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Competitive titrations of SULT1A1:TNP‐AMP reveals biological nucleotide affinities for a sulfotransferase
Author(s) -
Gaffney Kristen,
Macdonald Jane,
Martin Emily,
Beckmann Joe D.
Publication year - 2012
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.26.1_supplement.962.5
Subject(s) - nucleotide , chemistry , titration , affinities , gtp' , cofactor , biochemistry , stereochemistry , cytosol , enzyme , organic chemistry , gene
Cytosolic sulfotransferases (SULTs) sulfurylate xenobiotics, hormones, and neurotransmitters, with 3′‐phosphoadenosine‐5′‐phosphosulfate (PAPS) as the required cosubstrate. We are investigating the regulation of recombinant bovine SULT1A1, which prefers simples phenol substrates, by biological nucleotides. SULT1A1 inhibition by PAP, ATP, and (acyl)CoAs is known; however, it is unclear whether regulation by other nucleotides might occur. Kinetic evaluation of the many such possibilities would be difficult, so we have investigated 3′‐trinitrophenylated‐adenylates as possible displaceable fluorescent probes. Fluorimetric titrations revealed TNP‐AMP to have the lowest SULT affinity of these probes, making it most readily displaceable by biological nucleotides. Therefore, bSULT1A1 is first titrated with TNP‐AMP (K d = 0.50 μM), followed by competitive titration with a nucleotide of interest. The titrations were then numerically fit with an updated mathematical model for competition to obtain K d values. Competition (and K d values) observed to date are: PAP (0.22 μM), palmitoyl‐CoA (0.15 μM), octanoyl‐CoA (2.33 μM), malonyl‐CoA (38 μM), ATP (45 μM), GTP (61 μM), CTP (120 μM), UTP (164 μM), dATP (83 μM), dGTP (125 μM), and TTP (347 μM). Evaluation of additional nucleotides is ongoing.