Can microsecond domain motion affect enzymatic activity? A single-molecule FRET study
Final Report Abstract
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 enzyme adenylate kinase (AK), which catalyzes phosphotransfer between ATP to AMP. Using single-molecule FRET spectroscopy, my host Prof. Gilad Haran found that AK’s domain closure reaction is ultrafast, even faster than the overall turnover rate of the enzyme. In order to elucidate which impact these ultrafast motions have on enzymatic activity, we investigated the role of protein dynamics in the inhibition of the enzyme by its own substrate AMP. We show that inhibitory concentrations of AMP lead to a faster and more cooperative domain closure by ATP. The effect of AMP on activity and dynamics is modulated through mutations throughout the structure of the enzyme. The mutation of multiple evolutionary well-conserved residues reduces inhibition, suggesting that substrate inhibition is a functionally important feature in AK. Combining these insights, we developed a model that explains the complex activity of AK based on the experimentally observed opening and closing rates. We utilized this model to explain the surprising activation of the enzyme by urea. Urea commonly acts as a denaturant which abolishes the activity of a protein by unfolding it. However, minor concentrations can actually increase the activity of AK, and we were able to connect this behavior to its substrate inhibition. Urea influences the protein dynamics and nucleotide binding rates of the protein, causing a weaker inhibition by AMP and therefore a higher overall turnover. In a third project, we seek for a mechanistic understanding of our experimental observations. In collaboration with Prof. Wenfei Li, we showed that the repeated conformational transitions of AK are essential for the relaxation of incorrectly bound substrates into the catalytically competent conformation. Only if the enzyme can open and close very fast, substrates molecules that are incorrectly bound can find a way towards a configuration that allows for catalysis. Our findings on AK suggest that the relative occupancy of the different conformations and their fast interconversion are essential for maximizing catalytic activity. However, it is not clear yet whether this behavior is specific to AK or applies to other enzymes as well. In the enzyme phosphoglycerate kinase (PGK), which holds a key role in glycolysis, the catalytic reaction is accompanied by a large-scale hinge-bending motion, rendering it a good target for this evaluation. Multiple double-labeled, functional protein variants allowed us to detect potential domain movements from a multitude of angles. Within each domain, our smFRET results reveal only very limited movement. In contrast, when we studied the distances of the domains relative to each other, large-scale movements were detected. We can describe the dynamics of the protein in terms of 3 states, which population depends on the substrate conditions. The population shift upon substrate binding can be correlated very well to the protein's enzymatic activity. In summary, the ability to undergo fast conformational changes seems to be similarly crucial for PGK as for AK, suggesting that this is a widespread phenomenon that influences the activity of many proteins.
Publications
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Role of Repeated Conformational Transitions in Substrate Binding of Adenylate Kinase. The Journal of Physical Chemistry B, 126(41), 8188-8201.
Lu, Jiajun; Scheerer, David; Haran, Gilad; Li, Wenfei & Wang, Wei
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Allosteric communication between ligand binding domains modulates substrate inhibition in adenylate kinase. Proceedings of the National Academy of Sciences, 120(18).
Scheerer, David; Adkar, Bharat V.; Bhattacharyya, Sanchari; Levy, Dorit; Iljina, Marija; Riven, Inbal; Dym, Orly; Haran, Gilad & Shakhnovich, Eugene I.
