The objective of this project is two-fold: to elucidate the mechanism of action of the Fe-S enzyme aconitase through a combined approach of mutagenesis, crystallography, EPR and kinetic measurements; to engineer new metal binding sites and metal catalyzed activities by mutagenesis of residues in the active site of this Fe-S enzyme. The project interacts with others in the Metalloprotein Structure and Design Program Project by focusing on a protein which has an Fe-S cluster and is at the same time an enzyme. Mitochondrial aconitase employs a [4Fe-4S] cluster to catalyze the stereospecific dehydration/rehydration of citrate to isocitrate in basic energy metabolism. The active site is comprised of the cluster and 20 residues from 4 domains in the protein (3 His, 3 Asp, 1 Glu, 3 Ser, 4 Arg, 1 Gln, 2 Asn, 3 Cys) providing an unprecedented opportunity to introduce new metal sites, modify Fe-S cluster structure and properties, and redefine substrate specificity. Aconitase is the only Fe-S enzyme for which a crystal structure and site-directed mutations are available. It has recently been shown that a class of cytosolic proteins responsible for translational regulation of mRNA are aconitases. The cDNA for porcine mitochondrial aconitase has been cloned and sequenced, the recombinant protein has been over-expressed and crystallized, and 10 site-directed mutants of active site residues have been constructed, expressed, and studied.
The specific aim i s to construct, over-express and purify these and additional mutant enzymes in the P.I.'s laboratory for the purpose of supporting the crystallographic, spectroscopic and kinetic experiments. Two classes of mutations will be constructed: those which probe the mechanism of the wild type enzyme; and those which introduce novel properties and structures. In the first class there are two objectives. (1) To support the crystallographic work on the structure and mechanism of the enzyme. There is no funding at present for preparing mutant enzyme samples. (2) To provide samples of the mutant enzymes to H. Beinert and C. Kennedy for kinetic and EPR measurements. These experiments combined with knowledge of the crystal structures are essential to dissect the role of individual residues in the mechanism. In the second class there are two objectives. (1) to convert aconitase into an isopropylmalate isomerase by mutating side chains in contact with the gamma-carboxyl group of isocitrate. IPM isomerases catalyze an analogous dehydration/rehydration in leucine biosynthesis, and have a high degree of homology with aconitase. This experiment tests factors determining substrate specificity. (2) To construct a molecule capable of reversibly binding a fourth metal in the [3Fe-4S] cluster state as a function of pH or reductant added, i.e., an Fe sensitive molecular switch. this property is relevant to understanding the above mentioned RNA binding function.
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