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 proteinwater 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.solvation dynamics | enzyme catalysis | metalloenzymes I t has now been a century since the Michaelis-Menten (MM) steady-state theory provided a highly satisfactory description of the kinetic behavior of many enzymes (1, 2). The simplest kinetic mechanism with a single substrate assumes that an enzyme E combines with a substrate S to form an ES complex (known as the Michaelis complex), which undergoes an irreversible reaction to form the product P and the original enzyme. When the steady state is reached, it is assumed that the reaction rate is constant and follows the MM equation:This theory has been proven to be true for a wide variety of isolated enzymatic reactions in vitro (3) as well as more complicated reaction schemes involving multiple ligands and/or multisubunit enzymes (4). Recently, however, studies of enzyme dynamics together with single-molecule experiments have questioned the generality of existing kinetics steady-state laws for fluctuating enzyme systems (5-7). Steady-state kinetics were shown to depend on conformational transition rates of enzyme-substrate association, catalysis, and product release (6,8). In addition, the kinetics of simple or isolated reactions can differ from the kinetics of network reactions in a crowded environment as present in the cell (9, 10).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 (12-17), mediates molecular recognition (18-20), or acts as an adhesive to facilitat...

Label-free sensing of the binding state of MUC1 peptide and anti-MUC1 aptamer solution in fluidic chip by terahertz spectroscopy

“…In a previous study, the vibrational modes of water molecules at an active site were found to take along the distinctive fingerprints of the substrate in unique enzyme-substrate complexes [25]. This suggests the existence of a heterogeneous hydrogen bond network that assists molecular recognition and binding.…”

“…Based on existing anti-MUC1 antibodies (e.g., SM3 antibody), the SELEX library was synthesized to provide 4 25 different species of DNA for selection of anti-MUC1 aptamers [13]. Subsequently, an aptamer (anti-MUC1 S2.2) has been confirmed as a functional molecule, specifically binding to a common sequence (APDTRPAPG) of the VTR region of MUC1.…”

Perspective: Watching low-frequency vibrations of water in biomolecular recognition by THz spectroscopy

“…This was the first time study where simultaneously the fast structural as well the fast changes of the aqueous environment could be monitored in real time during enzyme catalysis. 48 Specifically, we investigated the solvation dynamics of human membrane type-1 matrix metalloproteinase (MT1-MMP) using kinetic THz absorption spectroscopy. For the experimental setup (see Fig.…”

“…6 Even more interestingly, we could show that these changes are distinct for distinct substrates, which have different biological functions and are almost absent in case of an enzyme-inhibitor complex. 48 Accompanying molecular dynamics simulation allowed a deeper insight into the underlying molecular mechanism: calculations of the typical hydrogen bond survival time for water molecules on three different locations, i.e., near the active site, near the separated enzyme and substrate, and near the substrate in the Michaelis complex, revealed a gradient of water dynamics near the active site of the metalloprotease MT1-MMP and water network dynamics during the Michaelis complex formation. Specifically, there is a sharp gradient of water dynamics near the active site prior to the binding, i.e., a "hydration funnel" (see Fig.…”

Probing conformational dynamics of an enzymatic active site by an in situ single fluorogenic probe under piconewton force manipulation

“…In recent years, it has been widely identified that conformational dynamics plays a crucial role in regulating enzymatic reactions (2,(4)(5)(6)(7). The advent of sitespecific mutagenesis, single-molecule (SM) spectroscopic technique, and molecular dynamics simulations have demonstrated complex dynamics involving multiple conformational states during a catalytic cycle (4,5,(7)(8)(9)(10)(11)(12)(13). Fundamentally, not only the enzymatic active site conformational dynamics but also the overall conformational fluctuation dynamics of the protein matrix, providing the required entropy, enthalpy, and corresponding energy landscape, has a profound impact on an enzyme to ultimately possess the power of enhancing reaction rates.…”