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Characterization of trinitrophenylated (TNP) adenosine nucleotide binding to bovine phenol sulfotransferase (bSULT1A1)
Author(s) -
Macdonald Jane A.,
Sawin Andrew,
Lapham David,
White Judy,
Turk Jeffrey A.,
Beckmann Joe D.
Publication year - 2008
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.22.1_supplement.792.15
Subject(s) - chemistry , stereochemistry , nucleotide , titration , affinities , isothermal microcalorimetry , sulfotransferase , binding site , enzyme , biochemistry , enthalpy , organic chemistry , physics , quantum mechanics , gene
Sulfate transfer from 3′‐phosphoadenosine‐5′‐phosphosulfate (PAPS) to various acceptors is catalyzed by sulfotransferases (SULTs). SULTs also bind other nucleotides, although measuring these affinities can be difficult. We have previously observed binding of TNP‐ATP to purified recombinant bSULT1A1 by fluorescence, and we have now investigated this in greater detail. First, a molecular model using human SULT1A1 coordinates has identified a feasible docking site for a 3′‐TNP group. Second, ionic strength increased the K d for TNP‐ATP from 1.1 nM at 16 mM I up to 185 nM at 516 mM I. This suggested an important role of charged phosphoryl groups in binding to the enzyme. Third, this role was confirmed by determination of K d values for TNP‐ATP (1.1 nM), TNP‐ADP (9.3 nM), and TNP‐AMP (175 nM). Next, the effect of temperature on TNP‐ATP binding was determined from 5–45°C, and greatest affinity was observed at 35°C. The positive Arrhenius plot slope from 5–35°C revealed enthalpy‐driven binding (ΔH = ca. −25 kJ/mol), which is being tested by titration microcalorimetry. Finally, K d values for biologically‐relevant nucleotide binding to bSULT1A1 are being determined by competitive titrations into SULT:TNP‐AD/TP complexes. These measurements will provide a meaningful view of SULT regulation and mechanism.