Isotope effects reveal that para-substituted benzylamines are poor reactivity probes of the quinoprotein mechanism for aromatic amine dehydrogenase

Biochemistry. 2007 Aug 14;46(32):9250-9. doi: 10.1021/bi7007239. Epub 2007 Jul 18.


Structure-activity correlations have been employed previously in the mechanistic interpretation of TTQ-dependent amine dehydrogenases using a series of para-substituted benzylamines. However, by combining the use of kinetic isotope effects (KIEs) and crystallographic analysis, in conjunction with structure-reactivity correlation studies, we show that para-substituted benzylamines are poor reactivity probes for TTQ-dependent aromatic amine dehydrogenase (AADH). Stopped-flow kinetic studies of the reductive half-reaction, with para-substituted benzylamines and their dideuterated counterparts, demonstrate that C-H or C-D bond breakage is not fully rate limiting (KIEs approximately unity). Contrary to previous reports, Hammett plots exhibit a poor correlation of structure-reactivity data with electronic substituent effects for para-substituted benzylamines and phenylethylamines. Crystallographic studies of enzyme-substrate complexes reveal that the observed structure-reactivity correlations are not attributed to distinct binding modes for para-substituted benzylamines in the active site, although two binding sites for p-nitrobenzylamine are identified. We identify structural rearrangements, prior to the H-transfer step, which are likely to limit the rate of TTQ reduction by benzylamines. This work emphasizes (i) the need for caution when applying structure-activity correlations to enzyme-catalyzed reactions and (ii) the added benefit of using both isotope effects and structural analysis, in conjunction with structure-reactivity relationships, to study chemical steps in enzyme reaction cycles.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Benzylamines / chemical synthesis
  • Benzylamines / chemistry*
  • Benzylamines / metabolism
  • Catalysis
  • Crystallography, X-Ray
  • Deuterium / chemistry
  • Deuterium / metabolism
  • Hydrogen Bonding
  • Isotope Labeling
  • Kinetics
  • Oxidoreductases Acting on CH-NH Group Donors / chemistry*
  • Oxidoreductases Acting on CH-NH Group Donors / metabolism
  • Phenethylamines / chemistry
  • Phenethylamines / metabolism
  • Quantitative Structure-Activity Relationship


  • Benzylamines
  • Phenethylamines
  • Deuterium
  • Oxidoreductases Acting on CH-NH Group Donors
  • aromatic amine dehydrogenase

Associated data

  • PDB/2HJ4
  • PDB/2HJB
  • PDB/2Q7Q