PowerPoint PresentationSlide 2Slide 3Slide 4Slide 5Slide 6Slide 7Slide 8Slide 9Slide 10Slide 11Slide 12Slide 13Slide 14Slide 15Lactate fermentation, lactate threshold and Cori cycleSlide 17Slide 18Slide 19Slide 20Slide 21Slide 22Slide 23Slide 24Glycolysis and gluconeogenesisYue [email protected] Lineberger Cancer CenterCatabolism can be divided into three major stagesGlucose enters the cell through specific transportersFirst step of glycolysis: trap the glucose inside the cell 1. G6P is not a substrate of the glucose transporter and therefore cannot pass through the membrane 2. Addition of the phosphoryl group destabilizes the glucose, facilitating its further metabolismGlycolysis includes 10 (or 9) reactions and can be divided into two (or 3) stagesTrap and destabilize glucose(2 ATPs used)6-C glucose is cleaved into two 3-C fragments ATP production(4 ATPs produced)123456789103 irreversible and regulatory stepson HK, PFK, PK. HKPKPFKThe net reaction of glycolysisGlucose + 2 Pi + 2ADP + 2NAD+  2 pyruvate + 2 ATP + 2 NADH + 2H+ + 2H2OHow is glycolysis regulated?Allosteric regulation of enzymesRegulation of an enzyme by the binding of a molecule (regulator) at a site different the active site and change the conformation (shape) of the active site, leading to the change of the activity of the enzyme, either positively or negatively.Regulation of glycolysis: allosteric inhibition of PFK1 by ATPThe enzyme activity increases as ATP/AMP ratio is lowered. i.e. glycolysis is stimulated as the energy is needed.ATP inhibits PFK by decreasing its affinity with its substrate, F6P, and AMP reverses the inhibition.PFK1PFKATP-to-ADP hydrolysis change the conformation of PFK1Webb et al. (2015) Nature, PMID: 25985179F-2,6-BP is the most potent activator of PFK and is able to activate PFK when ATP is highPFK1 F-2,6-BPHow is F-2,6-BP concentration regulated? Glycolysis can be regulated by the energy status via metabolitesF-2,6-BP concentration is regulated by phosphofructokinase 2 (PFK2)Fructose-6-phosphate(F6P)ATP ADPPhosphorylationXH2ODephosphorylationPXPFK2Fructose-2,6-bisphosphate(F2,6BP)How are two opposed activities of PFK2 (kinase vs vs phosphatase) regulated?PBy the phosphorylation on a single serine residue:Phosphorylation of Ser32 of PFK2 inhibits the kinase and activates the phosphatase.PFK2 is regulated in liver by phosphorylation and blood glucose concentrationF2,6BPF6PH2OPiF6PF2,6BPATPADPProtein kinase AFasting / glucose scarceDephosphorylationProtein phosphataseGlucagon riseF6PFed / glucose abundantNo PFK1 stimulationGlycolysis inactivePFK1StimulationGlycolysis activePXPhosphorylationXPFK2 PFK2Liver releases glucose to bloodBiosynthesisRegulation of PK in muscle by feedforward mechanismPFK1At REST(glycolysis inhibited)GlucoseG6PF6PF-1,6 BPPEPPyruvatePFK1PKATP/AMPDURING EXCISE(glycolysis stimulated)GlucoseG6PF6PF-1,6 BPPEPPyruvatePFK1PKATP/AMPFeedforward stimulationPKFeedforward regulation:The product of a earlier reaction causes change of an enzyme later in the pathway- O2 Lactate(Metabolic dead end)What happens to the pyruvate next depends on the presence of oxygen+ O2 TCA(Produce more ATP)Pyruvate produced at the end of glycolysis must be used so the pathway can continue.Two NAD+ are used up for the metabolism of each glucose. If NADH is not reoxidized, the glycolysis will be stoppedLactate fermentation, lactate threshold and Cori cycleAlso known as lactate inflection point (LIP) or anaerobic threshold (AT) Is the excise intensity at which lactate starts to accumulate in the blood stream (from 1 mM at rest to ~ 4 mM).  Is a useful measure for deciding excising intensityLactate threshold (LT)Gluconeogenesis synthesizes glucose from non-carbohydrates GlucoseG6PF6PF-1,6 BPG3P1,3-BPG3PG2PGPEPPyruvateHKLactateGPIPFKFru-1,6 PaseALDGAPDHPGKPGAMENOPKG6PLDHOxaloacetatePEPCKPCGlycolysisGluconeogenesisGlycerolAmino acidsAdipose TriacylglycerolsProlonged fast (> 24 hours)GKGK: glycerol kinase How to prevent gluconeogenesis in adipose? GK gene is not expressed in adipose tissueProteinsProlonged fastMuscleExcising- O2 Gluconeogenesis is not simply the reversal of glycolysis: there are 4 reactions unique to gluconeogenesisHow to prevent glycolysis and gluconeogenesis occurring simultaneously?Reciprocal regulation of glycolysis and gluconeogenesisGlucoseG6PF6PF-1,6 BPG3P1,3-BPG3PG2PGPEPPyruvateHKGPIALDGAPDHPGKPGAMENOG6POxaloacetatePCPEPCK PKPhosphoenol-PyruvatecarboxykinasePhosphofructo-kinasePyruvatekinaseFrutose1,6-bisphosphatasePFKFru-1,6 PaseSummary: glycolysis regulation and gluconeogenesis Liver functions to maintain blood glucose level in two ways: degrading glycogen to glucose (glycogenolysis) and synthesizing glucose from non-carbohydrate (gluconeogenesis).  Gluconeogenesis shares most enzymes with glycolysis, but is not simply the reversal of glycolysis. There are 4 reactions unique to gluconeogenesis. F-2,6-BP is the most potent activator of PFK1 and can override ATP inhibition and its concentration is controlled by bifunctional PFK2. PFK2 is phosphorylated when blood glucose is low and this will reduce F-2,6-BP and inactivate glycolysis in liver.  The most important control step of glycolysis is the regulation of phosphofructokinase (PFK1). PFK1 is allosterically inhibited by ATP and stimulated by AMP (by decreasing and increasing the affinity of PFK1 for its substrate, F6P). This allows cells to stimulate glycolysis when energy (ATP) is needed.  Pyruvate kinase (PK) catalyzes the last step of glycolysis, converting phosphoenolpyruvate (PEP) to pyruvate. What happens to pyruvate depends on the presence of oxygen.Glucose is not the only sugar we consumeGalactoseFructoseFructose can be converted into glycolytic intermediatesGalactose can be converted into G6P LactoseLactaseLactose intoleranceDecrease during normal developmentToo much lactose causing gas and diarrheaClassic GalactosemiaCaused by deficiency in galactose 1-phosphate uridyl transferase (GALT)High level accumulation of galactoseCausing vomiting, diarrhea, jaundice, cataracts …GalactoseGalactose 1-phosphateUDP-GalactoseGlucose 1-phosphateGlucose 6-phosphateUDP-GlucoseGALTGalactitolPXATP ADPPhosphorylationF2,6BPF6PH2OPiXF6PF2,6BPATPADPProtein kinase AFasting / glucose scarce(glycolysis inactive)H2ODephosphorylationPPhosphoprotein phosphataseGlucagon riseF6PFed / glucose abundant(Glycolysis active)No PFK1 stimulationGlycolysis inactivePFK1StimulationGlycolysis