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  Elucidation of the Catalytic Mechanism of a Miniature Zinc Finger Hydrolase

Ganguly, A., Luong, T. Q., Brylski, O., Dirkmann, M., Möller, D., Ebbinghaus, S., et al. (2017). Elucidation of the Catalytic Mechanism of a Miniature Zinc Finger Hydrolase. The Journal of Physical Chemistry B, 121(26), 6390-6398. doi:10.1021/acs.jpcb.7b05027.

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Ganguly, Abir1, Author              
Luong, Trung Quan2, Author
Brylski, Oliver3, Author
Dirkmann, Michael4, Author
Möller, David4, Author
Ebbinghaus, Simon3, Author
Schulz, Frank4, Author
Winter, Roland2, Author
Sanchez-Garcia, Elsa5, Author              
Thiel, Walter1, Author              
Affiliations:
1Research Department Thiel, Max-Planck-Institut für Kohlenforschung, Max Planck Society, ou_1445590              
2Fakultät für Chemie und Chemische Biologie, Physikalische Chemie I, Technische Universität Dortmund, 44227 Dortmund, Germany, ou_persistent22              
3Fakultät für Chemie und Biochemie, Physikalische Chemie II, Ruhr-Universität Bochum, 44780 Bochum, Germany, ou_persistent22              
4 Fakultät für Chemie und Biochemie, Organische Chemie I, Ruhr-Universität Bochum, 44780 Bochum, Germany, ou_persistent22              
5Research Group Sánchez-García, Max-Planck-Institut für Kohlenforschung, Max Planck Society, ou_1950289              

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 Abstract: To improve our mechanistic understanding of zinc metalloenzymes, we report a joint computational and experimental study of a minimal carbonic anhydrase (CA) mimic, a 22-residue Zn-finger hydrolase. We combine classical molecular dynamics (MD) simulations, quantum mechanics/molecular mechanics (QM/MM) geometry optimizations, and QM/MM free energy simulations with ambient and high-pressure kinetic measurements to investigate the mechanism of the hydrolysis of the substrate p-nitrophenylacetate (pNPA). The zinc center of the hydrolase prefers a pentacoordinated geometry, as found in most naturally occurring CAs and CA-like enzymes. Two possible mechanisms for the catalytic reaction are investigated. The first one is analogous to the commonly accepted mechanism for CA-like enzymes: a sequential pathway, in which a Zn2+-bound hydroxide acts as a nucleophile and the hydrolysis proceeds through a tetrahedral intermediate. The initial rate-limiting step of this reaction is the nucleophilic attack of the hydroxide on pNPA to form the tetrahedral intermediate. The computed free energy barrier of 18.5 kcal/mol is consistent with the experimental value of 20.5 kcal/mol obtained from our kinetics experiments. We also explore an alternative reverse protonation pathway for the hydrolase, in which a nearby hydroxide ion from the bulk acts as the nucleophile (instead of a zinc-bound hydroxide). According to QM/MM MD simulations, hydrolysis occurs spontaneously along this pathway. However, this second scenario is not viable in our system, as the tertiary structure of the hydrolase lacks a suitably positioned residue that would act as a general base and generate a hydroxide ion from a nearby bulk water molecule. Hence, our combined theoretical and experimental study indicates that the investigated minimal CA mimic retains the essential mechanistic features of CA-like enzyme catalysis. The high-pressure experiments show that its catalytic efficiency can be enhanced by applying hydrostatic pressure. According to the simulations, more drastic improvements might be afforded by mutations that make the reverse protonation pathway accessible.

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Language(s): eng - English
 Dates: 2017-06-052017-05-242017-06-242017-07-06
 Publication Status: Published in print
 Pages: 9
 Publishing info: -
 Table of Contents: -
 Rev. Type: Peer
 Identifiers: DOI: 10.1021/acs.jpcb.7b05027
 Degree: -

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Title: The Journal of Physical Chemistry B
  Other : J. Phys. Chem. B
Source Genre: Journal
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Publ. Info: Washington, D.C. : American Chemical Society
Pages: - Volume / Issue: 121 (26) Sequence Number: - Start / End Page: 6390 - 6398 Identifier: ISSN: 1520-6106
CoNE: https://pure.mpg.de/cone/journals/resource/1000000000293370_1