Isotope effects reveal that para-substituted benzylamines are poor reactivity probes of the quinoprotein mechanism for aromatic amine dehydrogenase.
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.
Hothi
Parvinder
P
Manchester Interdisciplinary Biocentre, Faculty of Life Sciences, University of Manchester, UK.
Roujeinikova
Anna
A
Khadra
Khalid Abu
KA
Lee
Michael
M
Cullis
Paul
P
Leys
David
D
Scrutton
Nigel S
NS
D001596:Benzylamines / Q000138:chemical synthesis / Q000737:chemistry* / Q000378:metabolism; D002384:Catalysis; D018360:Crystallography, X-Ray; D003903:Deuterium / Q000737:chemistry / Q000378:metabolism; D006860:Hydrogen Bonding; D007553:Isotope Labeling; D007700:Kinetics; D000587:Oxidoreductases Acting on CH-NH Group Donors / Q000737:chemistry* / Q000378:metabolism; D010627:Phenethylamines / Q000737:chemistry / Q000378:metabolism; D021281:Quantitative Structure-Activity Relationship
D001596:Benzylamines; D010627:Phenethylamines; D003903:Deuterium; D000587:Oxidoreductases Acting on CH-NH Group Donors; C037193:aromatic amine dehydrogenase