Probing the Transition State in Enzyme Catalysis by High-Pressure NMR Dynamics

Abstract

Protein conformational changes are frequently essential for enzyme catalysis, and in several cases, shown to be the limiting factor for overall catalytic speed. However, a structural understanding of corresponding transition states, needed to rationalize the kinetics, remains obscure due to their fleeting nature. Here, we determine the transition-state ensemble of the rate-limiting conformational transition in the enzyme adenylate kinase, by a synergistic approach between experimental high-pressure NMR relaxation during catalysis and molecular dynamics simulations. By comparing homologous kinases evolved under ambient or high pressure in the deep-sea, we detail transition state ensembles that differ in solvation as directly measured by the pressure dependence of catalysis. Capturing transition-state ensembles begins to complete the catalytic energy landscape that is generally characterized by structures of all intermediates and frequencies of transitions among them.

Figure 1 |. Pressure dependence of turnover for mesoAdk vs. piezoAdk to gain insight into transition states.

(a) Simplified Adk free-energy landscape showing only the rate-limiting step of lid-opening. The ground states (open and closed conformations of mesoAdk; core is shown in red, ATP- and AMP-lids in gold and nucleotides in grey) and pressure-dependent rates of opening and closing (k_open[P], k_close[P]) can be determined experimentally, but not the nature of the TSE (indicated by ‡). Dotted lines depict possible transition pathways with arrows indicting the possible TSE locations along the reaction coordinate. (b) MesoAdk and piezoAdk enzyme activity as a function of pressure measured using a coupled assay, described in methods, under saturating nucleotide concentrations at 20 °C. Insert shows the time traces of ATP production. Uncertainties of each point were calculated from the standard deviation of triplicate measurements. Enzymatic rates were fitted only to a first order pressure function (Eq. 1, red and blue lines) as the ln (k_cat) as the quadratic term was not significant (p < 0.01) as determined by F-test.

Figure 2 |. Dynamic and structural differences of Adk under pressure.

(a) ¹⁵N-TROSY CPMG relaxation dispersion NMR experiments at 20 °C for mesoAdk and piezoAdk saturated with Mg²⁺/ADP between 0.1-50 MPa. Representative residues for both enzymes are shown, each having a clear pressure dependence that results in a reduced exchange contribution with pressure. Residues shown are V196 and A176 for mesoAdk and piezoAdk, respectively. Uncertainties in R_2,eff were estimated from the variance in intensity of non-exchanging residues. (b) Sequence alignment of mesoAdk and piezoAdk (identity in blue, differences in red). (c) Sequence differences colored onto piezoAdk structure (4K46; identity in blue, non-identity in red, nucleotides in gray). (d) Structural alignment of mesoAdk (red) and piezoAdk (blue) performed using THESEUS. Residues that may induce ΔV^‡ are depicted in stick representation with several examples enlarged.

Figure 3 |. Putative transition-state ensemble identified from differential solvation in TMD simulations.

(a) Snapshots taken along the piezoAdk’s TMD trajectory from closed to open states. The dotted circle indicates where along the trajectory notable solvation differences are observed for mesoAdk and piezoAdk. (b-c) Close-up showing the lid-interface for mesoAdk (b) and piezoAdk (c) at onset of the TMD trajectories. Relevant side chains are labeled, water molecules are shown in red and white, and surface representation is used to show the boundary of solvent and protein. Insets of whole Adk are shown to illustrate the domain topology and salt-bridge location at each snapshot. (d-e) Close-up showing the difference in solvation for mesoAdk (d) and piezoAdk (e) upon initial lid-opening (dotted circle in a). For piezoAdk, an influx of water is observed due to separation of E157 and K50, whereas for mesoAdk, a direct salt bridge between K157 and D54 prevents hydration. (f) Double mutant K157E/Q160K mesoAdk enzyme activity as a function of pressure (same conditions and error analysis as in Fig. 1B). (g) Representative profiles (D61) for ¹⁵N TROSY-CPMG relaxation dispersion of K157E/Q160K mesoAdk saturated with Mg²⁺/ADP between 0.1-50 MPa. Corresponding ΔV^‡-closed calculated for lidopening from a global fit (Table 1) matches the ΔV^‡ observed from enzyme turnover (f). Errors calculated as described in Fig 2.

Figure 4 |. Triple mutation induces full pressure activation in mesoAdk.

(a-b) Close-up showing the Core/AMP-lid interface for mesoAdk (a) and piezoAdk (b) at start of TMD trajectories (same snapshot setup as Fig. 3). (c-d) Close-up illustrating the difference in solvation for mesoAdk (c) and piezoAdk (d) caused by a disrupted salt bridge in piezoAdk. In mesoAdk, E170 maintains dual interactions between the backbone of L58 and the charged group of K57. In contrast, piezoAdk’s V170 possesses neither interaction. Consequently, water molecules spill into the interface earlier in the opening trajectory. (e) Comparison of ΔV^‡ measured by enzyme turnover and NMR dynamics for mesoAdk, piezoAdk, and mutant forms of mesoAdk. Uncertainties were calculated as described in the legend of Table 1. (f) Residues mutated in mesoAdk variants are plotted onto the crystal structure (2ECK). Lid-interface mutations (green) increase the activation volume, while control mutants away from the interface (purple) had no effect. Nucleotides are shown in gray.