Structural basis for catalytically restrictive dynamics of a high-energy enzyme state

Abstract

An emerging paradigm in enzymology is that transient high-energy structural states play crucial roles in enzymatic reaction cycles. Generally, these high-energy or ‘invisible' states cannot be studied directly at atomic resolution using existing structural and spectroscopic techniques owing to their low populations or short residence times. Here we report the direct NMR-based detection of the molecular topology and conformational dynamics of a catalytically indispensable high-energy state of an adenylate kinase variant. On the basis of matching energy barriers for conformational dynamics and catalytic turnover, it was found that the enzyme's catalytic activity is governed by its dynamic interconversion between the high-energy state and a ground state structure that was determined by X-ray crystallography. Our results show that it is possible to rationally tune enzymes' conformational dynamics and hence their catalytic power—a key aspect in rational design of enzymes catalysing novel reactions.

Urea-Dependent Adenylate Kinase Activation following Redistribution of Structural States

“…[4][5][6] (denoted ''model 1'') well, yielding DG 0 fold and m fold values of 13.7 kJ mol À1 and 7.2 kJ mol À1 M À1 , respectively (Fig. 2).…”

“…In protein folding, the m value is proportional to the difference in SAS area between the folded ground state and transition state for the folding/unfolding process. Because the catalytic rate of Adk is limited by slow opening of the substrate-binding domains in the presence of bound nucleotides (5,6), the m act value will be proportional to the difference in SAS area between the closed state and the transition state associated with the opening reaction.…”

“…From a physical perspective, this result indicates that other aspects of Adk catalysis dampen the effect of binding affinity on the reaction rate, possibly via modulation of the energy of the transition state separating open and closed structures in the presence of bound substrate. This interpretation is based on the conclusion that the rate of Adk catalysis is limited by the free energy barrier for opening of the closed transition state in the presence of bound substrate (5,6). Nevertheless, the analysis provides experimental evidence of energetic coupling between Adk activity and ligand-binding affinity.…”

“…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.…”

Protein dynamics and function from solution state NMR spectroscopy

“…The catalytic model for Adk catalysis (Wolf-Watz et al 2004) contains an additional highenergy state corresponding to a substrate-bound open conformation. This state has now been directly observed by NMR spectroscopy (Kovermann et al 2015) and it was shown that the interconversion of this high-energy state with a ground state structure that has been characterized by x-ray crystallography is rate limiting for catalysis (as discussed further below).…”