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J Biol Chem, Vol. 275, Issue 17, 12463-12469, April 28, 2000
From the Research Institute for Food Science, Kyoto University,
Uji, Kyoto 611 0011, Japan
Iron release process of ovotransferrin N-lobe
(N-oTf) to anion/chelators has been resolved using kinetic and
mechanistic approach. The iron release kinetics of N-oTf were measured
at the endosomal pH of 5.6 with three different anions such as
nitrilotriacetate, pyrophosphate, and sulfate using stopped flow
spectrofluorimetric method, all yielding clear biphasic progress
curves. The two observed rate constants and the corresponding
amplitudes obtained from the double exponential curve fit to the
biphasic curves varied depending on the type and concentration of
anions. Several possible models for the iron release kinetic mechanism
were examined on the basis of a newly introduced quantitative equation.
Results from the curve fitting analyses were consistent with a dual
pathway mechanism that includes the competitive iron release from two different protein states, namely, X and Y, with the respective first
order rate constants of K1 and
K2 (X, domain closed holo N-oTf; Y, anion
induced different conformer of holo N-oTf). The reversible
interconversions of X to Y and Y to X are driven by the second order
rate constant k3 and the first order rate
constant K4, respectively. The obtained rate
constants were greatly variable for the three anions depending on the
synergistic or nonsynergistic nature. In the light of the anion-binding
sites of N-oTf located crystallographically, the compatible mechanistic
model that includes competitive anion binding to the iron coordination
sites and to a specific anion site is suggested for the dual pathway
iron release mechanism.
Anion-mediated Iron Release from Transferrins
THE KINETIC AND MECHANISTIC MODEL FOR N-LOBE OF
OVOTRANSFERRIN*
and
*
This work was supported in part by a grant-in-aid for
scientific research from the Ministry of Education, Science, Sports, and Culture of Japan.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
Recipient of a Postdoctoral Fellowship for Foreign Researchers of
the Japan Society for the Promotion of Science. Present address:
Dept. of Protein Chemistry and Technology, Central Food Technological
Research Institute, Mysore 570013, India.
§
To whom correspondence should be addressed: Research Inst. for Food
Science, Kyoto University, Uji, Kyoto 611 0011, Japan. Tel.:
81-774-38-3734; Fax: 81-774-38-3735.
Copyright © 2000 by The American Society for Biochemistry and Molecular Biology, Inc.
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