AndrewAlexanderSandbox1: Difference between revisions

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[[Image:Pyruvatekinasemechanism.gif|left]]

<ref>{{website1| title=Exercise-Induced Metabolic Acidosis: Where do the Protons come from?|url=http://www.sportsci.org/jour/0102/rar.htm|}}</ref>

<scene name='Keegan_Gelvoria_Sandbox_1/N_c_rainbow/null'>Pyruvate Kinase</scene> catalyzes the final reaction of glycolysis. It couples the free energy of PEP cleavage to the generation of ATP during the synthesis of the final product, pyruvate. ~~This reaction necessitates both K~~+ and ~~Mg2~~+ ~~cations, which has two steps~~. The first step is the nucleophilic attack of the PEP phosphorous atom by β-phosphoryl oxygen of ADP; this step displaces enolpyruvate while forming ATP. In the second step, enolpyruvate tautomerizes to pyruvate <ref>{{book |author=Voet, Donald; Voet, Judith C.; Pratt, Charlotte W.|title=Fundamentals of Biochemistry: Life at the Molecular Level|edition= 3|pages=502|}}</ref>. The formation of a high-energy intermediate by enolase in the 9th reaction of glycolysis allows for the synthesis of ATP in this reaction. Though the hydrolysis of 2PG is insufficient in driving the synthesis of ATP, the dehydration of 2PG allows for such a reaction to occur by forming a high-energy intermediate. The high potential of PEP reflects the large release of energy that occurs with the conversion of enolpyruvate to its keto tautomer, pyruvate <ref>{{book |author=Voet, Donald; Voet, Judith C.; Pratt, Charlotte W.|title=Fundamentals of Biochemistry: Life at the Molecular Level|edition= 3|pages=503|}}</ref>.

<scene name='Keegan_Gelvoria_Sandbox_1/N_c_rainbow/null'>Pyruvate Kinase</scene> catalyzes the final reaction of glycolysis. It couples the free energy of PEP cleavage to the generation of ATP during the synthesis of the final product, pyruvate.Pyruvate kinase cataylzes the transfer of phsphoryl group of PEP to ADP in the presence of two ions of MG2+ and one of K+. The first step is the nucleophilic attack of the PEP phosphorous atom by β-phosphoryl oxygen of ADP; this step displaces enolpyruvate while forming ATP. In the second step, enolpyruvate tautomerizes to pyruvate <ref>{{book |author=Voet, Donald; Voet, Judith C.; Pratt, Charlotte W.|title=Fundamentals of Biochemistry: Life at the Molecular Level|edition= 3|pages=502|}}</ref>. The formation of a high-energy intermediate by enolase in the 9th reaction of glycolysis allows for the synthesis of ATP in this reaction. Though the hydrolysis of 2PG is insufficient in driving the synthesis of ATP, the dehydration of 2PG allows for such a reaction to occur by forming a high-energy intermediate. The high potential of PEP reflects the large release of energy that occurs with the conversion of enolpyruvate to its keto tautomer, pyruvate <ref>{{book |author=Voet, Donald; Voet, Judith C.; Pratt, Charlotte W.|title=Fundamentals of Biochemistry: Life at the Molecular Level|edition= 3|pages=503|}}</ref>.

==Kinetics and Regulation==

The kinetics of pyruvate kinase depend on K+ concnentration. <ref>{{article |author=Oria-Hernandez, Jesus.; Cabrera, Nallely.|title=Pyruvate KKinase Revisited: The Activating effect of K+|volume=280|pages=37924-37929|}}</ref>.:

Without a high K+ concnetration, the kinetic mechanism of pyruvate kinase changes from random to ordered with phosphoenolpyruvate as the first substrate. Vmax with K+ was about 400 times higher than a wild type sample without K+. In the presence of K+, the affinities for phosphoenolpruvate and ADP were 2-6 times higher than in the abscence of K+. This shows that K+ is involved in the acquisition of the active conformation of the enzyme, allowing either phosphoenolpyruvate or ADP to bind independently, but without K+, ADP cannot bind to the enzyme until phosphoenolpyruvate forms a competent active site for an ordered mechanism. Wild type pyruvate kinase without K+ has an ordered rapid equilibrium kinetic mechanism that shows Vmax to be 0.8 +/- -.04 umol/min mg with a kcat of 3.2 s^-1. When the wild-type pyruvate kinase has K+, it is in a random rapid equilibrium kinetic mechanism with a Vmax of 299 +/- 11 umol/min mg with a kcat of 1182.

In the glycolytic cycle, there are three compounds that have a large negative ∆G which includes the reaction pyruvate kinase catalyzes. Due to these three steps regulating the overall activity of the cycle, they are generally irreversible in vivo. Through numerous studies, the activity of pyruvate kinase has been found to be regulated by these effectors <ref>{{article |author=Dann, Leighton G.; Britton, Hubert G.|title=Kinetics and Mechanism of Action of Muscle Pyruvate Kinase|volume=169|pages=39-54|}}</ref>.:

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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. This, then, assumes that the products inhibit the enzyme’s reaction by simply reversing the reaction. Both pyruvate and ATP have been shown to be non-competitive inhibitors of pyruvate kinase. <ref>{{article |author=Dann, Leighton G.; Britton, Hubert G.|title=Kinetics and Mechanism of Action of Muscle Pyruvate Kinase|volume=169|pages=39-54|}}</ref>.

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. This, then, assumes that the products inhibit the enzyme’s reaction by simply reversing the reaction. Both pyruvate and ATP have been shown to be non-competitive inhibitors of pyruvate kinase. <ref>{{article |author=Dann, Leighton G.; Britton, Hubert G.|title=Kinetics and Mechanism of Action of Muscle Pyruvate Kinase|volume=169|pages=39-54|}}</ref>. With or without K+, oxalate is a noncompetitive inhibitor of ADP-Mg2+. It will form a nonproductive ternary complex which diminishes Vmax without altering the binding of ADP-Mg2+.ref>{{article |author=Oria-Hernandez, Jesus.; Cabrera, Nallely.|title=Pyruvate KKinase Revisited: The Activating effect of K+|volume=280|pages=37924-37929|}}</ref>.:

==Pyruvate Kinase Deficiency==

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@@ Line 12: / Line 12: @@
 [[Image:Pyruvatekinasemechanism.gif|left]]
 <ref>{{website1| title=Exercise-Induced Metabolic Acidosis: Where do the Protons come from?|url=http://www.sportsci.org/jour/0102/rar.htm|}}</ref>
-<scene name='Keegan_Gelvoria_Sandbox_1/N_c_rainbow/null'>Pyruvate Kinase</scene> catalyzes the final reaction of glycolysis. It couples the free energy of PEP cleavage to the generation of ATP during the synthesis of the final product, pyruvate. This reaction necessitates both K+ and Mg2+ cations, which has two steps. The first step is the nucleophilic attack of the PEP phosphorous atom by β-phosphoryl oxygen of ADP; this step displaces enolpyruvate while forming ATP. In the second step, enolpyruvate tautomerizes to pyruvate <ref>{{book |author=Voet, Donald; Voet, Judith C.; Pratt, Charlotte W.|title=Fundamentals of Biochemistry: Life at the Molecular Level|edition= 3|pages=502|}}</ref>. The formation of a high-energy intermediate by enolase in the 9th reaction of glycolysis allows for the synthesis of ATP in this reaction. Though the hydrolysis of 2PG is insufficient in driving the synthesis of ATP, the dehydration of 2PG allows for such a reaction to occur by forming a high-energy intermediate. The high potential of PEP reflects the large release of energy that occurs with the conversion of enolpyruvate to its keto tautomer, pyruvate <ref>{{book |author=Voet, Donald; Voet, Judith C.; Pratt, Charlotte W.|title=Fundamentals of Biochemistry: Life at the Molecular Level|edition= 3|pages=503|}}</ref>.
+<scene name='Keegan_Gelvoria_Sandbox_1/N_c_rainbow/null'>Pyruvate Kinase</scene> catalyzes the final reaction of glycolysis. It couples the free energy of PEP cleavage to the generation of ATP during the synthesis of the final product, pyruvate.Pyruvate kinase cataylzes the transfer of phsphoryl group of PEP to ADP in the presence of two ions of MG2+ and one of K+. The first step is the nucleophilic attack of the PEP phosphorous atom by β-phosphoryl oxygen of ADP; this step displaces enolpyruvate while forming ATP. In the second step, enolpyruvate tautomerizes to pyruvate <ref>{{book |author=Voet, Donald; Voet, Judith C.; Pratt, Charlotte W.|title=Fundamentals of Biochemistry: Life at the Molecular Level|edition= 3|pages=502|}}</ref>. The formation of a high-energy intermediate by enolase in the 9th reaction of glycolysis allows for the synthesis of ATP in this reaction. Though the hydrolysis of 2PG is insufficient in driving the synthesis of ATP, the dehydration of 2PG allows for such a reaction to occur by forming a high-energy intermediate. The high potential of PEP reflects the large release of energy that occurs with the conversion of enolpyruvate to its keto tautomer, pyruvate <ref>{{book |author=Voet, Donald; Voet, Judith C.; Pratt, Charlotte W.|title=Fundamentals of Biochemistry: Life at the Molecular Level|edition= 3|pages=503|}}</ref>.
 ==Kinetics and Regulation==
+The kinetics of pyruvate kinase depend on K+ concnentration. <ref>{{article |author=Oria-Hernandez, Jesus.; Cabrera, Nallely.|title=Pyruvate KKinase Revisited: The Activating effect of K+|volume=280|pages=37924-37929|}}</ref>.:
+ Without a high K+ concnetration, the kinetic mechanism of pyruvate kinase changes from random to ordered with phosphoenolpyruvate as the first substrate. Vmax with K+ was about 400 times higher than a wild type sample without K+. In the presence of K+, the affinities for phosphoenolpruvate and ADP were 2-6 times higher than in the abscence of K+. This shows that K+ is involved in the  acquisition of the active conformation of the enzyme, allowing either phosphoenolpyruvate or ADP to bind independently, but without K+, ADP cannot bind to the enzyme until phosphoenolpyruvate forms a competent active site for an ordered mechanism. Wild type pyruvate kinase without K+ has an ordered rapid equilibrium kinetic mechanism that shows Vmax to be 0.8 +/- -.04 umol/min mg with a kcat of 3.2 s^-1. When the wild-type pyruvate kinase has K+, it is in a random rapid equilibrium kinetic mechanism with a Vmax of 299 +/- 11 umol/min mg with a kcat of 1182.
 In the glycolytic cycle, there are three compounds that have a large negative ∆G which includes the reaction pyruvate kinase catalyzes. Due to these three steps regulating the overall activity of the cycle, they are generally irreversible in vivo. Through numerous studies, the activity of pyruvate kinase has been found to be regulated by these effectors <ref>{{article |author=Dann, Leighton G.; Britton, Hubert G.|title=Kinetics and Mechanism of Action of Muscle Pyruvate Kinase|volume=169|pages=39-54|}}</ref>.:
@@ Line 27: / Line 30: @@
-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. This, then, assumes that the products inhibit the enzyme’s reaction by simply reversing the reaction. Both pyruvate and ATP have been shown to be non-competitive inhibitors of pyruvate kinase. <ref>{{article |author=Dann, Leighton G.; Britton, Hubert G.|title=Kinetics and Mechanism of Action of Muscle Pyruvate Kinase|volume=169|pages=39-54|}}</ref>.
+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. This, then, assumes that the products inhibit the enzyme’s reaction by simply reversing the reaction. Both pyruvate and ATP have been shown to be non-competitive inhibitors of pyruvate kinase. <ref>{{article |author=Dann, Leighton G.; Britton, Hubert G.|title=Kinetics and Mechanism of Action of Muscle Pyruvate Kinase|volume=169|pages=39-54|}}</ref>. With or without K+, oxalate is a noncompetitive inhibitor of ADP-Mg2+. It will form a nonproductive ternary complex which diminishes Vmax without altering the binding of ADP-Mg2+.ref>{{article |author=Oria-Hernandez, Jesus.; Cabrera, Nallely.|title=Pyruvate KKinase Revisited: The Activating effect of K+|volume=280|pages=37924-37929|}}</ref>.:
 ==Pyruvate Kinase Deficiency==