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ACONITASE: Structure and Function. Victoria Meliopoulos BCMB 8010 Fall 2007. Citrate (isocitrate) hydroxylase Important component of the citric acid cycle Equilibrium formed when citrate added to tissues
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ACONITASE:Structure and Function Victoria Meliopoulos BCMB 8010 Fall 2007
Citrate (isocitrate) hydroxylase Important component of the citric acid cycle Equilibrium formed when citrate added to tissues Theory of two aconitase enzymes – one to form cis-aconitate and one to form isocitrate Aconitase Figure: Reaction scheme of the aconitase mechanism. Citrate is dehydrogenated, forming cis-aconitate. The two carboxyl groups about the double bond are in the cis conformation. The substrate is then flipped about the double bond and rehydrated, forming isocitrate. Nomenclature for the carbon atoms is α, β, γ, starting from the top carbon in the double bond of cis-aconitate and working down. Carboxyl groups are excluded. Figure created in ChemDraw 8.0.
Purification • Initially very difficult – very unstable and difficult to isolate • Stabilized by the presence of Fe(II) and cysteine • Purification scheme uses (1): • Ethanol fractionation (x2) • Heat fractionation • Ammonium sulfate precipitation • pH manipulation 1. Morrison, J. F. (1954) Biochem. J. 56, 99 – 105.
Biological Aspects • Aconitase has been isolated from: • Bovine heart tissue (purification) • Porcine heart tissue (cDNA isolation) – J05224 (2) • Found in heart, liver, brain, kidney • Mitochondrial vs. cytosolic aconitase (3) • Coded by two different genes, but similar structures • Active sites are identical 2. Zheng, L., Andrews, P. C., Hermodson, M. A., Dixon, J. E., and Zalkin, H. (1990) J. Biol. Chem. 265, 2814 – 2821. 3. Robbins, A. H., and Stout, C. D. (1989) Proc. Natl. Acad. Sci. 86, 3639 – 3643.
4 domains 754 amino acids Iron-sulfur cluster at active site Structure Figure : General structure of aconitase, taken from bovine heart mitochondrial aconitase. The different colors represent the four domains of the protein. The figure was created using RasMol 2.7.3.1 and Microsoft PowerPoint 2003 to manipulate the file 1C96 from Lloyd, S. J., Lauble, H., Prasad, G. S., and Stout, C. D. (1999) Protein Science. 8, 2655 – 2662.
Mechanism Voet, D., and Voet, J. G. (2004) Biochemistry, 3rd ed., John Wiley & Sons, Hoboken, N. J.
Active Site Structure Figure: Illustration of the inactive (left) and active (right) iron-sulfur cluster of porcine heart aconitase. The atoms of the cluster are shown in yellow. Coordinating cysteine residues are represented in red. All other amino acids shown are within 3.5 Ǻ of the cluster and shown in green. These amino acids are Asn446, His167, His147, Ile145, and Ser357. The figure was created using RasMol 2.7.3.1 and Microsoft PowerPoint 2003 to manipulate the files 5ACN (left) and 6ACN (right) from Robbins et. al. (1989) Proc. Natl. Acad. Sci. 86, 3639 – 3643.
Catalytic Mechanism Figure: Methylisocitrate (MIC) bound to active site of bovine heart aconitase. The line illustrates the iron atom coordinated to the α-hydroxyl group. The general base, His101, is shown in green and the general acid, Ser642, is shown in red. The figure was created using RasMol 2.7.3.1 and Microsoft PowerPoint 2003 to manipulate the files 1AMI from Lauble, H., and Stout, C. D. (1995) Proteins 22, 1 -11.
