Kinetic studies on the removal of iron and aluminum from recombinant and site-directed mutant N-lobe half transferrins.
Kinetic studies have been conducted in pH 7.4 Hepes buffer at 25 degreesC on the removal of Fe(III) and Al(III) from the recombinant N-lobe half molecule of human serum transferrin (Tf/2N) and from the R124A, K206A, and K296A mutants of this protein. The rates of iron removal from Tf/2N by 3-hydroxypyridin-4-one (deferiprone) and nitrilotriacetic acid (NTA) are essentially identical with previous results on N-terminal monoferric transferrin (Tf-FeN). For both Tf/2N and Tf-FeN, iron removal by deferiprone follows simple saturation kinetics, while iron removal by NTA follows simple first-order kinetics. There is some discrepancy between the two proteins with respect to iron removal by PPi, but this may be due to differences in the chloride concentrations among different studies. The addition of Fe(NTA)2 to R124A at ambient bicarbonate concentrations forms the Fe-NTA-Tf ternary complex, but the usual Fe-CO3-Tf complex can be formed by adding ferrous ion in the presence of a larger excess of bicarbonate. This complex releases its iron very rapidly by a mechanism that is first-order with respect to the ligand. This suggests that the first-order component of metal release from transferrin involves the displacement of the synergistic carbonate anion. Since iron removal from K206A and K296A at pH 7.4 is extremely slow, studies have been conducted on the more labile Al3+ complexes of Tf/2N, K206A, and K296A. The removal of Al3+ from Tf/2N by PPi follows the same complex kinetic order with respect to the ligand concentration that is observed for iron removal, while the removal of Al3+ from both K206A and K296A reverts to a simple saturation process. The addition of perchlorate retards the removal of Al3+ from both K206A and K296A, suggesting that these lysine residues are not associated with the allosteric effects of inorganic anions on the rates of metal removal.
Li
Y
Y
Department of Chemistry, University of Missouri-St. Louis 63121, USA.
Harris
W R
WR
Maxwell
A
A
MacGillivray
R T
RT
Brown
T
T
D000409:Alanine / Q000235:genetics / Q000378:metabolism; D000535:Aluminum / Q000378:metabolism*; D000818:Animals; D001120:Arginine / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites / Q000235:genetics; D002460:Cell Line; D006224:Cricetinae; D000077543:Deferiprone; D006801:Humans; D007501:Iron / Q000378:metabolism*; D007668:Kidney; D007700:Kinetics; D008239:Lysine / Q000235:genetics / Q000378:metabolism; D046911:Macromolecular Substances; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011728:Pyridones / Q000494:pharmacology; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism*; D014168:Transferrin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D046911:Macromolecular Substances; D010446:Peptide Fragments; D011728:Pyridones; D011994:Recombinant Proteins; D014168:Transferrin; D000077543:Deferiprone; D001120:Arginine; D000535:Aluminum; D007501:Iron; D008239:Lysine; D000409:Alanine