Direct observation of ultrafast large-scale dynamics of an enzyme under turnover conditions

Abstract

The functional cycle of many proteins involves large-scale motions of domains and subunits. The relation between conformational dynamics and the chemical steps of enzymes remains under debate. Here we show that in the presence of substrates, domain motions of an enzyme can take place on the microsecond time scale, yet exert influence on the much-slower chemical step. We study the domain closure reaction of the enzyme adenylate kinase from Escherichia coli while in action (i.e., under turnover conditions), using single-molecule FRET spectroscopy. We find that substrate binding increases dramatically domain closing and opening times, making them as short as ∼15 and ∼45 µs, respectively. These large-scale conformational dynamics are likely the fastest measured to date, and are ∼100-200 times faster than the enzymatic turnover rate. Some active-site mutants are shown to fully or partially prevent the substrate-induced increase in domain closure times, while at the same time they also reduce enzymatic activity, establishing a clear connection between the two phenomena, despite their disparate time scales. Based on these surprising observations, we propose a paradigm for the mode of action of enzymes, in which numerous cycles of conformational rearrangement are required to find a mutual orientation of substrates that is optimal for the chemical reaction.

Keywords: adenylate kinase; enzyme dynamics; single-molecule fluorescence.

Fig. 1.

Single-molecule studies of the conformational dynamics of AK. (A) Structure of the open conformation of AK, with the LID domain in red and the NMPbind domain in blue (based on PDB ID code 4AKE). Donor (cyan) and acceptor (dark yellow) dyes attached to the CORE and LID domains, respectively, are depicted. (B) FRET efficiency histograms of AK in the absence of substrate (royal blue) and in the presence of saturating substrate concentrations (1 mM ATP, 1 mM AMP, and 160 µM ADP, orange), suggesting mostly open and closed conformations, respectively. In the presence of 2.5 μM ATP (with 1 mM AMP and 8.4 μM of ADP), a single broad peak is observed (gray), indicating fast exchange between open and closed conformations. The population with FRET efficiency < 0.2 is due to molecules without an active acceptor.

Fig. 2.

Domain closure dynamics under turnover conditions. (A–C) smFRET trajectories measured at increasing substrate concentration (ATP + ADP; Table S1): 4.2 μM (A), 16.8 μM (B), and 1.16 mM (C), with AMP at a fixed concentration of 1 mM. Top in each part shows donor (blue) and acceptor (light green) signals binned in 20 µs bins, and the Bottom shows the calculated FRET efficiency (orange), and the state of the system at each time bin, as calculated by the Viterbi algorithm based on the H²MM analysis (dark blue). (D) Closing and opening rates (cherry and green circles, respectively) as a function of substrate concentration, obtained from H²MM analysis of a series of smFRET experiments. Error bars represent standard errors of the mean. Continuous lines are fits to the model described in the text, from which closing and opening rate constants for the unbound and bound states of the enzyme were extracted. (E) Free energy profiles for the unbound (purple) and bound (gold) enzyme, calculated from the fitted rates (with an assumed preexponential factor of 1 μs in the Kramers rate expression) (38), indicate that the open conformation is more stable than the closed conformation in the unbound state, with the situation inverted in the bound state. The transition state free energy is reduced in the bound state, leading to faster domain closure dynamics. The curvatures and shapes of the free energy profiles were selected for visualization purposes only.

Scheme 1.

Kinetic model for domain closure of bound and unbound enzyme molecules. The terms in the scheme are defined in the text.

Fig. 3.

smFRET experiments shed light on mechanistic aspects of domain closure. (A) Substrate binding to both domains is required for domain closure. FRET efficiency histograms of AK in the absence of substrate (royal blue), with 50 μM ATP (red), with 50 μM ATP and 1 mM AMP (orange), and with 50 μM ATP, 1 mM AMP, and a suitable concentration of ADP to maintain equilibrium (dashed black line). (B) The two-substrate mimicking inhibitor, AP₅A, leads to a concentration dependence of the closing and opening rates (cherry and green, respectively) similar to ATP. (C) The nonhydrolyzable ATP analog, AMP-PNP, does not fully shift the equilibrium to the closed conformation even at a high concentration. At 50 μM AMP-PNP and 1 mM AMP (red), the FRET efficiency histogram is similar to the one without any substrate (royal blue), while at 1 mM AMP-PNP and 1 mM AMP (orange), the histogram has shifted only halfway to the position obtained with 1 mM ATP and 1 mM AMP and a suitable amount of ADP (dashed black line).

Fig. 4.

Point mutations affect the conformational dynamics of AK. The mutations K13M (A), T15A (B), and R123M (C) reduced the turnover rate to different degrees (Table 1), but fully or partially prevented switching from the unbound FEP to the bound FEP. Indeed, at saturating concentrations of the substrates (1 mM ATP and AMP together with 164 µM ADP for K13M and R123M; 10 mM ATP and 15 mM AMP together with 0.1 mM ADP for T15A), only a small population of the closed conformation was noted in FRET efficiency histograms (orange), compared with the wild-type enzyme (dashed black line). In green are FRET efficiency histograms of the apo mutant enzymes. Insets show the structure of AK with the mutated residues marked in color.