Tzvetan T. Mihaylov

Multinuclear diffusion NMR spectroscopy and DFT modeling: a powerful combination for unraveling the mechanism of phosphoester bond hydrolysis catalyzed by metal-substituted polyoxometalates.

A detailed reaction mechanism is proposed for the hydrolysis of the phosphoester bonds in the DNA model substrate bis(4-nitrophenyl) phosphate (BNPP) in the presence of the Zr(IV)-substituted Keggin type polyoxometalate (Et2NH2)8[{α-PW11O39Zr(μ-OH)(H2O)}2]⋅7 H2O (ZrK 2:2) at pD 6.4. Low-temperature (31)P DOSY spectra at pD 6.4 gave the first experimental evidence for the presence of ZrK 1:1 in fast equilibrium with ZrK 2:2 in purely aqueous solution. Moreover, theoretical calculations identified the ZrK 1:1 form as the potentially active species in solution. The reaction intermediates involved in the hydrolysis were identified by means of (1)H/(31)P NMR studies, including EXSY and DOSY NMR spectroscopy, which were supported by DFT calculations. This experimental/theoretical approach enabled the determination of the structures of four intermediate species in which the starting compound BNPP, nitrophenyl phosphate (NPP), or the end product phosphate (P) is coordinated to ZrK 1:1. In the proposed reaction mechanism, BNPP initially coordinates to ZrK 1:1 in a monodentate fashion, which results in hydrolysis of the first phosphoester bond in BNPP and formation of NPP. EXSY NMR studies showed that the bidentate complex between NPP and ZrK 1:1 is in equilibrium with monobound and free NPP. Subsequently, hydrolysis of NPP results in P, which is in equilibrium with its monobound form.

Hydrolytic Activity of Vanadate toward Serine-Containing Peptides Studied by Kinetic Experiments and DFT Theory

Hydrolysis of dipeptides glycylserine (Gly-Ser), leucylserine (Leu-Ser), histidylserine (His-Ser), glycylalanine (Gly-Ala), and serylglycine (Ser-Gly) was examined in vanadate solutions by means of (1)H, (13)C, and (51)V NMR spectroscopy. In the presence of a mixture of oxovanadates, the hydrolysis of the peptide bond in Gly-Ser proceeds under the physiological pH and temperature (37 °C, pD 7.4) with a rate constant of 8.9 × 10(-8) s(-1). NMR and EPR spectra did not show evidence for the formation of paramagnetic species, excluding the possibility of V(V) reduction to V(IV) and indicating that the cleavage of the peptide bond is purely hydrolytic. The pD dependence of k(obs) exhibits a bell-shaped profile, with the fastest hydrolysis observed at pD 7.4. Combined (1)H, (13)C, and (51)V NMR experiments revealed formation of three complexes between Gly-Ser and vanadate, of which only one complex, designated Complex 2, formed via coordination of amide oxygen and amino nitrogen to vanadate, is proposed to be hydrolytically active. Kinetic experiments at pD 7.4 performed by using a fixed amount of Gly-Ser and increasing amounts of Na(3)VO(4) allowed calculation of the formation constant for the Gly-Ser/VO(4)(3-) complex (K(f) = 16.1 M(-1)). The structure of the hydrolytically active Complex 2 is suggested also on the basis of DFT calculations. The energy difference between Complex 2 and the major complex detected in the reaction mixture, Complex 1, is calculated to be 7.1 kcal/mol in favor of the latter. The analysis of the molecular properties of Gly-Ser and their change upon different modes of coordination to the vanadate pointed out that only in Complex 2 the amide carbon is suitable for attack by the hydroxyl group in the Ser side chain, which acts as an effective nucleophile. The origin of the hydrolytic activity of vanadate is most likely a combination of the polarization of amide oxygen in Gly-Ser due to the binding to vanadate, followed by the intramolecular attack of the Ser hydroxyl group.

A Mechanistic Study of the Spontaneous Hydrolysis of Glycylserine as the Simplest Model for Protein Self‐Cleavage

A common feature of several classes of intrinsically reactive proteins with diverse biological functions is that they undergo self-catalyzed reactions initiated by an N→O or N→S acyl shift of a peptide bond adjacent to a serine, threonine, or cysteine residue. In this study, we examine the N→O acyl shift initiated peptide-bond hydrolysis at the serine residue on a model compound, glycylserine (GlySer), by means of DFT and ab initio methods. In the most favorable rate-determining transition state, the serine COO(-) group acts as a general base to accept a proton from the attacking OH function, which results in oxyoxazolidine ring closure. The calculated activation energy (29.4 kcal mol(-1) ) is in excellent agreement with the experimental value, 29.4 kcal mol(-1) , determined by (1) H NMR measurements. A reaction mechanism for the entire process of GlySer dipeptide hydrolysis is also proposed. In the case of proteins, we found that when no other groups that may act as a general base are available, the N→O acyl shift mechanism might instead involve a water-assisted proton transfer from the attacking serine OH group to the amide oxygen. However, the calculated energy barrier for this process is relatively high (33.6 kcal mol(-1) ), thus indicating that in absence of catalytic factors the peptide bond adjacent to serine is no longer a weak point in the protein backbone. An analogous rearrangement involving the amide N-protonated form, rather than the principle zwitterion form of GlySer, was also considered as a model for the previously proposed mechanism of sea-urchin sperm protein, enterokinase, and agrin (SEA) domain autoproteolysis. The calculated activation energy (14.3 kcal mol(-1) ) is significantly lower than the experimental value reported for SEA (≈21 kcal mol(-1) ), but is still in better agreement as compared to earlier theoretical attempts.

Reactivity of Dimeric Tetrazirconium(IV) Wells–Dawson Polyoxometalate toward Dipeptide Hydrolysis Studied by a Combined Experimental and Density Functional Theory Approach

Detailed kinetic studies on the hydrolysis of glycylglycine (Gly-Gly) in the presence of the dimeric tetrazirconium(IV)-substituted Wells-Dawson-type polyoxometalate Na14[Zr4(P2W16O59)2(μ3-O)2(OH)2(H2O)4] · 57H2O (1) were performed by a combination of (1)H, (13)C, and (31)P NMR spectroscopies. The catalyst was shown to be stable under a broad range of reaction conditions. The effect of pD on the hydrolysis of Gly-Gly showed a bell-shaped profile with the fastest hydrolysis observed at pD 7.4. The observed rate constant for the hydrolysis of Gly-Gly at pD 7.4 and 60 °C was 4.67 × 10(-7) s(-1), representing a significant acceleration as compared to the uncatalyzed reaction. (13)C NMR data were indicative for coordination of Gly-Gly to 1 via its amide oxygen and amine nitrogen atoms, resulting in a hydrolytically active complex. Importantly, the effective hydrolysis of a series of Gly-X dipeptides with different X side chain amino acids in the presence of 1 was achieved, and the observed rate constant was shown to be dependent on the volume, chemical nature, and charge of the X amino acid side chain. To give a mechanistic explanation of the observed catalytic hydrolysis of Gly-Gly, a detailed quantum-chemical study was performed. The theoretical results confirmed the nature of the experimentally suggested binding mode in the hydrolytically active complex formed between Gly-Gly and 1. To elucidate the role of 1 in the hydrolytic process, both the uncatalyzed and the polyoxometalate-catalyzed reactions were examined. In the rate-determining step of the uncatalyzed Gly-Gly hydrolysis, a carboxylic oxygen atom abstracts a proton from a solvent water molecule and the nascent OH nucleophile attacks the peptide carbon atom. Analogous general-base activity of the free carboxylic group was found to take place also in the case of polyoxometalate-catalyzed hydrolysis as the main catalytic effect originates from the -C═O···Zr(IV) binding.

Unraveling the Mechanisms of Carboxyl Ester Bond Hydrolysis Catalyzed by a Vanadate Anion

The mechanism of p-nitrophenyl acetate (pNPA) hydrolysis promoted by vanadate ions was investigated utilizing both density functional theory and ab initio methods. In accordance with experiments, suggesting pure hydrolytic ester bond cleavage involving a nucleophilic addition in the rate-limiting transition state, four possible B(AC)2 (acyl-oxygen bond cleavage) mode reaction pathways were modeled. Moreover, two alternative reaction modes were also considered. Geometry optimizations were carried out using B3LYP, BP86, and MPWB1K functionals, conjugated with a 6-31++G(d,p) basis set and a Stuttgart effective core potential (ECP) for the vanadium atom. Single-point calculations were performed utilizing M06, B3LYP-D, and BP86-D functionals as well as B2PLYP-D and MP2 methods with a 6-311++G(2d,2p) basis set (with and without ECP). To address bulk solvation effects, the universal solvation model (SMD) and the conductor-like polarizable continuum model were applied, using the parameters of water. All levels of theory predict the same reaction mechanism, B(AC)2-1, as the lowest-energy pathway on the potential energy surface for pNPA hydrolysis catalyzed by the H(2)VO(4)(-) ion in aqueous media. The B(AC)2-1 pathway passes through two transition states, the first associated with the nucleophilic addition of H(2)VO(4)(-) and the second with the release of p-nitrophenoxide ion (pNP(-)), linked with a tetrahedral intermediate state. The intermediate structure is stabilized via protonation of the acyl oxygen atom by the vanadate and formation of an intramolecular hydrogen bond. The first and second barrier heights are 24.9 and 1.3 kcal/mol respectively, as calculated with the SMD-M06 approach. The theoretically predicted B(AC)2-1 mechanism is in good agreement with the experiment.

Protein‐Assisted Formation and Stabilization of Catalytically Active Polyoxometalate Species

The effect of the protein environment on the formation and stabilization of an elusive catalytically active polyoxometalate (POM) species, K6 [Hf(α2 -P2 W17 O61 )] (1), is reported. In the co-crystal of hen egg-white lysozyme (HEWL) with 1, the catalytically active monomeric species is observed, originating from the dimeric 1:2 POM form, while it is intrinsically unstable under physiological pH conditions. The protein-assisted dissociation of the dimeric POM was rationalized by means of DFT calculations. The dissociation process is unfavorable in bulk water, but becomes favorable in the protein-POM complex due to the low dielectric response at the protein surface. The crystal structure shows that the monomeric form is stabilized by electrostatic and water-mediated hydrogen bonding interactions with the protein. It interacts at three distinct sites, close to the aspartate-containing hydrolysis sites, demonstrating high selectivity towards peptide bonds containing this residue.

Molecular Insight from DFT Computations and Kinetic Measurements into the Steric Factors Influencing Peptide Bond Hydrolysis Catalyzed by a Dimeric Zr(IV)-Substituted Keggin Type Polyoxometalate

Peptide bond hydrolysis of several peptides with a Gly-X sequence (X = Gly, Ala, Val, Leu, Ile, Phe) catalyzed by a dimeric Zr(IV)-substituted Keggin type polyoxometalate (POM), (Et2NH2)8[{α-PW11O39Zr(μ-OH)(H2O)}2]·7H2O (1), was studied by means of kinetic experiments and (1)H NMR spectroscopy. The observed rate of peptide bond hydrolysis was found to decrease with increase of the side chain bulkiness, from 4.44 × 10(-7) s(-1) for Gly-Gly to 0.81 × 10(-7) s(-1) for Gly-Ile. A thorough DFT investigation was performed to elucidate (a) the nature of the hydrolytically active species in solution, (b) the mechanism of peptide bond hydrolysis, and (c) the influence of the aliphatic residues on the rate of hydrolysis. Formation of substrate-catalyst complexes of the dimeric POM 1 was predicted as thermodynamically unlikely. Instead, the substrates prefer to bind to the monomerization product of 1, [α-PW11O39Zr(OH)(H2O)](4-) (2), which is also present in solution. In the hydrolytically active complex two dipeptide ligands are coordinated to the Zr(IV) center of 2. The first ligand is bidentate-bound through its amino nitrogen and amide oxygen atoms, while the second ligand is monodentate-bound through a carboxylic oxygen atom. The mechanism of hydrolysis involves nucleophilic attack by a solvent water molecule on the amide carbon atom of the bidentate-bound ligand. In this process the uncoordinated carboxylic group of the same ligand acts as a general base to abstract a proton from the attacking water molecule. The decrease of the hydrolysis rate with an increase in the side chain bulkiness is mostly due to the increased ligand conformational strain in the rate-limiting transition state, which elevates the reaction activation energy. The conformational strain increases first upon substitution of Hα in Gly-Gly with the aliphatic α substituent and second with the β branching of the α substituent.