The enzyme adenosine triphosphate phosphoribosyltransferase (ATP-PRT)
catalyses the first reaction of the histidine biosynthetic pathway. ATP-PRT
also represents a metabolic control point, directing the flux of metabolites
through this energetically expensive pathway. Two distinctly different forms
of ATP-PRT exist, the long form and the short form, which differ in the
presence of a C-terminal regulatory domain. In the short form, where this
domain is absent, it is functionally replaced by a regulatory protein, called
HisZ. ATP-PRT activity is modulated by two layers of regulation: active site
inhibition by adenosine monophosphate, which reflects cellular energy levels, and pathway end product feedback inhibition by histidine. In the long form ATP-PRT histidine binds to the allosteric site at the regulatory domain, but the exact nature of the inhibitory mechanism is still debated.
This thesis characterises a new member of the ATP-PRT long form from Campylobacter jejuni (CjeATP-PRT) and investigates the molecular mechanisms involved in the feed back inhibition by histidine.
Chapter 2 describes the characterisation of the CjeATP-PRT including
a detailed description of its crystal structure. The C. jejuni enzyme is similar
to the previously described enzymes of the ATP-PRT long form, but exists
only as hexameric species under experimental conditions, which contradicts
previous assumptions that the hexamer is exclusively inactive.
Chapter 3 investigates the catalytic apparatus of CjeATP-PRT by separating
the catalytic and regulatory domains of the enzyme for individual study. The isolated catalytic portion of the enzyme, the CjeATP-PRT Core mutant, forms a dimeric species with very limited catalytic capabilities but high substrate and product affnities. The CjeATP-PRT Core characteristics suggest that it exists in a permanently inhibited conformation, highlighting
the requirement of the regulatory domain not only for feedback regulation
but also for enzyme function. Additionally this supports the evolutionary
need for the recruitment of a regulatory apparatus.
In chapter 4 a potential intramolecular communication pathway from
the allosteric to the active site is probed by the generation of several single site mutations. One of these, CjeATP-PRT R216A, is completely insensitive to histidine inhibition, although this ligand is still able to bind at the allosteric
site, which is consistent with the involvement of R216 in the allosteric signal
communication. The catalytic abilities of CjeATP-PRT R216A are largely
impaired, leading to the assumption that this mutation causes a permanent
inhibitory response.
In summary this thesis supports the existence of a simple physical regulatorymechanism for the feedback inhibition of the ATP-PRT long form,
the change between two different hexamer conformations depending on the
presence of the allosteric effector.
| Identifer | oai:union.ndltd.org:canterbury.ac.nz/oai:ir.canterbury.ac.nz:10092/10799 |
| Date | January 2015 |
| Creators | Mittelstädt, Gerd Horst |
| Publisher | University of Canterbury. Chemistry |
| Source Sets | University of Canterbury |
| Language | English |
| Detected Language | English |
| Type | Electronic thesis or dissertation, Text |
| Rights | Copyright Gerd Horst Mittelstädt, http://library.canterbury.ac.nz/thesis/etheses_copyright.shtml |
| Relation | NZCU |
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