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BCMB8010 Enyzme Report:
The Structure and Function of Aconitase

Introduction

    Eukaryotes possess two forms of the protein aconitase: a mitochondrial and a cytosolic form (5).  The primary function of mitochondrial aconitase is enzymatic, converting citrate to isocitrate via a cis-aconitate intermediate in the Tricarboxylic Acid Cycle (3, 5).  The cytosolic aconitase protein, however, is ambifunctional (5).  When cellular iron levels are high, the cytosolic protein retains enzymatic aconitase function, as in the TCA cycle (5).  When iron levels are low, however, the protein functions to bind specific stem-loop sequences of mRNA called iron-responsive elements (IREs), and is thus called the iron-responsive element binding protein (IRP-BP) (5).  IREs are found at the 3’ ends of mRNAs encoding for proteins involved in iron transport into the cell (5).  Binding of IRE-BP at this site prevents the degradation of this mRNA, allowing the cell to import more iron in times of need (5).  IREs are also found at the 5’ ends of mRNAs, where IRE-BP binding prevents translation into protein (5).   Therefore, proteins involved in iron sequestration and/or utilization, such as ferritin, have IRE sequences at the 5’ end of mRNA (5).

    Aconitase enzyme activity depends of the presence of a [4Fe-4S] cluster (3).  Inactive aconitase has a [3Fe-4S] cluster and is activated upon addition of a fourth iron atom (Fea) (2).  This fourth iron atom has a slightly different electronic character than the others, and is the active site of the enzymatic reaction (2).  When substrate is bound, Fea is 6-coordinate, binding 3 S2- atoms of the cluster, as well as a carboxyl oxygen and hydroxyl group of the substrate and a solvent H2O molecule (4).  Coordination of the hydroxyl group makes it a better leaving group and facilitates the dehydration reaction that forms cis-aconitate from either citrate or isocitrate (1, 3).  Depending on which carboxyl oxygen coordinates the iron atom, cis-aconitate can then be converted to citrate or isocitrate upon addition of water (1).  In the TCA cycle, cis-aconitate is converted to isocitrate.

    Disassembly of the Fe-S cluster completely inactivates the mitochondrial aconitase and converts the cytosolic protein from aconitase function to RNA-binding function (5).  Removal of the Fe-S cluster brings about a conformational change that allows the IRE structure to fit at the active site, where three arginine residues form direct contacts with the mRNA (5).  The assembly and disassembly of the [4Fe-4S] cluster does not depend on the aconitase mechanism, and the mitochondrial protein is present to function in the TCA cycle, so it is unclear why the cytosolic protein retains its enzymatic acitivity (5).  One suggestion is that it is important for aconitase function to be regulated by iron, perhaps because citrate is involved in iron transport throughout the cell (5).

References:
(1)  Cleland W.W. and Kreevoy M.M. (1994) Science 254, 1887-1890.
(2)  Emptage M.H., Kent T.A., Kennedy M.C., Beinert H., and Munck E. (1983) Proc. Natl. Acad. Sci. USA 80, 4674-4678.
(3)  Garrett R.H. and Grisham C.M. (1999) Biochemistry, 2nd ed., Saunders College Publishing, New York, NY.
(4)  Kennedy M.C., Werst M., Telser J., Emptage M.H., Beinert H., and Hoffman B.M. (1987) Proc. Natl. Acad. Sci. USA 84, 8854-8858.
(5)  Philpott C.C. Klausner R.D., and Rouault T.A. (1994) Proc. Natl. Acad. Sci. USA 91, 7321-7325.

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