Illuminating the mechanistic roles of enzyme conformational dynamics

Abstract

Many enzymes mold their structures to enclose substrates in their active sites such that conformational remodeling may be required during each catalytic cycle. In adenylate kinase (AK), this involves a large-amplitude rearrangement of the enzyme's lid domain. Using our method of high-resolution single-molecule FRET, we directly followed AK's domain movements on its catalytic time scale. To quantitatively measure the enzyme's entire conformational distribution, we have applied maximum entropy-based methods to remove photon-counting noise from single-molecule data. This analysis shows unambiguously that AK is capable of dynamically sampling two distinct states, which correlate well with those observed by x-ray crystallography. Unexpectedly, the equilibrium favors the closed, active-site-forming configurations even in the absence of substrates. Our experiments further showed that interaction with substrates, rather than locking the enzyme into a compact state, restricts the spatial extent of conformational fluctuations and shifts the enzyme's conformational equilibrium toward the closed form by increasing the closing rate of the lid. Integrating these microscopic dynamics into macroscopic kinetics allows us to model lid opening-coupled product release as the enzyme's rate-limiting step.conformational equilibrium ͉ rate-limiting step ͉ single-molecule FRET ͉ adenylate kinase P roteins such as enzymes are flexible with a range of motions spanning from picoseconds for localized vibrations to seconds for concerted global conformational rearrangements (1). Despite their randomly fluctuating environment, in which stochastic collisions with solvent molecules drive changes in tertiary structure, enzymes have evolved to catalyze reactions efficiently and specifically. Indeed, conformational transitions have been postulated to play a central role in enzyme functions in a wide variety of ways, including direct contribution to catalysis (2), allosteric regulation (3), and large-scale conformational changes in response to ligand binding (4). Most of our current understanding of structural motions in solution comes from NMR experiments (5) as well as from molecular dynamics simulations (6), approaches that are best suited to study dynamics in the picoto millisecond time scales. Because catalysis in enzymes frequently occurs in the submillisecond to minute time regime, our current understanding of the relationship between enzyme function and conformational dynamics comes from NMR experiments involving relatively localized motions of active site forming loops on the submillisecond time scale (7-10). However, many enzymes contain active sites located in between domains in which large-amplitude, low-frequency domain motions are required to complete their Michaelis-Menten enzyme-substrate complexes. Even simple questions regarding these transitions remain generally unanswered: What is the number and range of conformational states accessible to enzymes during their catalytic cycle? How does the enzyme's conformation respond to interac...

Protein dynamics and function from solution state NMR spectroscopy

“…Adk undergoes a large conformational change in response to ligand binding that shuttles the protein into a closed and active conformation where phosphoryl transfer occurs (Rhoads & Lowenstein, 1968). Because of its favorable properties -specifically, its tendency to yield excellent NMR spectra (Ådén & Wolf-Watz, 2007;Schrank et al 2009), catalysis of a reversible reaction, and large conformational changes (Müller et al 1996;Müller & Schulz, 1992) -Adk has emerged as one of the principal model systems for analyzing the linkage between dynamics and enzymatic turnover (Arora & Brooks, 2007;Hanson et al 2007;Nagarajan et al 2011;Schrank et al 2009;Shapiro et al 2009Shapiro et al , 2000Shapiro & Meirovitch, 2006). In an extensive multi-approach effort, the Kern laboratory has shown that substrate-free Adk from the hyperthermophilic bacterium Aquifex aeolicus samples 'closed like' structures (Henzler-Wildman et al 2007).…”

Analysis of Trajectory Entropy for Continuous Stochastic Processes at Equilibrium

“…t 0 obs (1) Here, [X(t)] is the probability density of observing the trajectory X(t), [X(t)] is the probability density of reaching the same observation via a reference dynamics, and the integration X(t) is a path-integral over all continuous functions of X(t) realizable from the system dynamics. The definition of trajectory entropy in eq 1 is in fact the negative of Kullback−Leibler (KL) divergence that represents the extra information required for encoding [X(t)] relative to that for representing [X(t)].…”

Urea-Dependent Adenylate Kinase Activation following Redistribution of Structural States

“…Similarly, enzymatic activity is often dependent on conformational changes during their reaction cycles, and it has been established for a few enzymes that the dynamic interconversions between structural microstates are rate-limiting for turnover of substrate molecules (3,4). For instance, the rate of the reaction catalyzed by Escherichia coli adenylate kinase (Adk), is limited by slow reopening of substrate-binding subdomains in the presence of bound nucleotides (5)(6)(7), as illustrated in the following minimal reaction scheme. Here, E refers to Adk, S to substrate, P to product, and superscripts open and closed to Adk in open and closed states (Fig.…”