G. Michael Blackburn

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 (N eta 2) and aligns the electron density on N eta 2 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 N eta 2. This enables N eta 2 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.

Nucleic Acids in Chemistry and Biology4th EditionNucleic Acids in Chemistry and Biology4th EditionEdited byG. Michael BlackburnUniversity of Sheffield, UKEmail:

In this chapter we describe methods for the chemical synthesis of modified nucleosides and nucleotides. Because most naturally nucleosides and nucleotides are commercially available, syntheses have been directed at the generation of nucleosides containing modified bases and sugars and nucleotides in which oxygen atoms in the phosphate ester are replaced by other heteroatoms. The enzymatic processes associated with nucleotide metabolism are described and the roles of nucleotides in biology are discussed. Finally, the biological properties of nucleosides as antiviral and anticancer agents are highlighted.

The main DNA repair processes in humans involve direct repair (DR), base excision repair (BER), nucleotide excision repair (NER), interstrand crosslink repair (ICR) and base mismatch repair (BMR), as have been described in other chapters, in addition to homologous recombination (HR) and non-homologous end-joining (NHEJ). Studies on human repair systems have advanced rapidly, especially into UV damage, and recent studies have shown that human DNA polymerase η (Pol η) modulates susceptibility to skin cancer by promoting DNA synthesis past sunlight-induced cyclobutane pyrimidine dimers that have escaped nucleotide excision repair (NER). This bypass has low fidelity, meaning that in normal people, and especially in individuals with xeroderma pigmentosum who accumulate photodimers because they are NER-defective, the errors made by Pol η during dimer bypass may contribute to mutagenesis and to skin cancer. The depth of understanding that is now being achieved on the covalent modification of nucleic acids is awesome, both for adventitious (exogenous) and evolutionarily evolved (endogenous) modification. It is uncovering new questions and posing new challenges. A clear manifestation of this is the range of targets that have now been explored using cutting-edge methodologies that were unimaginable in earlier years. Above all, the advances made have brought us face-to-face with the amazing complexity of repair systems for our nucleic acids that supremely have made viable life on our planet.

Significance: Developed quantum mechanics/molecular mechanics (QM/MM) supercomputation is used to identify maturation of immunoglobulin (Ig) reactivity through optimization of amino acids in the catalytic core, focused on enhanced nucleophilic attack on phosphorus. A computationally optimized variant, l -Leu47Lys, delivers 340-fold reactivity enhancement for paraoxon relative to wild-type Ig-paraoxonase resulting from substrate-induced ionization of the Tyr37 nucleophile. Variant antibody stereoselectivity toward a P-chiral phenylphosphonate toxin results from starkly contrasting solvent-assisted or general base catalytic mechanisms relative to the parent Ig-paraoxonase. These mechanisms are defined by crystallographic and QM/MM analyses as resulting from changed substrate orientation and participating amino acid conformations within the catalytic site.

We report X‐ray crystallographic and ¹⁹F NMR studies of the G‐protein RhoA complexed with MgF₃⁻, GDP, and RhoGAP, which has the mutation Arg85′Ala. When combined with DFT calculations, these data permit the identification of changes in transition state (TS) properties. The X‐ray data show how Tyr34 maintains solvent exclusion and the core H‐bond network in the active site by relocating to replace the missing Arg85′ sidechain. The ¹⁹F NMR data show deshielding effects that indicate the main function of Arg85′ is electronic polarization of the transferring phosphoryl group, primarily mediated by H‐bonding to O^3G and thence to P^G. DFT calculations identify electron‐density redistribution and pinpoint why the TS for guanosine 5′‐triphosphate (GTP) hydrolysis is higher in energy when RhoA is complexed with RhoGAP_Arg85′Ala relative to wild‐type (WT) RhoGAP. This study demonstrates that ¹⁹F NMR measurements, in combination with X‐ray crystallography and DFT calculations, can reliably dissect the response of small GTPases to site‐specific modifications.

We report X‐ray crystallographic and ¹⁹F NMR studies of the G‐protein RhoA complexed with MgF₃⁻, GDP, and RhoGAP, which has the mutation Arg85′Ala. When combined with DFT calculations, these data permit the identification of changes in transition state (TS) properties. The X‐ray data show how Tyr34 maintains solvent exclusion and the core H‐bond network in the active site by relocating to replace the missing Arg85′ sidechain. The ¹⁹F NMR data show deshielding effects that indicate the main function of Arg85′ is electronic polarization of the transferring phosphoryl group, primarily mediated by H‐bonding to O^3G and thence to P^G. DFT calculations identify electron‐density redistribution and pinpoint why the TS for guanosine 5′‐triphosphate (GTP) hydrolysis is higher in energy when RhoA is complexed with RhoGAP_Arg85′Ala relative to wild‐type (WT) RhoGAP. This study demonstrates that ¹⁹F NMR measurements, in combination with X‐ray crystallography and DFT calculations, can reliably dissect the response of small GTPases to site‐specific modifications.

Procedures are proposed for the naming of individual atoms, P, O, F, N, and S in phosphate esters, amidates, thiophosphates, polyphosphates, their mimics, and analogues of transition states for enzyme-catalyzed phosphoryl transfer reactions. Their purpose is to enable scientists in very different fields, e.g. biochemistry, biophysics, chemistry, computational chemistry, crystallography, and molecular biology, to share standard protocols for the labelling of individual atoms in complex molecules. This will facilitate clear and unambiguous descriptions of structural results, as well as scientific intercommunication concerning them. At the present time, perusal of the Protein Data Bank (PDB) and other sources shows that there is a limited degree of commonality in nomenclature, but a large measure of irregularity in more complex structures. The recommendations described here adhere to established practice as closely as possible, in particular to IUPAC and IUBMB recommendations and to “best practice” in the PDB, especially to its atom labelling of amino acids, and particularly to Cahn-Ingold-Prelog rules for stereochemical nomenclature. They are designed to work in complex enzyme sites for binding phosphates but also to have utility for non-enzymatic systems. Above all, the recommendations are designed to be easy to comprehend and user-friendly.