QM/MM Study of the Reaction
Mechanisms of Biocatalyst

University of Tsukuba, Center for Computational Sciences
Division of Life Sciences
Megumi Kayanuma
2017/12/7

1Simulation of Biomolecules
accurate

QM (Quantum Mechanics)
chemical reaction, photochemistry, …

MM (Molecular Mechanics)
structure and dynamics of proteins

CS (Coarse-Grained)
Protein complexes
large scale
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Multiscale Modeling
QM/MM method• Quantum chemical model for region involved in chemical reaction• Classical model (MM) for other parts• Interaction between QM and MM regions

𝐻𝑒𝑓𝑓 = 𝐻𝑄𝑀 + 𝐻𝑀𝑀 + 𝐻𝑄𝑀/𝑀𝑀MMQM

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Chemical Reactions

Energytransition state

ΔE‡

reactant

ΔEproduct
reaction coordinate

Catalysts stabilize the transition state or create new reaction path→ accelerate the reaction
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Analysis of Reaction using QM Method
 How the reaction proceed?

Energy (kJ/mol) Which reaction step require the highest energy?
 How catalysts stabilize the transition states?
0 water
1 water
2 water

M. Kayanuma et al., Chem. Phys. Lett., 687 (2017) 178
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Biocatalyst
 Biochemistry
 Catalytic chemistry
Application
 Industry
 Bioremediation
Merits Turnover number Reaction rate Structural and stereochemical selectivity
 Mild condition (temperature, pH, etc.)

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Nitrile Hydratase (NHase) catalyze hydration of nitrile to the corresponding amide
R-CN + H2O → R-CONH2
 chemical industry (e.g. production of acrylamide) metal ion (Fe(III) or Co(III)) at the active site
 unique coordination structure (one -SO and one -SO2)

ONO FeSO
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N OSS7

Reaction Mechanism of NHase
 Time-resolved crystal structures of a mutant (βR56K) suggested
the involvement of a cyclic intermediate
Y. Yamanaka et al., Angew. Chem. Int. Ed. 54 (2015) 10763

 However, the subsequent mechanism was not elucidated
Disulfide intermediate formation and proton transfer from βR56
K. H. Hopmann, Inorg. Chem. 53 (2014) 2760

Water attack of the cyclic intermediate and amide formation
Y. Yamanaka et al., Angew. Chem. Int. Ed. 54 (2015) 10763

Water attack of the cyclic intermediate and imidic acid formation
K. M. Light et al., Chem. Sci. 6 (2015) 6280

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Previous Theoretical Studies
Use cluster model of the active site
(protein environment was treated as polarizable model)
K. H. Hopmann, Inorg. Chem. 53 (2014) 2760

activesite

activesite

polarizablemodel

Do some specific amino acid residues affect the reaction?
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Aime



Reveal the reaction mechanism of nitrile hydratase (NHase)
subsequent to the cyclic intermediate using hybrid quantum
mechanics/molecular mechanics (QM/MM) method
Analyze the effects of protein environment on the active site

MMβArg56

αCys114QM
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Method

QM: B3LYPLANL2DZ and 6-31G(d)(6-311++G(d,p) for energy calculation)
MM: AMBER99
Initial structure: PDBID 3A8O
βTyr72
Substrate: CH3CN
Water: 10×10×10 nm3
Ions: Na+×24
Cutoff: 9 Å(no cutoff for energy calculation)
αSer113
Program: NWChem 6.3
Computer: COMA @ Univ. Tsukuba

QM regeon
βTyr37
CH3CN
βArg56

αCys114FeH2O

αCys112
αC109
βArg141

Energies are relative values to the 5-coordinated structure
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11Results: Reaction Mechanism
Cyclic intermediate

Disulfide intermediate

Imidic acid intermediate

Amide product2017/12/712Conflict with Experiment?

Previous and present theoretical studies
Formation of the disulfide intermediate

βArg56

Experimental study of a mutant (βR56K)
No change in the position of the S atoms→ Disulfide intermediate seemed improbable

Why disulfide intermediate is not observed inthe experiment of βR56K mutant?

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13Effect of Arg56
protonated cyclic intermediate
Arg56

ΔE-18.3 kJ/mol

Lys56

ΔE+19.2 kJ/mol

WT

R56K

disulfide intermediate

×constraint (S-S)

βArg56 is required for the formation of disulfide intermediate
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14Water attack Reaction path via imidic acid formation has lower reaction barrier Isomerization of imidic acid might occur outside the protein

disulfide intermediate

amide productor isomerization
occur outside protein

imidic acid intermediate
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15Effects of Protein Environment
Previous theoretical study using active site models:
proton transfer from βArg56 after disulfide intermediate formation
K. H. Hopmann, Inorg. Chem. 53 (2014) 2760

βArg56

Deprotonation of βArg56 was not favorable in protein environment
Considering the effects of protein environment is important
2017/12/716Conclusion
We revealed the reaction mechanism of NHase subsequent tothe formation of cyclic intermediate using QM/MM method

protontransferβTyr37βTyr72stabilize disulfide
intermediate
βArg56
αCys114αSer113FeαCys112
αC109
βArg141

M. Kayanuma et al., J. Phys. Chem. B, 120 (2016) 3259
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Acknowledgement
University of Tsukuba
 Prof. Yasuteru Shigeta
 Assist. Prof. Mitsuo Shoji
Akita University
 Prof. Masafumi Odaka
Tokyo University of Agriculture and Technology
 Prof. Masafumi Yohda

This research used computational resources of COMA provided byInterdisciplinary Computational Science Program in Center for
Computational Sciences, University of Tsukuba."

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