Active site citrate synthase

Citrate, prochirality of, 1156 Citrate synthase, active site of, 1046 function of, 1045 mechanism of action of, 1043, 1047... 

A reduction of muscarinic receptor sites in the ventral horn has also been found to be related to this neurodegenerative disorder (Whitehouse et al., 1983b), while the observation of decreased citrate synthase activity of isolated anterior horn cells has led to the proposal that neuronal ACh production may be depressed (Hayashi and Tsubaki, 1982). 

Citrate synthase in mammals is a dimer of 49-kD subunits (Table 20.1). On each subunit, oxaloacetate and acetyl-CoA bind to the active site, which lies in a cleft between two domains and is surrounded mainly by a-helical segments (Figure 20.6). Binding of oxaloacetate induces a conformational change that facilitates the binding of acetyl-CoA and closes the active site, so that the reactive carbanion of acetyl-CoA is protected from protonation by water. 

Figure 26.9 X-ray crystal structure of citrate synthase. Part (a) is a space-filling model and part (b) is a ribbon model, which emphasizes the a-helical segments of the protein chain and indicates that the enzyme is dimeric that is, it consists of two identical chains held together by hydrogen bonds and other intermolecular attractions. Part (cl is a close-up of the active site in which oxaloacetate and an unreactive acetyl CoA mimic are bound.

Acifluorfen, synthesis of, 683 Acrolein, structure of, 697 Acrylic acid, pKa of, 756 structure of. 753 Activating group (aromatic substitution), 561 acidity and, 760 explanation of, 564-565 Activation energy, 158 magnitude of, 159 reaction rate and, 158-159 Active site (enzyme), 162-163 citrate synthase and, 1046 hexokinase and, 163... 

Citrate is isomerized to isocitrate by the enzyme aconitase (aconitate hydratase) the reaction occurs in two steps dehydration to r-aconitate, some of which remains bound to the enzyme and rehydration to isocitrate. Although citrate is a symmetric molecule, aconitase reacts with citrate asymmetrically, so that the two carbon atoms that are lost in subsequent reactions of the cycle are not those that were added from acetyl-CoA. This asymmetric behavior is due to channeling— transfer of the product of citrate synthase directly onto the active site of aconitase without entering free solution. This provides integration of citric acid cycle activity and the provision of citrate in the cytosol as a source of acetyl-CoA for fatty acid synthesis. The poison fluo-roacetate is toxic because fluoroacetyl-CoA condenses with oxaloacetate to form fluorocitrate, which inhibits aconitase, causing citrate to accumulate. 

In a very broad overview of the structural categories one can state several statistical correlations with type of function. Hemes are almost always bound by helices, but never in parallel a//3 structures. Relatively complex enzymatic functions, especially those involving allosteric control, are occasionally antiparallel /3 but most often parallel a//3. Binding and receptor proteins are most often antiparallel /3, while the proteins that bind in those receptor sites (i.e., hormones, toxins, and enzyme inhibitors) are most apt to be small disulfide-rich structures. However, there are exceptions to all of the above generalizations (such as cytochrome cs as a nonhelical heme protein or citrate synthase as a helical enzyme), and when one focuses on the really significant level of detail within the active site then the correlation with overall tertiary structure disappears altogether. For almost all of the dozen identifiable groups of functionally similar proteins that are represented by at least two known protein structures, there are at least... 

One interesting feature here is that both acetyl-CoA and oxaloacetic acid have the potential to form enolate anions, and that oxaloacetic acid is actually more acidic than acetyl-CoA, in that there are two carbonyl groups flanking the methylene. That citrate synthase achieves the aldol reaction as shown reflects that the enzyme active site must have a basic residue appropriately positioned to abstract a proton from acetyl-CoA rather than oxaloacetic acid, thus allowing acetyl-CoA to act as the nucleophile. 

Figure 4.6 The active site of citrate synthase, complexed with acetyl-CoA and the substrate analog L-malate (PDB 3CSq. Arg329, together with NE2(His320) and ND1(His236), contributes to a second oxyanion hole for the tetrahedral oxyanion that is generated by the nucleophilic attack of enolized acetyl-CoA on...

Figure 4.6 displays the structure of the complexed active site of citrate synthase complexed with L-malate and acetyl-CoA. This active site is rather solvent exposed... 

Citrate synthase from mitochondria has been crystallized and visualized by x-ray diffraction in the presence and absence of its substrates and inhibitors (Fig. 16-8). Each subunit of the homodimeric enzyme is a single polypeptide with two domains, one large and rigid, the other smaller and more flexible, with the active site between them. Oxaloacetate, the first substrate to bind to the enzyme, induces a large conformational... 

One of the simplest biochemical addition reactions is the hydration of carbon dioxide to form carbonic acid, which is released from the zinc-containing carbonic anhydrase (left, Fig. 13-1) as HC03-. Aconitase (center, Fig. 13-4) is shown here removing a water molecule from isocitrate, an intermediate compound in the citric acid cycle. The H20 that is removed will become bonded to an iron atom of the Fe4S4 cluster at the active site as indicated by the black H20. An enolate anion derived from acetyl-CoA adds to the carbonyl group of oxaloacetate to form citrate in the active site of citrate synthase (right, Fig. 13-9) to initiate the citric acid cycle. 

Figure 13-9 Active site of pig citrate synthase. (A) Stereoscopic view with a molecule of citrate in the active site.200 Courtesy of Stephen J. Remington. (B) Interpretive view of the enolate anion of acetyl-CoA and oxaloacetate bound in the active site. Based on work by Kurz et al.2W...

Citrate synthase catalyzes the condensation reaction by bringing the substrates into close proximity, orienting them, and polarizing certain bonds. Two histidine residues and an aspartate residue are important players (Figure 1711). One of the histidine residues (His 274) donates a proton to the carbonyl oxygen of acetyl CoA to promote the removal of a methyl proton by Asp 375. Oxaloacetate is activated by the transfer of a proton from His 320 to its carbonyl carbon atom. The concomitant attack of the enol of acetyl CoA on the carbonyl carbon of oxaloacetate results in the formation of a carbon-carbon bond. The newly formed citryl CoA induces additional structural changes in the enzyme. The active site becomes completely enclosed. His 274 participates again as a proton donor to hydrolyze the thioester. Coenzyme A leaves the enzyme, followed by citrate, and the enzyme returns to the initial open conformation. 

The sequences of citrate synthases from the eukaryotes pig heart and kidney, Arabidopsis thaliana and Saccharomyces cerevisiae, and from the eubacteria Escherichia coli, Rickettsia prowazekii, Acinetobacter anitratum, Acetobacter aceti and Pseudomonas aeruginosa have been determined (see the literature [85,88] for references). In addition, a high-resolution X-ray crystallographic structure is available for the pig heart enzyme [89,90]. This has allowed the identification of 12 residues which are critical for substrate binding and catalysis multiple sequence alignments [91] have indicated that the majority of these 12 active site residues are conserved between all eukaryotic and eubacterial citrate synthases. 

Enzymes exert their catalytic activity by bringing reactant molecu together, holding them in the orientation necessary for reaction, and viding any necessary acidic or basic sites to catalyze specific steps, look, for example, at citrate synthase, an enzyme that catalyzes the aid like addition of acetyl CoA to oxaloacetate to give citrate (Section 23.1 This reaction is the first step in the so-called citric a ld cycle, in which ace groups produced by degradation of food molecules are metabolic burned to yield CO2 and H2O. We ll look at the details of the citric cycle in Section 29.5. 

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