Continuous fluorescence assays for reactions involving adenine

Ross S. Firestone, Scott A. Cameron, Peter C. Tyler, Rodrigo G. Ducati, Adam Z. Spitz, Vern L. Schramm

Research output: Contribution to journalArticle

6 Scopus citations

Abstract

5′-Methylthioadenosine phosphorylase (MTAP) and 5′-methylthioadenosine nucleosidase (MTAN) catalyze the phosphorolysis and hydrolysis of 5′-methylthioadenosine (MTA), respectively. Both enzymes have low KM values for their substrates. Kinetic assays for these enzymes are challenging, as the ultraviolet absorbance spectra for reactant MTA and product adenine are similar. We report a new assay using 2-amino-5′-methylthioadenosine (2AMTA) as an alternative substrate for MTAP and MTAN enzymes. Hydrolysis or phosphorolysis of 2AMTA forms 2,6-diaminopurine, a fluorescent and easily quantitated product. We kinetically characterize 2AMTA with human MTAP, bacterial MTANs and use 2,6-diaminopurine as a fluorescent substrate for yeast adenine phosphoribosyltransferase. 2AMTA was used as the substrate to kinetically characterize the dissociation constants for three-transition-state analogue inhibitors of MTAP and MTAN. Kinetic values obtained from continuous fluorescent assays with MTA were in good agreement with previously measured literature values, but gave smaller experimental errors. Chemical synthesis from ribose and 2,6-dichloropurine provided crystalline 2AMTA as the oxalate salt. Chemo-enzymatic synthesis from ribose and 2,6-diaminopurine produced 2-amino-S-adenosylmethionine for hydrolytic conversion to 2AMTA. Interaction of 2AMTA with human MTAP was also characterized by pre-steady-state kinetics and by analysis of the crystal structure in a complex with sulfate as a catalytically inert analogue of phosphate. This assay is suitable for inhibitor screening by detection of fluorescent product, for quantitative analysis of hits by rapid and accurate measurement of inhibition constants in continuous assays, and pre-steady-state kinetic analysis of the target enzymes.

Original languageEnglish (US)
Pages (from-to)11860-11867
Number of pages8
JournalAnalytical Chemistry
Volume88
Issue number23
DOIs
StatePublished - 2016

ASJC Scopus subject areas

  • Analytical Chemistry

Fingerprint Dive into the research topics of 'Continuous fluorescence assays for reactions involving adenine'. Together they form a unique fingerprint.

  • Cite this

    Firestone, R. S., Cameron, S. A., Tyler, P. C., Ducati, R. G., Spitz, A. Z., & Schramm, V. L. (2016). Continuous fluorescence assays for reactions involving adenine. Analytical Chemistry, 88(23), 11860-11867. https://doi.org/10.1021/acs.analchem.6b03621