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Mutational analysis of CjeATP-PRT identified one particularly interesting mutation site, arginine R216. The mutation to alanine resulted in a mutant enzyme that lacks the allosteric regulation by histidine. The mutated residue is positioned at the interface between helixα8, of its own chain, and helixα10 of the neighbouring chain. This interface undergoes significant changes upon binding of histidine, moving closer together and forming new contacts includ- ing hydrogen bonding interactions with R216. The lack of these contacts in the R216A mutant has been shown to not adversely affect histidine binding. R216 must therefore play a crucial role in the molecular communication of the inhibitory signal.

The kinetic properties of the CjeATP-PRT R216A and CjeATP-PRT Core mutants are in very close agreement. The very similar catalytic turn over rate and the strongly increased sensitivity to AMP and the reaction product PR-ATP are indicative of this fact. The binding of histidine to the R216A mutant was confirmed by ITC, but lacks the initial strong exother- mic signal observed for the wild type, which may be associated with a re- arrangement of the enzyme complex altering the properties of the histidine binding sites. This conformational or energetic change is absent in CjeATP- PRT R216A. Preliminary crystallisation results also indicate that the R216A mutant structure might adopt the histidine inhibited conformation in the presence of ATP, unlike the wild type enzyme. Taken together these findings imply the existence of a permanently or predominantly inhibited conforma- tion of the catalytic core of CjeATP-PRT R216A, as seen for the CjeATP- PRT Core dimer. It is therefore hypothesised that the mutation of R216 has detached the ACT domain motion from the catalytic dimer motion, which effectively broke the transmission of the regulatory signal along this path- way resulting in a fixed “false” signal. The catalytic core is receiving a constant histidine binding signal, resulting in permanently reduced catalytic efficiency, while the “disconnected” ACT domain still allows for effect-less histidine binding.

With only limited crystallographic evidence from the generated mu- tants, little can be inferred about the exact nature of the ATP-PRT inhibi- tion. The AMP bound structure of EcoATP-PRT (1H3D) clearly adopts the open hexamer conformation, leaving doubts about whether the inhibitory ef- fect is solely conformational. The inhibitors AMP and histidine both increase theTmofCjeATP-PRT, while the substrate ATP does not. This leads to the conclusion that the inhibitory signal rigidifies the complex, which supports a change in dynamics. More evidence was brought about by the direct compar- ison of the ATP bound structures of CjeATP-PRT wild type and Core. The conformations of the single chains are very similar and ATP adopts the same binding mode, but the activity of both species is undoubtedly different. This is indicative of the alteration of essential enzyme dynamics by the removal of the ACT domain and consequently also by the break of the R216 contact point.

However, the crystallographic observations on the wild type enzyme support a conformational driven regulatory signal transmission, which in- volves large domain movements that are communicated via hinge points. These movements in turn force the dimer interfaces into different arrange- ments altering the catalytic properties of the ATP-PRT active sites. R216 sits on one of the crucial connection points between domains and a hypo- thetical communication pathway focussing on similar connections is shown in Figure 5.2). The pathway naturally includes the residues of the histidine binding site in the ACT domain and travels along inter-subunit interactions. The α1-β2 loop is thought to be another important connection point. In the histidine bound, closed conformation the loop protrudes into the active site of a neighbouring chain. Therefore the loop may serve as a “sensor” for the binding of the active site inhibitor AMP. This interaction could be the reason for the increased AMP binding affinity of the wild type enzyme in the presence of histidine and thereby explain the synergistic inhibitory effect. Regarding the effects of AMP binding on histidine binding, the presence of AMP enables loop interactions, which might lower the energy required for the conformational change of the hexamer. The C-terminal side of the α1-

Figure 5.2: Hypothetical inhibitory signal communication pathway. Car- toon representation of three adjacent chains in the CjeATP-PRT crystal structure 4YB6 with the third chain (dark green) only partially shown. A schematic pathway (solid red lines) between the allosteric and active sites (black circles) is superim- posed onto the three dimensional image, highlighting potential key contacts in the transmission of the regulatory signal. The dashed red line depicts the lost interactions between theα1-β2 loop and helix α7 at the dimer interface.

β2 loop in turn is part of the open dimer interface interacting closely with residues of helix α7. A connection that is completely removed in the closed conformation. This might also direct the inhibitory signal flow from the dimer interface to the active site.

This is not the first report of uncoupled activity and feedback regulation in a long form ATP-PRT. The complete truncation of the regulatory domain of CglATP-PRT generated a mutant that is not responsive to histidine and also has a reduced specific activity. These results are largely consistent with the findings on the CjeATP-PRT enzyme presented in this thesis and vali- date that similar mechanisms for the inhibitory signal transmission exist in all long form ATP-PRT enzymes. Furthermore the debilitating effect of the ACT domain removal was able to be partially compensated by a single site mutant, S143F. This mutation did not only increase the specific activity of the CglATP-PRT truncation mutant, but it also removed the feedback in- hibition of histidine in the full length CglATP-PRT enzyme.98 The site of this randomly generated mutation, and the equivalentCjeATP-PRT residue A158, is positioned in the α6 helix at the dimer interface. From a crystal- lographic view point this mutation site has no obvious involvement in any regulatory pathway other than its position close to the dimer interface. The change to a large hydrophobic side chain most likely disrupts the position- ing of the two chains relative to each other. This leads to the assumption that this conformational change positively alters the catalytic abilities of the enzyme by “fixing” the dimer interface in the active conformation, directly opposite to the effect caused by the CjeATP-PRT R216A mutation.

Overall the presented results strongly support a simple physical mech- anism for the allosteric regulation of long form ATP-PRT enzymes by histi- dine. Two different hexamer conformations exist that differ greatly in their exhibited catalytic properties. In the absence of histidine, the equilibrium between the two states favours the active conformation, whereas histidine binding stabilises the inactive conformation and drives the equilibrium in the other direction. Due to the architecture of the enzyme and the nature of the ACT domain interfaces, the conformational change always affects the entire hexamer, with all chains adopting the same conformation. These de- scribed characteristics are consistent with the concerted MWC model67 _of