Antlion Strategy for Enzyme Specificity

Abstract

The role of protein conformational dynamics in enzyme specificity and efficiency has held the fascination of enzymologists since the original debates over “lock and key” versus “induced-fit” mechanisms. Recent data has shown that the large, substrate-induced conformational changes are an important governor of enzyme specificity. Molecular dynamics simulations hold the promise for atomically detailed analysis of induced-fit mechanisms, but the large spatial and temporal scales are a challenge. Therefore, conformational transitions and induced fits on the millisecond scale have been studied infrequently and represent a major frontier in computer simulation. Here we investigated induced-fit mechanisms using Directional Milestoning applied to HIV reverse transcriptase for both correct and mismatched base pairs. The predicted rate and free energy profiles agree with available experimental data, including new single molecule kinetic measurements. The substrate-induced conformational change proceeds through a transition-state with motions of up to 25 A in approximately 250 μs. The induced-fit mechanism affords specificity based upon a kinetic rather than a thermodynamic selection, which we liken to the manner in which an antlion captures its prey by digging a hole in the sand and waiting. Ants fall into the hole and are slow to escape and so they are eaten, whereas larger insects, which might see the antlion as prey, rapidly climb out of the hole. The substrate-induced conformational change is rapid and affords fast sampling of the bound nucleotide. A correct nucleotide leads to tight binding and alignment of catalytic residues to promote catalysis while a mismatched base precludes the formation of the tight binding state and the rapid opening of the specificity loop affords release of the mismatch.