the reversibility and specificity of the transamination in AATase.

Dipeptide hydrolysis by the dinuclear zinc enzyme human renal dipeptidase: Mechanistic insights from DFT calculations

The reaction mechanism of the dinuclear zinc enzyme human renal dipeptidase is investigated using hybrid density functional theory. This enzyme catalyzes the hydrolysis of dipeptides and beta-lactam antibiotics. Two different protonation states in which the important active site residue Asp288 is either neutral or ionized were considered. In both cases, the bridging hydroxide is shown to be capable of performing the nucleophilic attack on the substrate carbonyl carbon from its bridging position, resulting in the formation of a tetrahedral intermediate. This step is followed by protonation of the dipeptide nitrogen, coupled with C-N bond cleavage. The calculations establish that both cases have quite feasible energy barriers. When the Asp288 is neutral, the hydrolytic reaction occurs with a large exothermicity. However, the reaction becomes very close to thermoneutral with an ionized Asp288. The two zinc ions are shown to play different roles in the reaction. Zn1 binds the amino group of the substrate, and Zn2 interacts with the carboxylate group of the substrate, helping in orienting it for the nucleophilic attack. In addition, Zn2 stabilizes the oxyanion of the tetrahedral intermediate, thereby facilitating the nucleophilic attack.

DFT Study on the Mechanism of Escherichia coli Inorganic Pyrophosphatase

Escherichia coli inorganic pyrophosphatase (E-PPase) is a tetranuclear divalent metal dependent enzyme that catalyzes the reversible interconversion of pyrophosphate (PPi) and orthophosphate (Pi), with Mg(2+) conferring the highest activity. In the present work, the reaction mechanism of E-PPase is investigated using the hybrid density functional theory (DFT) method B3LYP with a large model of the active site. Our calculated results shed further light on the detailed reaction mechanism. In particular, the important residue Asp67, either protonated or unprotonated, was taken into account in the present calculations. Our calculations indicated that a protonated Asp67 is crucial for the reverse reaction to take place; however, it is lost sight of in the forward reaction. The bridging hydroxide is shown to be capable of performing nucleophilic in-line attack on the substrate from its bridging position in the presence of four Mg(2+) ions. During the catalysis, the roles of the four magnesium ions are suggested to provide a necessary conformation of the active site, facilitate the nucleophile formation and substrate orientation, and stabilize the trigonal bipyramid transition state, thereby lowering the barrier for the nucleophilic attack.