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Molecular Biology

, Volume 51, Issue 6, pp 830–839 | Cite as

Peculiarities of the Mechanism of Interactions of Catalytic Antibodies with Organophosphorus Substrates

  • Yu. A. Mokrushina
  • S. O. Pipiya
  • A. V. Stepanova
  • O. G. Shamborant
  • V. D. Knorre
  • I. V. Smirnov
  • A. G. Gabibov
  • I. I. Vorobiev
Current Trends in the Application of Monoclonal Antibodies Special Issue
  • 34 Downloads

Abstract

Catalytic antibodies are a promising model for creating highly specific biocatalysts with predetermined activity. However, in order to realize the directed change or improve their properties, it is necessary to understand the basics of catalysis and the specificity of interactions with substrates. In the present work, a structural and functional study of the Fab fragment of antibody A5 and a comparative analysis of its properties with antibody A17 have been carried out. These antibodies were previously selected for their ability to interact with organophosphorus compounds via covalent catalysis. It has been established that antibody A5 has exceptional specificity for phosphonate X with bimolecular reaction rate constants of 510 ± 20 and 390 ± 20 min–1M–1 for kappa and lambda variants, respectively. 3D-Modeling of antibody A5 structure made it possible to establish that the reaction residue L-Y33 is located on the surface of the active site, in contrast to the A17 antibody, in which the reaction residue L-Y37 is located at the bottom of a deep hydrophobic pocket. To investigate a detailed mechanism of the reaction, A5 antibody mutants with replacements L-R51W and H-F100W were created, which made it possible to perform stopped-flow kinetics. Tryptophan mutants were obtained as Fab fragments in the expression system of the methylotrophic yeast species Pichia pastoris. It has been established that the effectiveness of their interaction with phosphonate X is comparable to the wild-type antibody. Using the data of the stopped-flow kinetics method, significant conformational changes were established in the phosphonate modification process. The reaction was found to proceed using the induced-fit mechanism; the kinetic parameters of the elementary stages of the process have been calculated. The results present the prospects for the further improvement of antibody-based biocatalysts.

Keywords

catalytic antibodies organophosphorus compounds induced-fit mechanism 

Abbreviations

CDR

complementarity determining region

FRW

framework region of the antibody variable domain

VH

variable domain of heavy chain

VL

variable domain of light chain

QM/MM

hybrid quantum mechanics/molecular mechanics modeling

OP

organophosphates

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Copyright information

© Pleiades Publishing, Inc. 2017

Authors and Affiliations

  • Yu. A. Mokrushina
    • 1
  • S. O. Pipiya
    • 1
  • A. V. Stepanova
    • 1
  • O. G. Shamborant
    • 1
  • V. D. Knorre
    • 1
  • I. V. Smirnov
    • 1
    • 2
  • A. G. Gabibov
    • 1
    • 2
  • I. I. Vorobiev
    • 1
    • 3
  1. 1.Shemyakin–Ovchinnikov Institute of Bioorganic ChemistryRussian Academy of SciencesMoscowRussia
  2. 2.Institute of Fundamental Medicine and BiologyKazan (Volga Region) Federal University, KazanRepublic of TatarstanRussia
  3. 3.Institute of Bioengineering, Research Center of BiotechnologyRussian Academy of SciencesMoscowRussia

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