Urea-Dependent Adenylate Kinase Activation following Redistribution of Structural States

Abstract

Proteins are often functionally dependent on conformational changes that allow them to sample structural states that are sparsely populated in the absence of a substrate or binding partner. The distribution of such structural microstates is governed by their relative stability, and the kinetics of their interconversion is governed by the magnitude of associated activation barriers. Here, we have explored the interplay among structure, stability, and function of a selected enzyme, adenylate kinase (Adk), by monitoring changes in its enzymatic activity in response to additions of urea. For this purpose we used a 31 P NMR assay that was found useful for heterogeneous sample compositions such as presence of urea. It was found that Adk is activated at low urea concentrations whereas higher urea concentrations unfolds and thereby deactivates the enzyme. From a quantitative analysis of chemical shifts, it was found that urea redistributes preexisting structural microstates, stabilizing a substrate-bound open state at the expense of a substrate-bound closed state. Adk is rate-limited by slow opening of substrate binding domains and the urea-dependent redistribution of structural states is consistent with a model where the increased activity results from an increased rate-constant for domain opening. In addition, we also detected a strong correlation between the catalytic free energy and free energy of substrate (ATP) binding, which is also consistent with the catalytic model for Adk. From a general perspective, it appears that urea can be used to modulate conformational equilibria of folded proteins toward more expanded states for cases where a sizeable difference in solvent-accessible surface area exists between the states involved. This effect complements the action of osmolytes, such as trimethylamine N-oxide, that favor more compact protein states.

Structural Basis for GTP versus ATP Selectivity in the NMP Kinase AK3

“…Linear free energy relationship between substrate turnover and binding affinities. A linear free energy relationship between substrate turnover and binding affinity has previously been established for AK eco . The ΔΔ G values are calculated relative to wild-type AK eco .…”

“…In GTP the free electron pairs at the O 6 oxygen serves as hydrogen bond acceptors, while the hydrogens on the N 6 atom of ATP serves as hydrogen bond donors. A linear free energy relationship between substrate turnover and binding affinity has previously been established for AK eco 53 . The ΔΔG values are calculated relative to wild-type AK eco .…”

Molecular mechanism of ATP versus GTP selectivity of adenylate kinase

“…Therefore, ATP will accumulate as GTP is consumed and the resulting Adk activity would be compromised by phosphorylation of AMP from this "secondary" ATP pool. To accurately measure the Adk activity with GTP as the phosphate donor, we used a 31 P real-time NMR assay (25,26) that is suitable for quantification of k cat for phosphoryl donor compounds other than ATP (SI Appendix, Fig. S1).…”

Moving beyond static snapshots: Protein dynamics and the Protein Data Bank

“…At present there are over 1200 papers with adenylate kinase in the title via PubMed. Enzymology (34,35), genetics (36), crystallography (37)(38)(39)(40), solution scattering (41), NMR (42,43), MD simulations (44,45), thermodynamics and unfolding (46,47), single molecule studies (48,49), hydrogen exchange mass spectrometry (43), various spectroscopies, mutagenesis (50,51), evolution (52,53), phylogeny (54), and bioinformatics (55), and others have all been employed to gain insight regarding the connections between structure, dynamics, and function. There is not a single model for a pathway commensurate with all the studies.…”