Adenine phosphoribosyl transferase is a ubiquitous enzyme which salvages
endogenous adenine, via the nucleotide AMP, for use by the cell. This activity, in
conjunction with other interconnected purine salvage mechanisms is an energy-efficient
way for the cell to satisfy its purine requirements. APRT is a target molecule in certain
human diseases, for chemotherapeutics, and in vivo mutagenesis studies. There is little
known about structure-function relationships in APRT. In the absence of solved three-dimensional
crystal structures, we have explored structure-function relationships in APRT
by sequence comparison, in vitro mutagenesis and kinetic analysis, protein crosslinking,
and in vivo selection of mutant enzymes with altered substrate affinities. Chinese hamster
APRT shares identifiable sequence similarities to all other phosphoribosyl transferases, and
many other nucleotide binding proteins, in regions which probably serve closely similar
functions across diverse protein families. Predicted secondary structures of CHO APRT
are very similar to other APRT molecules, and to a lesser degree to other phosphoribosyl
transferases. Residues of part of the generalized nucleotide binding motif of APRT were
found to have specific roles in binding substrate, which can be extrapolated to the same
functional elements in other nucleotide binding proteins. In addition, mutants identified by
selection for altered substrate affinities are widely dispersed in the primary sequence.
Although APRT is thought to exist as a dimer in its native context, certain mutants of
APRT which have impaired ability to form dimers appear to have near-wildtype activity. / Graduate
| Identifer | oai:union.ndltd.org:uvic.ca/oai:dspace.library.uvic.ca:1828/8280 |
| Date | 15 June 2017 |
| Creators | Ford, Barry Noel |
| Contributors | Glickman, Barry W. |
| Source Sets | University of Victoria |
| Language | English, English |
| Detected Language | English |
| Type | Thesis |
| Rights | Available to the World Wide Web |
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