Figure 1.
Enzyme structures can be categorized according to the fate of the bound nucleoside triphosphate (NTP).
A) Phosphoryl transfer in which the O3β―Pγ bond is cleaved. B) Reactions in which the Pα―O3α 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.
Figure 2.
Models used to test the dependence of hyperconjugation and O3β―Pγ bond length on enzyme-ligand interactions.
N-methylacetamide (A) was used to model a (neutral) protein backbone amide hydrogen bond to O3β, 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 Oγ 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 O3β―Pγ bond length. Hydrogen bonds are shown with dashed red lines. White = hydrogen, gray = carbon, red = oxygen, blue = nitrogen, orange = phosphorus.
Figure 3.
Mean number of enzyme-ligand interactions.
Interactions are shown with nonbridging β- (A), bridging β- (B) and bridging α-oxygens (C) in the O3β―Pγ cleaving-, Pα―O3α 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.
Figure 4.
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).
Mean numbers of hydrogen bonds were measured for non-bridging (a & c) and bridging (b & d) oxygens. The catalytic group is composed of both O3β—Pγ and Pα—O3α 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).
Figure 5.
Hyperconjugation in methyl triphosphate, a model for ATP.
Electron density is transferred from a lone pair orbital on one of the γ-oxygens (Oγ), into the antibonding orbital (σ*) of the O3β—Pγ bond. Red lettering indicates the atoms involved in the hyperconjugative interaction and the red arrow represents electron density transfer.
Figure 6.
The O3β—Pγ bond length and hyperconjugation increase with decreasing D•••O3β hydrogen bond length in Structure1.
Calculated E(2) energy is of the n(Oγ)→σ*(O3β—Pγ) hyperconjugative interaction. D is the N—H hydrogen bond donor in N-methylacetamide.
Figure 7.
Effects of a hydrogen bond at O3β on orbital and interaction energies in Structure1.
Shortening the hydrogen bond between N-methylacetamide and methyl triphosphate: A) decreases the orbital energy of the σ*(O3β—Pγ) anti-bonding orbital; while (B) leaving unchanged both the n(Oγ) donor orbital energies and (C) Fi,j, a measure of the overlap between the n(Oγ) lone pair orbitals and σ*(O3β—Pγ). D denotes hydrogen bond donor. σ* denotes σ*(O3β—Pγ).
Figure 8.
Impact of protonation of the γ-oxygens.
Protonation at the γ-oxygens decreases the effects of the O3β hydrogen bond on both A) the magnitude of hyperconjugation and B) the O3β—Pγ bond length. Changes are calculated relative to corresponding structures without a D•••O3β hydrogen bond. The ability of interactions with the O3β to increase the scissile bond depends substantially on the presence of strong intrinsic hyperconjugation in the ligand.
Figure 9.
Impact of (secondary) interactions with γ-oxygens on O3β—Pγ bond elongation induced by (primary) O3β interactions.
Secondary interactions with γ-oxygens have modest impact (much smaller than the direct effects characterized by Summerton et al. [16]) and are substantial only for charged donors.
Figure 10.
Impact of a hydrogen bond with a non-bridging β-oxygen.
An interaction between N-methylacetamide and a non-bridging β-oxygen of methyl triphosphate decreases (A) the hyperconjugation and (B) scissile bond length. Interactions with the non-bridging β-oxygen (filled diamonds, Structure 7) are compared to those for O3β (open diamonds, Structure 1). Changes are measured relative to methyl triphosphate absent a hydrogen bond.
Figure 11.
Saturation of NTP oxygen lone pairs by enzyme or solvent interactions in O3β—Pγ-cleaving active sites.
Bars denote standard errors.
Figure 12.
Arginine kinase active site mutation.
Selected interactions of the nucleotide in the crystal structure of the transition state analog complex of Horseshoe Crab Arginine Kinase (AK, PDB ID 1M15) [48]. Carbon = green, nitrogen = dark blue, oxygen = red and phosphorus = orange. In AK, Arg280 contacts the O3β oxygen of ADP, an α-oxygen and the Asp324 side chain. In ATP, the O3β 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.