On the use of the experimentally determined enzyme inhibition constant as a measure of absolute binding affinity

ABSTRACT Defined as a state function representing an inhibitor’s absolute affinity for its target enzyme, the experimentally determined enzyme inhibition constant ( K i ) is widely used to rank order binding affinities of different inhibitors for a common enzyme or different enzymes for a common inhibitor and to benchmark computational approaches to predicting binding affinity. Herein, we report that adsorption of bis(7)-tacrine to the glass container surface increased its K i against Electrophorus electricus acetylcholinesterase ( ee AChE) to 3.2 ± 0.1 nM (n = 5) compared to 2.9 ± 0.4 pM (n = 5) that was determined using plastic containers with other assay conditions kept the same. We also report that, due to binding or “adsorption” of bis(7)-tacrine to the inactive ee AChE, the bis(7)-tacrine K i increased from 2.9 ± 0.4 pM (n = 5) to 734 ± 70 pM (n = 5) as the specific ee AChE activity decreased from 342 U/mg to 26 U/mg while other assay conditions were kept the same. These results caution against using K i s to rank order binding potencies, define selectivity, or benchmark computational methods without knowing detailed assay conditions. Abbreviations K i enzyme inhibition constant AChE acetylcholinesterase ee AChE Electrophorus electricus AChE ATCh acetylthiocholine chloride bis(7)-tacrine 1,7- N -heptylene-bis-9,9'-amino-1,2,3,4-tetrahydro-acridinium dihydrochloride DTNB 5,5’-dithiobis(2-nitrobenzoic acid) SEA specific enzyme activity tacrine 9-amino-1,2,3,4-tetrahydroacridinium monohydrochloride.