Continuous Fluorescence Assays for Reactions Involving Adenine

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.

Direct Detection of Products from S-Adenosylmethionine-Dependent Enzymes Using a Competitive Fluorescence Polarization Assay

“…Previous kinetic characterization of MTA and AdoHcy for HpMTAN has shown these substrates to possess low μ M affinity by measuring the spectral change of the reaction through formation of adenine. 25,31 More recently, steady-state kinetic analysis using a luciferase-based assay for Staphylococcus aureus MTAN indicated negative cooperativity between the two monomers demonstrating K m values for MTA of 100 and 900 nM for the first and second active sight, respectively. 32 The binding isotherm of AdoHcy-TAMRA presented here represents the affinity measured for the first active site binding event, which affords the high sensitivity of the developed FP assay.…”

Heat Capacity Changes for Transition-State Analogue Binding and Catalysis with Human 5′-Methylthioadenosine Phosphorylase

“…Phosphorolysis generates the fluorescent 2,6-diaminopurine product to allow accurate quantitation of product formation. 42 Pre-steady-state experiments were carried out on an Applied Photophysics model SX20 stopped-flow instrument, which has a dead time of approximately 2 ms. Each experiment was an average of at least 12 measurements under identical conditions. $$\begin{array}{c}left\ln k=\ln \frac{k_{\mathrm{normalb}}T}{h}-\left[\frac{\mathrm{normal\Delta }{H}_{T_0}^{‡}+\mathrm{normal\Delta }{C}_{\mathrm{p}}^{‡}false(T-T_{0}false)}{RT}\right]\\ +\left[\frac{\mathrm{normal\Delta }{S}_{T_0}^{‡}+\mathrm{normal\Delta }{C}_{\mathrm{p}}^{‡}false(\ln T-\ln T_{0}false)}{R}\right]\end{array}$$…”

“…Pre-steady-state kinetics were characterized using 2-amino-5′-methylthioadenosine as an alternative substrate. Phosphorolysis generates the fluorescent 2,6-diaminopurine product to allow accurate quantitation of product formation . Pre-steady-state experiments were carried out on an Applied Photophysics model SX20 stopped-flow instrument, which has a dead time of approximately 2 ms. Each experiment was an average of at least 12 measurements under identical conditions. …”

Synthesis of bis-Phosphate Iminoaltritol Enantiomers and Structural Characterization with Adenine Phosphoribosyltransferase

“…45 Enzyme activity was assessed by use of a recently published direct and sensitive fluorimetric assay, where 2,6-diaminopurine replaced adenine, the natural substrate (Figure 2). 46…”

“…Enzyme activity assays for ScAPRT were performed at 25 °C in 50 mM HEPES at pH 7.5 in 250 μ L total reaction volumes, and each individual initial rate was the average of triplicate measurements. Reaction mixtures contained 2 mM MgCl 2 , 250 μ M 2,6-diaminopurine (2,6-DAP, a fluorescent and easily quantitated product), 46 250 μ M PRPP, and 1 mM DTT and were initiated by the addition of ScAPRT (100 nM). DAP consumption was monitored by a change in fluorescence (Ex: 280 nm/Em: 345 nm) over the course of 1 h using a SpectraMax M5 plate reader (Molecular Devices), at variable concentrations of either D-DIAB or L-DIAB (0− 200 μ M).…”