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ATP as an Allosteric Modulator and Chelator of Fe 3+ from Fe 3+ ‐transferrin. Is ATP a Major Fe 3+ Carrier
Author(s) -
Cowart Richard E.,
Shaffer Tyler B.,
O'Hara Ryan D.,
Campbell Jacquelyn A.,
SchneiderTugan Lori L.,
Binz Sara K.,
Pope Mitchell A.,
Gregory Robert B.
Publication year - 2018
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.2018.32.1_supplement.799.1
Subject(s) - transferrin , chemistry , allosteric regulation , chelation , pyrophosphate , adenosine triphosphate , adenosine monophosphate , kinetics , biochemistry , biophysics , stereochemistry , adenosine , inorganic chemistry , enzyme , biology , physics , quantum mechanics
The objectives of this study were to examine two chelators in the removal of Fe 3+ from Fe 3+ ‐transferrin. One was pyrophosphate (PPi) as it is a reasonably effective chelator in this reaction, and the other was ATP in that it consists of PPi attached to adenosine monophosphate. We reasoned that ATP might bind to a nucleotide binding site if present and in close proximity to the metal center, to enhance Fe 3+ removal and give insight into the mechanism of Fe 3+ release. In this study we used uv/vis spectroscopy to examine the removal of Fe 3+ from Fe 3+ ‐transferrin by time‐course and Vi reactions. At acidic pH we determined association constants of Fe 3+ ‐transferrin alone, and Fe 3+ ‐transferrin in the presence of ATP. We also employed molecular modeling to identify and characterize a putative ATP binding site. Our results show that sigmoidal kinetics were obtained as a function of pH when ATP was the chelator. At pH 7.5 the kinetic data showed there was no removal of Fe 3+ by ATP. ATP was found to exert positive heterotropic effects at acidic pH and resulted in the increase in the association constant of Fe 3+ ‐transferrin. These effects were not seen with PPi. We concluded that there were two destabilizations of Fe 3+ ‐transferrin necessary for Fe 3+ release; the first due to a low pH induced conformational change in the protein and the second due to ATP, also at low pH, where ATP served as an allosteric modulator. Essentially 100% of Fe 3+ was released from both the N‐ and C‐lobes at pH 5.0 in the presence of ATP in ~ 3 minutes as determined by time‐course reaction. Molecular modeling studies identified a putative 25 amino acid ATP binding channel in the N‐lobe of human serum transferrin. These conserved amino acids were identified on each open face of the NI/NII subdomains of apo‐transferrin with the channel forming upon NI/NII subdomain closure as Fe 3+ is acquired. At neutral pH ATP bound approximately 9 Å from Fe 3+ in the metal center. At acidic pH the protein conformational change positioned ATP immediately adjacent to Fe 3+ for rapid chelation. In that ATP is an extracellular, allosteric signaling molecule a mechanism of Fe 3+ release from Fe 3+ ‐transferrin is proposed. Here, extracellular ATP is acquired by transferrin in the serum at neutral pH where chelation does not occur. Upon binding to the transferrin receptor and internalization into an acidic endosome, a pH induced conformational change along with positive heterotropic effects occur to allow ATP to efficiently chelate Fe 3+ . We conclude that transferrin is an allosteric protein, ATP is an allosteric modulator and effective chelator, and that ATP is likely a major Fe 3+ carrier in higher organisms. Support or Funding Information State of Iowa grant GIVF This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .