The Biochemistry of Citrate
Synthase
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Juliana Pernik |
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As the first enzyme of the citric acid cycle, Citrate Synthase catalyzes the condensation of oxaloacetate and acetyl-CoA to yield citrate and CoA. The age, ubiquity, and metabolic importance of this enzyme enhance its interest as an experimental subject. The enzyme demonstrates the connection between changes in protein conformation and the chemical catalysis&emdash;the forming and breaking of covalent bonds in the substrates. Citrate synthase first adjusts its conformation in order to facilitate the condensation reaction of oxaloacetate (OAA) and acetyl coenzyme A (AcCo A ) to produce the thioester intermediate, citryl CoA. The enzyme changes its conformation at least once more to hydrolize the thioester intermediate and produce citrate.
Our lab focuses on an enzyme originating in the Archae-bacteria Thermoplasma acidipilum, TpCS. This TpCS has a unique fluorescence spectra that differfor every important intermediate in the mechanism. These spectra report on the changes in protein conformation. The enzyme contains four tryptophan residues, which are located in several different regions of the molecule. Interestingly, none of these is located immediately in the active site. In order to study these, each has been replaced one at a time with phenylalanine (F), a non-fluorescent amino acid with similar chemical properties. W17F, W115F, and W245F showed stabilities and activities similar to those of the wild type. Oddly, W348F decreased in both its stability and activity.
Our lab then searched for an appropriate alternative to phenylalanine for W348. W348 has different interactions with other residues in the open and closed forms of the enzyme. This we have witnessed from X-ray structures. In the open form, it has significant cation-pi interaction with R344. In the closed form, it makes a new hydrogen bond with the alcoholic side chain of S192, but no cation-pi interaction. Model studies show that F can make neither of these interactions, which may account for that mutant's instability. Tyrosine is predicted to form at least some of the same interactions as tryptophan, but perhaps in a slightly different manner. Both cation-pi and hydrogen bonds could be present in the open form. Since the conformation changes are essential to the enzyme's catalytic strategy, perturbation of the conformation equilibrium would be a significant result.
In order to create the appropriate mutation, I performed a PCR reaction. This process inserted a small point mutation into the gene sequence of the enzyme, resulting in a tyrosine residue instead of a tryptophan. The PCR, as usual, left a small cut in the DNA; I transformed a super competent strain of bacteria, XL-1 blue, to repair this nick. To assure that the PCR reaction created the necessary mutation, I then sent the DNA for sequencing.
Meanwhile, I transformed MG1655, an expressing strain, with a plasmid containing the new gene. These particular bacteria are able to express the gene mutation and produce the desired enzyme. To purify the enzymes, we use affinity chromatography. The dye molecule attached to the column matrix is thought to be recognized by the enzyme as similar to its substrate. Our W348Y mutant failed to do so. The mutant enzyme is active and has approximately the correct molecular weight. Much more detailed study, however, is necessary to discover how it differs from the wild type, as detected by its failure to bind to the affinity dye. The exciting possibility exists, as can be rationalized from the model studies, that this mutation perturbs the conformational equilibrium. Much more work will have to be done to characterize this mutation.
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Natural Sciences Learning Center
Washington University - Biology
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