Uses the HPLC to analyze the activity of your CoA biosynthetic enzymes (PanK, PPAT, DPCK), based on the characteristic HPLC shift to lower retention time on addition of negative charge to the CoA intermediate.
- 250 mM ATP (151 mg/mL in 1M Tris-HCl pH 8)
- 0.5 M KPO4 (pH 7.4)
- 1M KCl
- 2M MgCl2
- CoA Biosynthetic Enzymes (His or MBP)
- 200 mM KC12 (coumarin-pantetheine)
Master Mix (1 mL - enough for 33x30 uL rxns)
- 420 uL H20
- 400 uL 0.5M KPO4 (final conc 200 mM)
- 80 uL 1M KCl (final conc 80 mM)
- 80 uL 0.25M ATP (final conc 20 mM)
- 20 uL 2M MgCl2 (centrifuge after adding - final conc 40 mM)
- 5 uL 0.2M KC12 (final conc. 1 mM)
For a 30 uL reaction:
- add 30 uL Master mix see recipe above) to 1.5 mL epi tubes (x4)
- to each epi add:
- 2.5 uL H2O
- 0.5 uL PanK + 1.5 uL H2O
- 0.5 uL PanK + 1 uL PPAT + 1 uL H2O
- 0.5 uL PanK + 1 uL PPAT + 1 uL DPCK
- incubate RT 30 min.
- during this time turn on HPLC - equilibrate column (Burdick and Jackson OD5 w/ guard column front loaded - 25 cmx4.6 mm) with buffer
- use method JLM_BUFA.M - uses buffer A = 25 mM KPO4 pH 6.55, buffer B = acetonitrile (NO TFA)
- 0-5 min isocratic A, 5-20 min gradient to 45% B, 20-28 min gradient to 100% B, 28-29 min gradient to 100%A, 29-35 min 100%A1
- transfer rxns to HPLC tubes
- 10 uL reactions - monitor @ 220 nm, 254 nm (adenine), 360 nm (coumarin)
- coumarin pantetheine: 20.88 min
- coumarin phosphopantetheine: 17.15 min
- coumarin dephospho-CoA: 16.02 min
- coumarin CoA: 14.68 min
- this is Jordan's mix - I've seen a lot off diff. reagent/enzyme conc work in this assay (see references) - key is to find one that works for you
- other conditions you may have luck with:
- omit KCl in KPO4 buffer (Worthington special)
- works fine in Tris-Cl - must include KCl
- reaction tolerates pH 6.5-8.0 - 7.4 seems optimal - CoaE won't turnover positively charged CoA analogues (i.e. amino pantetheine) unless at pH 9 or higher
- for prep scale (> 1 mL) chemoenzymatic CoA synthesis I have used the following conditions in the past: 400 mM Tris 7.4, 50 mM ATP, 4 mM pantetheine analogue, 20 mM KCl, 15 mM MgCl2, 60 uL/mL each CoA biosynthetic enzyme (PPAT, DPCK, PanK)
- always add MgCl2 last to master mix or will precipitate in neutral/basic buffered sol'n
- 25 mM pH 6.55 gives good peaks - traditional 0.05% TFA mobile phases can give peak broadening/tailing due to pH matching the pKa of the CoA phosphate (pKa~ 2)
Relevant papers and books
- Bibart RT, Vogel KW, and Drueckhammer DG. Development of a Second Generation Coenzyme A Analogue Synthon. J Org Chem. 1999 Apr 16;64(8):2903-2909.
- Nazi I, Koteva KP, and Wright GD. One-pot chemoenzymatic preparation of coenzyme A analogues. Anal Biochem. 2004 Jan 1;324(1):100-5.
- Dai M, Feng Y, and Tonge PJ. Synthesis of crotonyl-oxyCoA: a mechanistic probe of the reaction catalyzed by enoyl-CoA hydratase. J Am Chem Soc. 2001 Jan 24;123(3):506-7.
- van Wyk M and Strauss E. One-pot preparation of coenzyme A analogues via an improved chemo-enzymatic synthesis of pre-CoA thioester synthons. Chem Commun (Camb). 2007 Jan 28(4):398-400. DOI:10.1039/b613527g |
- Strauss E and Begley TP. The antibiotic activity of N-pentylpantothenamide results from its conversion to ethyldethia-coenzyme a, a coenzyme a antimetabolite. J Biol Chem. 2002 Dec 13;277(50):48205-9. DOI:10.1074/jbc.M204560200 |
- Worthington AS and Burkart MD. One-pot chemo-enzymatic synthesis of reporter-modified proteins. Org Biomol Chem. 2006 Jan 7;4(1):44-6. DOI:10.1039/b512735a |
- Meier JL and Burkart MD. Chapter 9. Synthetic probes for polyketide and nonribosomal peptide biosynthetic enzymes. Methods Enzymol. 2009;458:219-54. DOI:10.1016/S0076-6879(09)04809-5 |
or instead, discuss this protocol.