Correlated structural kinetics and retarded solvent dynamics at the metalloprotease active site

Abstract

Solvent dynamics can play a major role in enzyme activity, but obtaining an accurate, quantitative picture of solvent activity during catalysis is quite challenging. Here, we combine terahertz spectroscopy and X-ray absorption analyses to measure changes in the coupled water-protein motions during peptide hydrolysis by a zinc-dependent human metalloprotease. These changes were tightly correlated with rearrangements at the active site during the formation of productive enzyme-substrate intermediates and were different from those in an enzyme–inhibitor complex. Molecular dynamics simulations showed a steep gradient of fast-to-slow coupled protein-water motions around the protein, active site and substrate. Our results show that water retardation occurs before formation of the functional Michaelis complex. We propose that the observed gradient of coupled protein-water motions may assist enzyme-substrate interactions through water-polarizing mechanisms that are remotely mediated by the catalytic metal ion and the enzyme active site.

Terahertz optical measurements of correlated motions with possible allosteric function

“…It is tempting to suggest that the decrease observed is associated with a net blue shift in the vibrations due to the binding, as suggested by the early calculations of Karplus and coworkers (Bruccoleri et al 1986). However, the net decrease observed may be a reflection more of what is happening at the surface of the protein rather than the long-range motions (Grossman et al 2011;Luong et al 2011). The data illustrate several key features of THz absorbance measurements of proteins.…”

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

“…To identify specific structural pathways, we measured proteolysis of the model substrates under pre-steady-state conditions using a stopped-flow apparatus. A surplus of substrate (1:20 E:S ratio) was used to avoid enzyme and substrate diffusion processes (11). In this setup, the FRET substrates are mixed with MT1-MMP, and the changes in fluorescence intensities are recorded ( Fig.…”

“…Using an integrated spectroscopic approach combining stopped-flow X-ray absorption spectroscopy (XAS) and kinetic terahertz (THz) absorption spectroscopy (KITA), we showed that the proteinwater-coupled motions at the enzyme active site were retarded during the formation of the Michaelis complex within the reaction steady state (11). However, in vivo, the substrates of metalloproteinases are often much more complex.…”

“…Moreover, within the classical description, the contribution of the solvent in effecting catalysis is almost completely neglected, despite its potential role in mediating enzyme-substrate interactions (11). Importantly, the solvent plays an active role in various protein reactions (12)(13)(14)(15)(16)(17), mediates molecular recognition (18)(19)(20), or acts as an adhesive to facilitate binding (21).…”

“…Recently, we reported the correlation between enzyme structural kinetics and protein-water-coupled motions during the formation of the Michaelis complex between a zinc-dependent protease [membrane type 1 matrix metalloproteinase (MT1-MMP)] and a short (6-mer) peptide substrate (11). Using an integrated spectroscopic approach combining stopped-flow X-ray absorption spectroscopy (XAS) and kinetic terahertz (THz) absorption spectroscopy (KITA), we showed that the proteinwater-coupled motions at the enzyme active site were retarded during the formation of the Michaelis complex within the reaction steady state (11).…”