Characterizing Active Site Conformational Heterogeneity along the Trajectory of an Enzymatic Phosphoryl Transfer Reaction

Abstract

States along the phosphoryl transfer reaction catalyzed by the nucleoside monophosphate kinase UmpK were captured and changes in the conformational heterogeneity of conserved active site arginine side‐chains were quantified by NMR spin‐relaxation methods. In addition to apo and ligand‐bound UmpK, a transition state analog (TSA) complex was utilized to evaluate the extent to which active site conformational entropy contributes to the transition state free energy. The catalytically essential arginine side‐chain guanidino groups were found to be remarkably rigid in the TSA complex, indicating that the enzyme has evolved to restrict the conformational freedom along its reaction path over the energy landscape, which in turn allows the phosphoryl transfer to occur selectively by avoiding side reactions.

Assessment of enzyme active site positioning and tests of catalytic mechanisms through X-ray-derived conformational ensembles

“…Our ensemble data provided evidence against each model for KSI. Similarly, prior NMR data suggested that most of the substrate positioning needed to form a new bond occurs in forming the enzyme•substrate complex of the UMP/CMP kinase reaction (Zeymer et al, 2016). Future ensemble determinations with additional enzymes will test the generality of these conclusions.…”

Arginine Side‐Chain Hydrogen Exchange: Quantifying Arginine Side‐Chain Interactions in Solution

“…Moreover, small changes in temperature and particularly in pH affect the rates substantially, thus generally allowing for a large range of arginine side‐chains to be characterised. It is envisaged that the new method serves as a particularly valuable tool to characterise active sites in enzymes,, protein‐protein or protein‐nucleic acid interactions, and phase separation, where arginine residues are expected to play a crucial role for biological function.…”

“…Whilst a knowledge of protein backbone behaviour is often imperative to understand many aspects of protein functions, it is typically the side‐chains that directly mediate activity and participate in the important and functional interactions. Arginine residues are often crucial to many biological interaction interfaces because the flexible arginine side‐chain, consisting of a chain of aliphatic carbon atoms and a terminal guanidinium group, is capable of an array of hydrogen‐bonds and other interactions …”

“…Arginine residues are often crucial to many biological inter-action interfaces because the flexible arginine side-chain, consisting of a chain of aliphatic carbon atoms and a terminal guanidinium group, is capable of an array of hydrogen-bonds and other interactions. [21][22][23] The arginine guanidinium group has a very high pK a of 14 [24] and the delocalised positive charge is therefore present under all physiologically relevant pH values. [25] Each arginine guanidinium group has a large delocalised p-system for cationp and pÀp interactions [26,27] as well as five guanidinium protons that are available for hydrogen-bonding and salt-bridging, but generally labile and able to exchange with the bulk solvent.…”

Determining isoleucine side-chain rotamer-sampling in proteins from 13C chemical shift

“…Protein side chains have the ability to sample several conformations, which is important for many biological processes. Side-chain motions are often de-correlated from the backbone, [1][2][3] making it important to be able to specifically characterise them. The structure and conformational sampling of side chains are often derived from a combination of several NMR measurements, such as nuclear Overhauser effects (NOEs), three-bond scalar couplings, 4,5 residual dipolar couplings and spin-relaxation measurements.…”