Modeling of Peptide Hydrolysis by Thermolysin. A Semiempirical and QM/MM Study

Antonczak, Serge; Monard, Gérald; Ruiz‐López, Manuel F.; Rivail, Jean‐Louis

doi:10.1021/ja981650u

Cited by 76 publications

(49 citation statements)

References 65 publications

(46 reference statements)

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“…Protonation states of charged residues in the enzyme complex were determined via H++ program42 and by carefully examining local hydrogen bond networks. In all TLN models, His142 and His146 was identified as singly protonated, whereas His231 was doubly protonated, which are the same as previous studies43,44. For HDAC8 models, His180 was determined as singly protonated on δ site, while His142 and His143 were determined as singly protonated on ε site.…”

Section: Methodssupporting

confidence: 66%

Flexibility of Catalytic Zinc Coordination in Thermolysin and HDAC8: A Born−Oppenheimer ab Initio QM/MM Molecular Dynamics Study

Wang

et al. 2009

J. Chem. Theory Comput.

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AbstracsThe different coordination modes and fast ligand exchange of zinc coordination has been suggested to be one key catalytic feature of the zinc ion which makes it an invaluable metal in biological catalysis. However, partly due to the well known difficulties for zinc to be characterized by spectroscopy methods, evidence for dynamic nature of the catalytic zinc coordination has so far mainly been indirect. In this work, Born-Oppenheimer ab initio QM/MM molecular dynamics simulation has been employed, which allows for a first-principle description of the dynamics of the metal active site while properly including effects of the heterogeneous and fluctuating protein environment. Our simulations have provided direct evidence regarding inherent flexibility of the catalytic zinc coordination shell in Thermolysin (TLN) and Histone Deacetylase 8 (HDAC8). We have observed different coordination modes and fast ligand exchange during the picosecond's timescale. For TLN, the coordination of the carboxylate group of Glu166 to Zinc is found to continuously change between monodentate and bidentate manner dynamically; while for HDAC8, the flexibility mainly comes from the coordination to a non-amino-acid ligand. Such distinct dynamics in the zinc coordination shell between two enzymes suggests that the catalytic role of Zinc in TLN and HDAC8 is likely to be different in spite of the fact that both catalyze the hydrolysis of amide bond. Meanwhile, considering that such Born-Oppenheimer ab initio QM/MM MD simulations are very much desired but are widely considered to be too computationally expensive to be feasible, our current study demonstrates the viability and powerfulness of this state-of-the-art approach in simulating metalloenzymes.Zinc is relatively abundant in biological materials. Approximately 10% of the total human proteome have been identified to bind with zinc in vivo from a bioinformatics investigation1 and they play very crucial roles in all forms of life2 -6 . For mononuclear zinc enzymes, a typical metal coordination environment contains three amino acid side chain ligands (His, Glu, Asp and Cys) and one/two small molecule(s). 3, 7 , 8 The flexibility of zinc coordination, which allows different coordination modes and fast ligand exchange, has been suggested to be one key catalytic feature of the zinc ion which makes it an invaluable metal in biological catalysis.9 However, partly due to the well known difficulties for zinc to be characterized by spectroscopy methods 10,11 , evidence for dynamic nature of the catalytic zinc coordination has so far mainly In order to provide deep insights into the dynamics and flexibility of the zinc catalytic site, which would be essential in characterizing their catalytic mechanisms and rational design of novel inhibitors for zinc enzymes, we have carried out DFT QM/MM Born-Oppenheimer molecular dynamics (BOMD) simulations on TLN and HDAC8. Although semi-empirical QM/MM BOMD simulations of some zinc-dependent enzymes have been carried out 29-32, one main concern is the acc...

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Section: Methodssupporting

confidence: 66%

Flexibility of Catalytic Zinc Coordination in Thermolysin and HDAC8: A Born−Oppenheimer ab Initio QM/MM Molecular Dynamics Study

Wang

et al. 2009

J. Chem. Theory Comput.

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“…The very simple model used here to represent this reaction does not take into account finer effects such as polarization of the substrate (due the metal center and its ligands [37] ) or a stabilizing role due to molecular recognition. All these contributions could energetically favor the feasibility of the reactions described above, but keeping in mind that electron transfer is often the most energetic Our results have also shown that quercetin oxygenolysis should proceed by 1,3-cycloaddition, despite the fact that 1,2-cycloadditions are less energy demanding.…”

Section: Discussionmentioning

confidence: 99%

Oxygenolysis of Flavonoid Compounds: DFT Description of the Mechanism for Quercetin

et al. 2004

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Flavonoids are naturally occurring phenol derivatives present in substantial amounts in a large variety of plants, fruits and vegetables daily eaten by humans. Most of these compounds exhibit several interesting biological activities, such as antiradical and antioxidant actions. Indeed, by complexation with specific enzymes, flavonoids are notably liable to metabolize molecular dioxygen. On the basis of experimental results describing oxygenolysis of the flavonoid quercetin, activated by the enzyme quercetin 2,3-dioxygenase (2,3-QD),ur attention has focused on the role of metal center in the activation of the substrate quercetin. Thus, in the present study, by means of DFT calculations at the B3LYP/ 6-31(+)G* level on model molecular systems, we describe different mechanisms for dioxygen metabolization by quercetin. Stationary points are described, and energetic and structural analyses along the reaction paths are reported. Our calculations show that the copper cation must act as an oxidant towards the substrate and that the reaction proceeds through a 1,3-cycloaddition.

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“…Their results 55 suggest that the rate determining step is the formation of a tetrahedral intermediate arising from the attack of a Zn-coordinated OH group on the carbonyl of the substrate. A semiempirical and QM/MM study of peptide hydrolysis by thermolysin was performed by Rivail 56 and co-workers while Giessner and Jacob 57 employed MNDO (with special Zn parameterization) to calculate the interactions between zinc and different ligands at the thermolysin active site. Goldblum and co-workers performed a semi-empirical QM study on the alternative mechanisms for zinc proteinases 58–60.…”

Section: Introductionmentioning

confidence: 99%

On the origin of the catalytic power of carboxypeptidase A and other metalloenzymes

Kilshtain

Warshel

2009

Proteins

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Zinc metalloenzymes play a major role in key biological processes and Carboxypeptidase-A (CPA) is a major prototype of such enzymes. The present work quantifies the energetics of the catalytic reaction of CPA and its mutants using the EVB approach. The simulations allow us to quantify the origin of the catalytic power of this enzyme and to examine different mechanistic alternatives. The first step of the analysis used experimental information to determine the activation energy of each assumed mechanism of the reference reaction without the enzyme. The next step of the analysis involved EVB simulations of the reference reaction and then a calibration of the simulations by forcing them to reproduce the energetics of the reference reaction, in each assumed mechanism. The calibrated EVB was then used in systematic simulations of the catalytic reaction in the protein environment, without changing any parameter. The simulations reproduced the observed rate enhancement in two feasible general acid-general base mechanisms (GAGB-1 and GAGB-2), although the calculations with the GAGB-2 mechanism underestimated the catalytic effect in some treatments. We also reproduced the catalytic effect in the R127A mutant. The mutation calculations indicate that the GAGB-2 mechanism is significantly less likely than the GAGB-1 mechanism. It is also found, that the enzyme loses all its catalytic effect without the metal. This and earlier studies show that the catalytic effect of the metal is not some constant electrostatic effect, that can be assessed from gas phase studies, but a reflection of the dielectric effect of the specific environment.

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Modeling of Peptide Hydrolysis by Thermolysin. A Semiempirical and QM/MM Study

Cited by 76 publications

References 65 publications

Flexibility of Catalytic Zinc Coordination in Thermolysin and HDAC8: A Born−Oppenheimer ab Initio QM/MM Molecular Dynamics Study

Flexibility of Catalytic Zinc Coordination in Thermolysin and HDAC8: A Born−Oppenheimer ab Initio QM/MM Molecular Dynamics Study

Oxygenolysis of Flavonoid Compounds: DFT Description of the Mechanism for Quercetin

On the origin of the catalytic power of carboxypeptidase A and other metalloenzymes

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