This Perspective addresses the unresolved, and still hotly contested, question of how enzymes transition from stable enzyme-substrate (ES) complexes to successful, femtosecond barrier crossings. By extending Marcus theory to enzyme-catalyzed reactions, we argue that environmental reorganization of the protein scaffold, together with associated water molecules, achieves the intersection of reactant and product potential energy surfaces. After discussing the experimentally demonstrated importance of reduced activation enthalpy in enzyme-catalyzed transformations, we describe new methodologies that measure the temperature dependence of (i) time-averaged hydrogen/deuterium exchange into backbone amides and (ii) time-dependent Stokes shifts to longer emission wavelengths in appended chromophores at the protein/water interface. These methods not only identify specific pathways for the transfer of thermal energy from solvent to the reacting bonds of bound substrates but also suggest that collective thermally activated protein restructuring must occur very rapidly (on the ns–ps time scale) over long distances. Based on these findings, we introduce a comprehensive model for how barrier crossing takes place from the ES complex. This exploits the structural preorganization inherent in protein folding and subsequent conformational sampling, which optimally positions essential catalytic components within ES ground states and correctly places reactive bonds in the substrate(s) relative to embedded energy transfer networks connecting the protein surface to the active site. The existence of these anisotropic energy distribution pathways introduces a new dimension into the ongoing quest for improved de novo enzyme design.

DNA-joining by ligase and polymerase enzymes has provided the foundational tools for generating recombinant DNA and enabled the assembly of gene and genome-sized synthetic products. Xenobiotic nucleic acid (XNA) analogues of DNA and RNA with alternatives to the canonical bases, so-called 'unnatural' nucleobase pairs (UBP-XNAs), represent the next frontier of nucleic acid technologies, with applications as novel therapeutics and in engineering semi-synthetic biological organisms. To realise the full potential of UBP-XNAs, researchers require a suite of compatible enzymes for processing nucleic acids on a par with those already available for manipulating canonical DNA. In particular, enzymes able to join UBP-XNA will be essential for generating large assemblies and also hold promise in the synthesis of single-stranded oligonucleotides. Here, we review recent and emerging advances in the DNA-joining enzymes, DNA polymerases and DNA ligases, and describe their applications to UBP-XNA manipulation. We also discuss the future directions of this field which we consider will involve two-pronged approaches of enzyme biodiscovery for natural UBP-XNA compatible enzymes, coupled with improvement by structure-guided engineering.

Advances in X-ray crystallography and cryogenic electron microscopy (cryo-EM) offer the promise of elucidating functionally relevant conformational changes that are not easily studied by other biophysical methods. Here we show that 3D variability analysis (3DVA) of the cryo-EM map for wild-type (WT) human asparagine synthetase (ASNS) identifies a functional role for the Arg-142 side chain and test this hypothesis experimentally by characterizing the R142I variant in which Arg-142 is replaced by isoleucine. Support for Arg-142 playing a role in the intramolecular translocation of ammonia between the active site of the enzyme is provided by the glutamine-dependent synthetase activity of the R142 variant relative to WT ASNS, and MD simulations provide a possible molecular mechanism for these findings. Combining 3DVA with MD simulations is a generally applicable approach to generate testable hypotheses of how conformational changes in buried side chains might regulate function in enzymes.

Arginine phosphorylation plays numerous roles throughout biology. Arginine kinase (AK) catalyzes the delivery of an anionic phosphoryl group (PO₃^–) from ATP to a planar, trigonal nitrogen in a guanidinium cation. Density functional theory (DFT) calculations have yielded a model of the transition state (TS) for the AK-catalyzed reaction. They reveal a network of over 50 hydrogen bonds that delivers unprecedented pyramidalization and out-of-plane polarization of the arginine guanidinium nitrogen (Nη2) and aligns the electron density on Nη2 with the scissile P–O bond, leading to in-line phosphoryl transfer via an associative mechanism. In the reverse reaction, the hydrogen-bonding network enforces the conformational distortion of a bound phosphoarginine substrate to increase the basicity of Nη2. This enables Nη2 protonation, which triggers PO₃^– migration to generate ATP. This polarization–pyramidalization of nitrogen in the arginine side chain is likely a general phenomenon that is exploited by many classes of enzymes mediating the post-translational modification of arginine.