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In this study, mechanisms of hydrolysis of all four chemically diverse cleavage sites of human serum albumin (HSA) by [Zr(OH)(PW11O39)]4− (ZrK) have been investigated using the hybrid two‐layer QM/MM (ONIOM) method. These reactions have been proposed to occur through the following two mechanisms: internal attack (IA) and water assisted (WA). In both mechanisms, the cleavage of the peptide bond in the Cys392‐Glu393 site of HSA is predicted to occur in the rate‐limiting step of the mechanism. With the barrier of 27.5 kcal/mol for the hydrolysis of this site, the IA mechanism is found to be energetically more favorable than the WA mechanism (barrier = 31.6 kcal/mol). The energetics for the IA mechanism are in line with the experimentally measured values for the cleavage of a wide range of dipeptides. These calculations also suggest an energetic preference (Cys392‐Glu393, Ala257‐Asp258, Lys313‐Asp314, and Arg114‐Leu115) for the hydrolysis of all four sites of HSA. © 2018 Wiley Periodicals, Inc.

In this DFT study, the substrate promiscuity of the binuclear [Fe­(II)-Zn­(II)] core containing glycerophosphodiesterase (GpdQ) from Enterobacter aerogenes has been investigated through the hydrolysis of three chemically diverse groups of substrates: i.e., phosphomono-, phosphodi-, and phosphotriesters. The hydrolysis of these substrates is studied by comparing stepwise, concerted, and substrate-assisted mechanisms. Both the stepwise and concerted mechanisms occur with similar barriers, while the energetics for the substrate-assisted mechanism are significantly less favorable. Irrespective of the mechanism, active site residue His217 plays a critical role, in agreement with structural, kinetics, and spectroscopic data, but the transition state of the reaction depends on the identity of the substrate (dissociative for the triester paraoxon, associative for the monoester 4-nitrophenyl phosphate (NPP), and in-between for the diesters glycerol-3-phosphoethanolamine (GPE) and bis­(4-nitrophenyl)­phosphate (BNPP)). In good agreement with available kinetic and spectrophotometric data, the calculations highlight the preference of GpdQ for diester substrates, followed by tri- and monoesters. For substrates with two different types of scissile bonds (paraoxon and GPE) a clear preference for the bond with the stronger electron withdrawing leaving group was observed. The extensive agreement between experimental data and DFT calculations enhances the understanding of the mechanism of GpdQ-catalyzed hydrolysis and paves the way for the rational design of optimized catalysts for the hydrolysis of different types of phosphoesters.

Interactions of the catalytically active binuclear form of glycerophosphodiesterase (GpdQ) with chemically diverse substrates, i.e. phosphomono-, phosphodi-, and phosphotriester have been investigated using molecular dynamics (MD) simulations.

In this study, mechanisms of phosphodiester hydrolysis catalyzed by six di- and tetravalent metal-cyclen ( M-C ) complexes ( Zn-C, Cu-C, Co-C, Ce-C, Zr-C and Ti-C ) have been investigated using DFT calculations. The activities of these complexes were studied using three distinct mechanisms: (1) direct attack ( DA ), (2) catalyst-assisted ( CA ), and (3) water-assisted ( WA ). All divalent metal complexes ( Zn-C, Cu-C and Co-C ) coordinated to the BNPP substrate in a monodentate fashion and activated its scissile phosphoester bond. However, all tetravalent metal complexes ( Ce-C, Zr-C , and Ti-C ) interacted with BNPP in a bidentate manner and strengthened this bond. The DA mechanism was energetically the most feasible for all divalent M-C complexes, while the WA mechanism was favored by the tetravalent complexes, except Ce-C . The divalent complexes were found to be more reactive than their tetravalent counterparts. Zn-C catalyzed the hydrolysis with the lowest barrier among all M-C complexes, while Ti-C was the most reactive tetravalent complex. The activities of Ce-C and Zr-C , except Ti-C , were improved with an increase in the coordination number of the metal ion. The structural and mechanistic information provided in this study will be very helpful in the development of more efficient metal complexes for this critical reaction.

The hydrolysis of extremely stable peptide and phosphoester bonds by metalloenzymes is of great interest in biotechnology and industry. However, due to various shortcomings only a handful of these enzymes have been used for industrial applications. Therefore, in the last two decades intensive scientific efforts have been made in rational development of small molecules to imitate the activities of natural enzymes. Despite these efforts, their currently available synthetic analogues are inferior in terms of selectivity, catalytic rate, and turnover and the designing of efficient artificial metalloenzymes remains a distant goal. This is a challenging area of research that necessitates a rigorous integration between experiments and theory. The realization of this goal requires knowledge of the catalytic activities of both enzymes and their existing analogues and an effective fusion of that knowledge. This article reviews several studies in which a plethora of computational techniques have been successfully employed to investigate the functioning of two chemically promiscuous mono‐ and binuclear metalloenzymes (insulin degrading enzyme and glycerophosphodiesterase) and two synthetic analogues. These studies will help us derive fundamental principles of peptide and phosphoester hydrolysis and pave the way to design efficient small molecule catalysts for these reactions. This article is categorized under: Structure and Mechanism > Reaction Mechanisms and Catalysis

In this study, chemical promiscuity of a binuclear metallohydrolase Streptomyces griseus aminopeptidase (SgAP) has been investigated using DFT calculations. SgAP catalyzes two diverse reactions, peptide and phosphoester hydrolyses, using its binuclear (Zn–Zn) core. On the basis of the experimental information, mechanisms of these reactions have been investigated utilizing leucine p-nitro aniline (Leu-pNA) and bis­(4-nitrophenyl) phosphate (BNPP) as the substrates. The computed barriers of 16.5 and 16.8 kcal/mol for the most plausible mechanisms proposed by the DFT calculations are in good agreement with the measured values of 13.9 and 18.3 kcal/mol for the Leu-pNA and BNPP hydrolyses, respectively. The former was found to occur through the transfer of two protons, while the latter with only one proton transfer. They are in line with the experimental observations. The cleavage of the peptide bond was the rate-determining process for the Leu-pNA hydrolysis. However, the creation of the nucleophile and its attack on the electrophile phosphorus atom was the rate-determining step for the BNPP hydrolysis. These calculations showed that the chemical nature of the substrate and its binding mode influence the nucleophilicity of the metal bound hydroxyl nucleophile. Additionally, the nucleophilicity was found to be critical for the Leu-pNA hydrolysis, whereas double Lewis acid activation was needed for the BNPP hydrolysis. That could be one of the reasons why peptide hydrolysis can be catalyzed by both mononuclear and binuclear metal cofactors containing hydrolases, while phosphoester hydrolysis is almost exclusively by binuclear metallohydrolases. These results will be helpful in the development of versatile catalysts for chemically distinct hydrolytic reactions.

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