Structure function relationships in Chinese hamster adenine phoshoribosyl transferase




Ford, Barry Noel

Journal Title

Journal ISSN

Volume Title



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.



Hamsters, Adenine