Arginine kinase active site mutation.

<p>Selected interactions of the nucleotide in the crystal structure of the transition state analog complex of Horseshoe Crab Arginine Kinase (AK, PDB ID 1M15) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108310#pone.0108310-Yousef2" target="_blank">[48]</a>. Carbon = green, nitrogen = dark blue, oxygen = red and phosphorus = orange. In AK, Arg₂₈₀ contacts the O_3β oxygen of ADP, an α-oxygen and the Asp₃₂₄ side chain. In ATP, the O_3β oxygen bridges to the γ-phosphate which is mimicked by nitrate in this transition state analog complex. Hydrogen bonds are shown with red dotted lines.</p

Saturation of NTP oxygen lone pairs by enzyme or solvent interactions in O_3β—P_γ-cleaving active sites.

<p>Bars denote standard errors.</p

Mean number of enzyme-ligand interactions.

<p>Interactions are shown with nonbridging β- (A), bridging β- (B) and bridging α-oxygens (C) in the O_3β―P_γ cleaving-, P_α―O_3α cleaving- and non-catalytic-NTP-binding sites. The resolution cutoff of structures used is 2.7 Å, and water is not included. Bars show standard errors.</p

Common Hydrogen Bond Interactions in Diverse Phosphoryl Transfer Active Sites

<div><p>Phosphoryl transfer reactions figure prominently in energy metabolism, signaling, transport and motility. Prior detailed studies of selected systems have highlighted mechanistic features that distinguish different phosphoryl transfer enzymes. Here, a top-down approach is developed for comparing statistically the active site configurations between populations of diverse structures in the Protein Data Bank, and it reveals patterns of hydrogen bonding that transcend enzyme families. Through analysis of large samples of structures, insights are drawn at a level of detail exceeding the experimental precision of an individual structure. In phosphagen kinases, for example, hydrogen bonds with the O_3β of the nucleotide substrate are revealed as analogous to those in unrelated G proteins. In G proteins and other enzymes, interactions with O_3β have been understood in terms of electrostatic favoring of the transition state. Ground state quantum mechanical calculations on model compounds show that the active site interactions highlighted in our database analysis can affect substrate phosphate charge and bond length, in ways that are consistent with prior experimental observations, by modulating hyperconjugative orbital interactions that weaken the scissile bond. Testing experimentally the inference about the importance of O_3β interactions in phosphagen kinases, mutation of arginine kinase Arg₂₈₀ decreases k_cat, as predicted, with little impact upon K_M.</p></div

Effects of a hydrogen bond at O_3β on orbital and interaction energies in Structure1.

<p>Shortening the hydrogen bond between N-methylacetamide and methyl triphosphate: A) decreases the orbital energy of the σ*(O_3β—P_γ) anti-bonding orbital; while (B) leaving unchanged both the n(O_γ) donor orbital energies and (C) F_i,j, a measure of the overlap between the n(O_γ) lone pair orbitals and σ*(O_3β—P_γ). D denotes hydrogen bond donor. σ* denotes σ*(O_3β—P_γ).</p

Enzyme structures can be categorized according to the fate of the bound nucleoside triphosphate (NTP).

<p>A) Phosphoryl transfer in which the O_3β―P_γ bond is cleaved. B) Reactions in which the P_α―O_3α bond is cleaved and C) Structures where the bound NTP does not undergo a chemical reaction. Red lettering indicates the atoms in the scissile bond and red arrows depict the transfer of electrons in going from reactants to products.</p

Impact of (secondary) interactions with γ-oxygens on O_3β—P_γ bond elongation induced by (primary) O_3β interactions.

<p>Secondary interactions with γ-oxygens have modest impact (much smaller than the direct effects characterized by Summerton et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108310#pone.0108310-Summerton1" target="_blank">[16]</a>) and are substantial only for charged donors.</p

Non-catalytic (blue) active sites have a preference positively charged hydrogen bond donors (a–b) whereas in catalytic (red) active sites neutral interactions are favored (c–d).

<p>Mean numbers of hydrogen bonds were measured for non-bridging (a & c) and bridging (b & d) oxygens. The catalytic group is composed of both O_3β—P_γ and P_α—O_3α structure sets. Positive donors include Lys, Arg, and His side chains and neutral donors include Asn, Gln, Trp, Ser Thr,Tyr, Cys side chains, the nucleotide O2′ and O3′ oxygens and all backbone nitrogens (except Pro).</p

Models used to test the dependence of hyperconjugation and O_3β―P_γ bond length on enzyme-ligand interactions.

<p>N-methylacetamide (A) was used to model a (neutral) protein backbone amide hydrogen bond to O_3β, using methyl triphosphate to model an NTP nucleotide. Structures 2 (B) and 3 (C) were used to investigate the effects of protonation at the γ-oxygens. Additional active site hydrogen bonds, represented in Structures 4 through 6 (D–F), were used to assess the secondary effects of different types of O_γ hydrogen bonds. Acetamide (E) 1-propylaminium (F) were used to model asparagine and lysine side chains respectively. Structure 7 (G) was used to investigate the impact of hydrogen bonding at a nonbridging β-oxygen on hyperconjugation and O_3β―P_γ bond length. Hydrogen bonds are shown with dashed red lines. White = hydrogen, gray = carbon, red = oxygen, blue = nitrogen, orange = phosphorus.</p