Channeling in purine biosynthesis : efforts to detect interactions between PurF and PurD and characterization of the FGAR-AT complex

Purine biosynthesis has been used as a paradigm for the study of metabolism of unstable molecules. Both phosphoribosylamine (PRA) and N5-carboxyaminoimidazole ribonucleotide (N5-CAIR) have estimated half-lives in vivo of seconds. In order to avoid metabolite decomposition, one strategy cells could employ is channeling-the direct transfer of a metabolite between enzyme active sites without diffusion into the bulk media. While kinetic evidence for channeling of PRA has been reported between phosphoribosylpyrophosphate amidotransferase (PurF) and glycinamide ribonucleotide synthetase (PurD), no evidence for a PurF:PurD complex has been found. In an effort to detect this complex, stopped-flow fluorescence spectroscopy was used to detect changes in PurF fluorescence that may result from interaction with PurD. Critical to the success of these experiments was incorporation of tryptophan analogs (4-fluorotryptophan and 7-azatryptophan) into the proteins in order to increase signal specificity for PurF. No evidence for a PurF:PurD interaction was found under any of the conditions tested. The implication of this finding is discussed with regard to the PurF:PurD channeling model. Like all amidotransferase enzymes (ATs), channeling of NH3 between glutaminase and AT active sites has been implicated in the formylglycinamide ribonucleotide amidotransferase (FGAR-AT).

Department dc:contributor.department

Massachusetts Institute of Technology. Dept. of Chemistry.

Grantor dc:publisher

Massachusetts Institute of Technology

Year dc:date.issued

2006

Author dc:creator

• Hoskins, Aaron A. (Aaron Andrew)

Advisor dc:contributor.advisor

• Jo Anne Stubbe.

• Chemistry.

Statement dc:rights

• M.I.T. theses are protected by copyright. They may be viewed from this source for any purpose, but reproduction or distribution in any format is prohibited without written permission. See provided URL for inquiries about permission.

Licence dc:rights.uri

• http://dspace.mit.edu/handle/1721.1/34491
• http://dspace.mit.edu/handle/1721.1/7582

Language dc:language.iso

eng

Identifier URI

http://dspace.mit.edu/handle/1721.1/34491

OAI identifier oai:identifier

oai:dspace.mit.edu:1721.1/34491