Allosteric communication between ligand binding domains modulates substrate inhibition in adenylate kinase

Abstract

Enzymes play a vital role in life processes; they control chemical reactions and allow functional cycles to be synchronized. Many enzymes harness large-scale motions of their domains to achieve tremendous catalytic prowess and high selectivity for specific substrates. One outstanding example is provided by the three-domain enzyme adenylate kinase (AK), which catalyzes phosphotransfer between ATP to AMP. Here we study the phenomenon of substrate inhibition by AMP and its correlation with domain motions. Using single-molecule FRET spectroscopy, we show that AMP does not block access to the ATP binding site, neither by competitive binding to the ATP cognate site nor by directly closing the LID domain. Instead, inhibitory concentrations of AMP lead to a faster and more cooperative domain closure by ATP, leading in turn to an increased population of the closed state. The effect of AMP binding can be modulated through mutations throughout the structure of the enzyme, as shown by the screening of an extensive AK mutant library. The mutation of multiple conserved residues reduces substrate inhibition, suggesting that substrate inhibition is an evolutionary well conserved feature in AK. Combining these insights, we developed a model that explains the complex activity of AK, particularly substrate inhibition, based on the experimentally observed opening and closing rates. Notably, the model indicates that the catalytic power is affected by the microsecond balance between the open and closed states of the enzyme. Our findings highlight the crucial role of protein motions in enzymatic activity.

Elucidating Dynamics of Adenylate Kinase from Enzyme Opening to Ligand Release

“…Significantly, a similar hybrid mode of ligand binding has been proposed by previous simulations by Matsunaga et al, and these results are supported by experiments by Ådén et al, who have extensively studied the surface of ligand binding using ATP as a probe molecule . In addition, our results have implications for understanding the allosteric communication between AMP binding and ATP lid closure rates, which is recently proposed by Scheerer et al In particular, they observed increased rates of ATP lid closure in the presence of AMP, while AMP did not interfere with the binding of ATP to its cognate binding pocket. In a related study, Lu et al proposed that repeated conformational transitions are required for proper substrate binding .…”

“…They also noted different rates of opening between the apo- and holo-state enzymes, with the opening in the holo-state being faster. More recent sm-FRET studies further confirmed this result with the suggestion of multiple conformational transitions before establishing the correct binding poses of reaction substrates . Similarly, Zheng and Cui, utilizing molecular dynamics (MD) simulations and Markov state modeling (MSM), reported opening rates of an apo-state AK that were orders of magnitude faster than the catalytic turnover rate .…”

“…More recent sm-FRET studies further confirmed this result with the suggestion of multiple conformational transitions before establishing the correct binding poses of reaction substrates. 48 Similarly, Zheng and Cui, utilizing molecular dynamics (MD) simulations and Markov state modeling (MSM), reported opening rates of an apo-state AK that were orders of magnitude faster than the catalytic turnover rate. 40 Our own studies have corroborated these results by showing that the time scales of AK opening differ between the reactant and product states; between the two states, the opening in the product state is faster.…”

Allostery can convert binding free energies into concerted domain motions in enzymes

“…30,33,50,56 The apo-enzyme showed one main current level that occasionally switched to a deeper state (M1, IRES M1 = 49.2 ± 0.1 %, kon M1 = 4.66 ± 0.72 s -1 and koff M1 = 55.2 ± 15.8 s -1 , Figure 2A, Figure S3). Previous work indicated that the enzyme might spontaneously close 33,36,41,45,51,53,[67][68][69][70] S2), most likely, representing all possible domain configurations that AK can adopt within the nanopore.…”

“…Structural studies have shown that the LID and NMP domains undergo major conformation changes [31][32][33] , closing over the active site to protect the ligands from hydrolysis. 34 Many studies have attempted to elucidate how dynamics and conformational changes in AK are associated with molecular recognition, catalysis and allostery 33,[35][36][37][38][39][40][41][42][43][44][45][46][47][48][49][50][51] .…”