Enzymatic turnover of macromolecules generates long-lasting protein-water-coupled motions beyond reaction steady state.

Abstract

The main focus of enzymology is on the enzyme rates, substrate structures, and reactivity, whereas the role of solvent dynamics in mediating the biological reaction is often left aside owing to its complex molecular behavior. We used integrated X-ray- and terahertz- based time-resolved spectroscopic tools to study protein-water dynamics during proteolysis of collagen-like substrates by a matrix metalloproteinase. We show equilibration of structural kinetic transitions in the millisecond timescale during degradation of the two model substrates collagen and gelatin, which have different supersecondary structure and flexibility. Unexpectedly, the detected changes in collective enzyme-substrate-water-coupled motions persisted well beyond steady state for both substrates while displaying substrate-specific behaviors. Molecular dynamics simulations further showed that a hydration funnel (i.e., a gradient in retardation of hydrogen bond (HB) dynamics toward the active site) is substrate-dependent, exhibiting a steeper gradient for the more complex enzyme-collagen system. The long-lasting changes in protein-water dynamics reflect a collection of local energetic equilibrium states specifically formed during substrate conversion. Thus, the observed long-lasting water dynamics contribute to the net enzyme reactivity, impacting substrate binding, positional catalysis, and product release.