Bicarbonate and the cofactor biotin are involved in this activation, which requires the expenditure of ATP. In animals, glucose cannot be generated from acetyl CoA and fatty acids. The bypassing of the glycolytic step catalyzed by pyruvate kinase is actually accomplished in a two-step process catalyzed by two separate enzymes, pyruvate carboxylase and phosphoenolpyruvate carboxykinase PEPCK. Oxaloacetate OA is the common metabolite of these enzymes, the product of the former and substrate of the latter in gluconeogenesis.
Pyruvate carboxylase [ EC 6. Pyruvate carboxylase is a member of a group of biotin-dependent carboxylase enzymes also discussed below , which also includes acetyl CoA carboxylase [ EC 6. The latter two enzymes play important roles in fatty acid metabolism.
All the biotin-dependent carboxylase reactions require ATP hydrolysis as well as participation of the biotin prosthetic group. The general reaction catalyzed by the biotin-dependent carboxylases is shown below. The significance of biotin is that it acts as a carrier of CO 2 , which is activated to be transferred to pyruvate in the carboxylation reaction catalyzed by pyruvate carboxylase.
The biotin-dependent carboxylases utilize a common mechanism to generate carboxybiotin. The substrate for carboxylation of biotin is actually bicarbonate, rather than CO 2. This intermediate is activated to transfer CO 2 to biotin, forming carboxybiotin. Pyruvate carboxylase has a dual metabolic role.
When citric acid cycle intermediates are utilized as biosynthetic precursors for example, succinate is a precursor for heme biosynthesis , the amount of OA will no longer be sufficient to match the input of acetyl CoA.
Thus, in generating OA from pyruvate, pyruvate carboxylase catalyzes an anaplerotic reaction. In this context, it makes sense that pyruvate carboxylase is allosterically activated by acetyl CoA, since excess acetyl CoA signals the need for more OA. Of course, the OA generated is also an obligatory intermediate in the first stage of gluconeogenesis, the conversion of pyruvate to PEP. Recall that the pyruvate kinase reaction of glycolysis produces ATP and is exergonic.
This tells us that the gluconeogenic conversion of pyruvate to PEP will require the input of a significant amount of energy. In accordance with this expectation, the pyruvate carboxylase reaction, which is the first step in this conversion, requires ATP. How does pyruvate carboxylase work? The mechanism consists of two stages, each of which occurs at a different subsite of the enzyme: i formation of carboxybiotin, described above, and ii transfer of activated CO 2 from carboxybiotin to pyruvate.
The first stage occurs at the ATP-bicarbonate site, where formation of caboxybiotin occurs via a carboxyphosphate intermediate. This step is thought to involve release of CO 2 from carboxyphosphate, followed by nucleophilic attack of the N1 nitrogen of biotin on the CO 2 carbon atom.
The second stage occurs when the carboxybiotin swings - about the long linker arm - into the pyruvate subsite, where it carboxylates pyruvate at C3. In this stage, the biotin cofactor apparently functions not only as a source of CO 2 , but also assists in generation of the enolate form of pyruvate.
Energetically, the favorable decarboxylation helps drive the formation of the enol phosphate, which has a significantly higher standard free energy of hydrolysis than the phosphoanhydride bonds of ATP or GTP.
Furthermore, we see that the conversion of pyruvate to PEP consumes two high energy phosphate bonds. In some species, it is mitochondrial, and in others it is cytosolic, while in still others notably including humans it is roughly equally distributed in both locations. The individual reactions of gluconeogenesis and the enzymes that catalyze them. Through site directed mutagenisis studies, Lys is suggested to be involved in both activator binding and in the allosteric transition mechanism.
These domain interfaces are critical for the trasnition. Tey couple changes in the tertiary and quaternary structures in fructose 1,6 biphosphate binding sites for pyruvate kinase. A phosphate-binding site for the allosteric activator is created by residues encoded by a region of the gene that corresponds to spliced exons of mammalian isozymes [9].
FBP activation induces several conformational changes among active-site sidechains through a mechanism that is most likely to involve significant domain motions. The conformational differences observed between the active sites of inactive and fully active Pyruvate Kinase enzymes is in agreement with the thermodynamic mechanism of allosteric activation through a metal relay that increases the affinity of the enzyme for its phosphoenolpyruvate substrate.
As indicated earlier, phosphoenolpyruvate can enhance the activity of the reaction by adding into the enzyme because it is the rate limiting step. The enzyme follows hyperbolic kinetics. Experiments found that no incorporation was found in the reaction, indicating a random, rapid dissociation of the products.
Both pyruvate and ATP have been shown to be non-competitive inhibitors of pyruvate kinase [11]. Pyruvate kinase deficiency is the most frequent enzyme abnormality of glycolysis that causes hemolytic anemia.
In cells that lack mitochondria, this deficiency is especially harmful, because these cells must use anaerobic glycolysis as their sole source of energy because the TCA cycle is not available. Red blood cells, in a state of pyruvate kinase deficiency, rapidly become deficient in ATP and can undergo hemolysis. This is transmitted as an autosomal recessive trit.
The severity of hemolysis is extremely variable such as a mild case to life-threatening neonatal anaemia requiring transfusions. Over one hundred eighty different mutations have been discovered in relation to this deficiency with most being autosomal recessive, but a few strands are autosomal dominant. The deficiency causes red blood cells to deform into echinocytes on peripheral blood smears.
This causes the buildup of reaction intermediates which can also increase the level of 2,3-bisphosphoglycerate in the cells.
This causes a rightward shift in the hemoglobin oxygen saturation curve, which means that there is a decreased oxygen affinity for the hemoglobin and earlier oxygen unloading than under normal conditions [13]. Pyruvate kinase 3D structures. For additional information, see: Carbohydrate Metabolism.
Category : Topic Page. Pyruvate Kinase From Proteopedia. Jump to: navigation , search. Show: Asymmetric Unit Biological Assembly.
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