Mixed and non-competitive enzyme inhibition: underlying mechanisms andmechanistic irrelevance of the formal two-site model

The formal mechanism of linear mixed and non-competitive enzyme inhibition implies the binding ofinhibitors to both the active site of the free enzyme in competition with the substrate, and to an allostericsite on the enzyme-substrate complex. However, it is evident from a review of the scientific literature thatthe two-site mechanism is frequently mistaken as the actual underlying mechanism of mixed inhibition. Inthis study, we conducted a comprehensive assessment of the mechanistic relevance of this type of inhib-ition using a statistical approach. By combining a statistical analysis of the inhibition cases documented inthe BRENDA database with a theoretical investigation of inhibition models, we conclude that mixed inhibi-tors exclusively bind to the active site of enzymes. Hence ruling out any implication of allosteric sites anddepriving the two-site model of any mechanistic relevance.