Glycolysis & Gluconeogenesis

What are the potential ramifications of ingesting large amounts of fructose?

1) Hepatic triacylglycerol production facilitated 2) Rate of lipogenesis increased = increased fat production 3) Insulin and leptin production not stimulated = from increase in fat production = insulin isn't released or has hard time interacting with its receptor 4) LDL particle size reduced 5) Insulin resistance promoted

Which enzymes in gluconeogenesis pathway are regulated and what regulates them?

1) Phosphoenolpyruvate carboxykinase: Negative regulator: ADP 2) Pyruvate carboxylase: Positive regulator: Acetyl CoA Negative regulator: ADP Glucagon promotes transcription of pyruvate carboxylase 3) Fructose 1,6-bisphosphatase: Positive regulator: Citrate Negative regulators: Fructose 1,6-bisphosphate, AMP Glucagon promotes transcription of fructose 1,6-bisphosphatase 4) Glucose 6-phosphatase: Positive regulator: glucose 6-phosphate Glucagon promotes transcription of glucose 6-phosphatase

4 unique, irreversible reactions in gluconeogenesis

1) Pyruvate carboxylase 2) Phosphoenolpyruvate carboxykinase 3) Fructose 1,6-bisphosphatase 4) Glucose 6-phosphatase

Identify steps of gluconeogenesis that differentiates this metabolic process from glycolysis

4 unique, irreversible reactions Pyruvate carboxylase Phosphoenolpyruvate carboxykinase Fructose 1-6-bisphosphatase Glucose 6-phosphatase

Pyruvate carboxylase: Positive allosteric modulator

Acetyl CoA

Glycolysis Stage 1: Key control point for glycolysis

After glucose 6-phosphate is formed, if going down glycolytic pathway---is isomerase that converts glucose 6-phosphate to fructose 6-phosphate (isomerization takes place) Fructose 6-phosphate is then acted upon by phosphofructokinase, utilizing a molecule of ATP, to phosphorylate fructose 6-phosphate to give fructose 1,6-bisphosphate This is now the committed pathway, once this reaction takes place = now committed to go down the glycolytic pathway This becomes a key control point for glycolysis = major point at which glycolysis is regulated = at this point in the pathway We have already consumed 2 molecules of ATP---for a pathway that is supposed to generate ATP = have just consumed it and not generated any at this point Slide 19 Glycolysis

What is the polyol pathway and what is its implication in the complication of diabetes?

Conversion of glucose to sorbitol & ultimately fructose. In diabetic with hyperglycemia = not being acted upon by normal metabolic processes---enzyme aldose reductase will react & form sorbitol This can create osmotic effects in the cells More importantly, consumes NADPH during reaction NADPH is very important in minimizing oxidative stress = reactive oxidative species being formed = damage taking place within cells---happens with uncontrolled diabetes = no NADPH to combat the oxidative stress, because used up in the polyol pathway

What is the Cori Cycle and what is its importance?

Cori Cycle is the when the gluconeogenic pathway provides other tissues with glucose. If one organ is doing glycolysis an another is doing gluconeogenesis = beneficial One organ can help supply energy needs of another When running, muscle cells are going to be very active in terms of using glucose When get to pyruvate, can go into aerobic oxidation/glycolysis or when oxygen is limited---can go into anaerobic glycolysis and form lactate Lactate can be released into systemic circulation to cardiac muscle Also used by muscle & converted to pyruvate---goes into oxidative phosphorylation pathway Or pyruvate can go into liver & gluconeogenic pathway---make glucose & out to systemic circulation & picked up by muscles to use Liver can provide metabolic needs of muscle to sustain activity. Similar scenario in starvation situations

What is fermentation? What is the importance of lactic acid fermentation with respect to the glycolytic process?

Fermentation occurs in muscles with limited oxygen, to change pyruvate into lactate. After the formation of lactate, it can be used in the glycolytic pathway to create ATP

Describe how glucagon stimulates fructose 1,6-bisphosphatase activity, thereby enhancing gluconeogenesis

First, glucagon enhances the expression of fructose 1,6-bisphosphatase. Second, glucagon activates protein kinase A, which phosphorylates the bifunctional enzyme PFK2/FBPase2. This leads to activation of FBPase2 (fructose bisphosphatase2) activity, which results in the breakdown of negative regulator of fructose 1,6-bisphosphatase, fructose 2,6-bisphosphate

Pyruvate kinase: Positive allosteric regulator

Fructose 1,6-bisphosphate

What are the general means by which galactose and fructose are brought into the glycolytic pathway? What is the ramification of where fructose enters the glycolytic cycle?

Galactose is brought into the glycolytic pathway at glucose 6-phosphate. Fructose in the adipose tissue is converted by hexokinase to fructose 6-phosphate, and in the liver tissue it goes through the fructose 1-phosphate pathway---becoming glyceraldehyde or dihydroxyacetone, and then glyceraldehyde 3-phosphate. The ramifications of where fructose enters the pathway are (since is below the regulatory step of phosphofructokinase) even if phosphofructokinase is shut off, glyceraldehyde and dihydroxyacetone are still developed, and glyceraldehyde 3-phosphate is still being formed, which can form acyl glycerol (fats) and acyl CoA (fatty acids)

Bifunctional Enzyme Synthesizes and Degrades Fructose 2,6-bisphosphate

Get fructose 2,6-bisphosphate by fructose 6-phosphate Carried out by an enzyme (molecule) that has 2 enzymatic activities One is phosphofructokinase 2 = phosphorylates fructose 6-phosphate to give fructose 2,6-bisphosphate Another is fructose phosphatase 2 = removes phosphate group from fructose 2,6-bisphosphate and gives fructose 6-phosphate This system is regulated = either turning on kinase or phosphatase activity This is how form the regulator that regulates the phosphofructokinase = key committed regulatory step in glycolytic pathway Slide 24 Glycolysis

Hexokinase: Negative allosteric modulator

Glucose 6-phosphate

Conversion of Glucose to Other Cell Constituents

Glucose also serves as precursor for other important constituents that our cells need Glycolysis pathway shown Intermediates can off-shoot into making amino acids like serine, and then subsequently glycine and cysteine Or glycerol 3 phosphate---important along with the fatty acids = makes triacylglycerols/fats When get into tricarboxylic acid cycle, the intermediates of many other amino acids are derived from intermediates of TCA cycle Glucose also serves as a function in terms of producing other important biochemical molecules Slide 6 Glycolysis

Glycolysis & Gluconeogenesis: Need to know

How these 2 processes are regulated---need to really understand Need to know steps that are regulated----allosterically and by hormones (insulin and glucagon) Understand the transporters that talked about, their role & how relates to secretion of insulin & role within the liver in terms of metabolization of glucose Biotin, UDP-glucose = carrier molecules----know what they are doing These are very key points that will be on exam

Skeletal muscle: Insulin actions

Increase glucose uptake Increase glycogen synthesis Increase protein synthesis

What is the biochemical basis of galactosemia and what are its clinical ramifications?

Inherited deficiency in galactose 1-phosphate uridyl transferase Cannot handle galactose from diet Manifestations: Diarrhea Liver enlargement & jaundice Retarded mental development Cataracts

What does glucose 6-phosphatase do?

It transforms glucose 6-phosphate to a phosphate ion and glucose, releasing glucose & transporting it out of liver cells

GLUT 1

Located: All tissues Basal glucose uptake Km is 1 mM BBB tissues

Regulation of Glycolysis in Muscle

Muscle at rest vs muscle at exercise At rest, muscle doesn't need ATP = ATP stores are plentiful & glycolysis is inhibited ATP, being plentiful, see phosphofructokinase and pyruvate kinase being inhibited That being the case, fructose 6-phosphate will build up---which can go back to become glucose 6-phosphate & if that builds up--will inhibit hexokinase At exercise, things will change ATP that has been there, will be diminished & AMP will have higher concentrations Energy charge in the muscle has changed---lowered it AMP will activate phosphofructokinase = will increase the concentrations of fructose 1,6-bisphosphate within the muscle cell = in turn will activate pyruvate kinase Slide 34 Glycolysis

Skeletal muscle: Glucagon actions

None

Reciprocal Regulation of Gluconeogenesis and Glycolysis

Regulate those steps that are unique to gluconeogenesis 1st step: Pyruvate carboxylase = that is regulated by acetyl CoA positively, and ADP negatively ADP is reflection of energy charge in cell = large amounts of ADP around = means little amount of ATP ---- under those conditions, do not want to consume energy to make glucose, want to metabolize glucose to make ATP Makes sense that concentrations of ADP around, will slow down gluconeogenesis = do so by allosterically inhibiting pyruvate carboxylase ADP also does that with next step, phosphoenolpyruvate carboxykinase Slow down gluconeogenesis when have low energy charge = those circumstances---cell needs energy, and to get energy, needs to metabolize glucose Acetyl CoA = positive modulator of pyruvate carboxylase This is because pyruvate going to oxaloacetate forms a unique role: 1) starts process of gluconeogenesis 2) oxaloacetate also component of citric acid cycle, process in aerobic pathway---take pyruvate to acetyl CoA and further metabolize it to release the maximum amount of energy & ATP formation in our cells Acetyl CoA = when plenty around---want to make sure it can also go into citric acid cycle By forming oxaloacetate, which is a component of the cycle is one of the reasons why Acetyl CoA positively affects this reaction Just like in glycolytic pathway, where phosphofructokinase was very important, the corresponding fructose 1,6-bisphosphatase site is also very key point of regulation in gluconeogenesis Some of the components that have an effect on phosphofructokinase have an opposite effect on fructose 1,6-bisphosphate Citrate negatively effects phosphofructokinase, positively effects fructose 1,6-phosphatase AMP positively effects phosphofructokinase, negatively effects fructose 1,6-phosphatase In liver cell, both glycolysis and gluconeogenesis are thermodynamically favorable processes, but not useful for cell to have both going on at same time When one is going on, the other needs to be dampened down---this is a way to regulate that Fructose 2,6-bisphosphate has positive effect on phosphofructokinase, and negative effect on fructose 1,6-bisphosphatase AMP as example: low energy charge----indication that cell needs ATP = expect gluconeogenic process to be turned off & glycolysis to be turned on Citrate = indication of building block availability = when have a lot of citrate around = have more than enough building blocks, so can start making glucose (gluconeogenesis) Whereas when citrate is low = indication that need building blocks---need to metabolize glucose (glycolysis) Importance of this is that fructose 1,6-bisphosphatase is a target for antidiabetic drugs Designing mimics of AMP that would negate this process In diabetes, even though have hyperglycemia --- body thinks is not getting enough glucose, either because insulin is not being released or insulin is not acting appropriately (insulin resistance) Diabetes = gluconeogenic pathway is turned on by glucagon = this just aggravates hyperglycemia because the role of the liver is to start spewing out glucose under gluconeogenic conditions Blocking that process is a means of treating some of the symptoms of diabetes, hyperglycemia Whether talking about glycolysis or gluconeogenesis = fructose 2,6 bisphosphate is key allosteric regulator in each case One case = stimulates (glycolysis) Other case = inhibits (gluconeogenesis) Control & synthesis of that allosteric modulator becomes very important Slide 24 Glycolysis: Conversion of fructose 6-phosphate to fructose 2,6-bisphosphate by allosteric modulator This is done by a molecule that possesses 2 enzymatic activities (kinase and phosphatase) How this system is regulated is done by turning on either phosphatase or kinase activity Factors that control that are both hormonal as well as substrate concentrations Slide 24 and 53 Glycolysis

What is gluconeogenesis and in which tissues does it primarily occur?

Synthesis of glucose from non-carbohydrate precursors (lactic acid---product of anaerobic glycolysis, amino acids, glycerol---from breakdown of triacylglycerols/fats), it converts pyruvate to glucose Sites: Liver = major, Kidney = minor

What is the important ramification of the pathway by which fructose enters into the glycolytic pathway in the liver?

The entry of fructose into the glycolytic pathway in the liver occurs after the key regulatory step in the glycolytic pathway, ie: phosphofructokinase

How are glucokinase allosteric activators useful as antidiabetic agents?

They bind to an allosteric site on glucokinase, activating the enzyme---increasing the affinity of glucose, making it work at lower concentrations, increasing Vmax (increases rate of reaction)---treats hypoglycemia and insulin resistance

Gluconeogenesis takes place during intense exercise, which seems counterintuitive. Why and how would an organism synthesize glucose and at the same time use glucose to generate energy?

This is an example of inter-organ cooperation that allows the organism to shift part of the metabolic burden from muscle to liver. Glycolysis proceeds to lactate in active skeletal muscle during intense exercise because of insufficient oxygen for complete oxidation. The lactate is released into the blood and subsequently absorbed by the liver, where it is converted by gluconeogenesis into glucose. This glucose is released into the blood stream and becomes available to muscle cells for continued exercise

What is the general function of cofactor carboxybiotin?

To transform pyruvate to oxaloacetate

Key things to remember from this lecture

Transporters Glucokinase Phosphofructokinase and hexokinase = regulatory effects on glycolytic pathway

The Cori Cycle

Using gluconeogenic pathway in liver that utilizes the lactate from muscle/RBCs to provide glucose that can then be used by muscle/RBCs in glycolysis Helping or assisting one tissue in its needs in terms of metabolism

What factors control the synthesis and degradation of fructose 2,6-bisphosphate?

When kinase is active & phosphatase is inactive---fructose 6-phosphate is transformed to fructose 2,6-bisphosphate. Large amount of fructose 6-phosphate = stimulatory effect on kinase Large amount of fructose 2,6-bisphosphate = stimulatory effect on phosphatase. Glucagon is promoting gluconeogenesis Glucagon stimulates protein kinase A when blood glucose is scarce---changing phosphofructokinase to fructose 1,6-bisphosphatase Insulin stimulates phosphoprotein phosphatase----changing fructose 1, 6-bisphosphatase to phosphofructokinase

Glucokinase only activated when

high levels of glucose in blood

Glucagon promotes transcription of

phosphatase and pyruvate carboxylase enzymes = gluconeogenic enzymes So favors the anabolic reaction creating glucose

Insulin promotes transcription of

phosphofructokinase & pyruvate kinase transcription = glycolytic enzymes So favors the breaking down of glucose

What are the metabolic fates of glucose 6-phosphate?

1) Glycolysis: Glucose 6-phosphate to fructose 6-phosphate to pyruvate 2) Pentose phosphate pathway: Glucose 6-phosphate to 6-phosphogluconate to pentose phosphates 3) Glucose 6-phosphate to Glucose 1-phosphate to glycogen storage 4) Can be formed into UDP-glucuronic acid to form glucuronides in liver metabolism of various drugs

Potential Ramifications of Ingesting Large Amounts of Fructose

1) Hepatic triacylglycerol production facilitated 2) Rate of lipogenesis increased---increased fat formation Problem with that, is when increase fat---affects insulin = insulin resistance is now enhanced Increase in fat/fatty acids has an effect on insulin being released, and insulin being able to interact with its receptor to exert its effects 3) Insulin and leptin production not stimulated 4) Low-density lipoprotein particle size reduced 5) Insulin resistance promoted Fructose primarily enters glycolytic pathway in the liver Liver is the primary place where fructose will be metabolized

What are the major actions of insulin, glucagon, GLP-1, and GIP in metabolism?

1) Insulin: signals the need to remove glucose from the blood for storage as glycogen or conversion into fat. a) In adipose tissue: Increase glucose uptake, increase lipogenesis, decrease lipolysis. b) Muscle: increase glucose uptake, increase glycogen synthesis, increase protein synthesis. c) Liver: Decrease gluconeogenesis (biosynthesis of glucose), increase glycogen synthesis, increase lipogenesis 2) Glucagon: When blood glucose level is low, glucagon triggered cAMP cascade leads to phosphorylation of pyruvate kinase---which diminishes its activity = favors gluconeogenesis a) Adipose tissue: Increase lipolysis b) Muscle: No effect c) Liver: Increase gluconeogenesis, increase glycogenolysis (breakdown of glucose) 3) GLP-1 (Glucagon-like peptide 1): signal molecule that induces feelings of satiety in the brain. a) Pancreas: Beta cells: increase insulin release & biosynthesis b) Pancreas: Alpha cells: decrease glucagon secretion c) Brain: Increase satiety, decrease food intake d) Stomach: decrease gastric emptying, decrease gastric acid secretion 4) GIP (Gastric inhibitory polypeptide): a) Adipose tissue: Increase lipolysis b) Pancreas: Beta cells: increase insulin release and biosynthesis

Diverse Fates of Pyruvate

1) Pyruvate can serve as a building block for alanine---by transamination 2) Carboxylation of pyruvate to give oxaloacetate = important component of gluconeogenesis & also citric acid cycle 3) Process of regenerating NAD+ is going to occur in the process of forming Acetyl CoA by oxidative decarboxylation Generate Acetyl CoA, which goes into citric acid cycle and oxidative phosphorylation This constitutes aerobic glycolysis, because these processes require oxygen This process regenerates NAD+ 4) Other process that can take place is anaerobic glycolysis This is caused by the reduction of pyruvate to lactate (also called fermentation) Some of our very pathogenic substances are also anaerobes---operate under no oxygen This has some benefits---if no oxygen, can still sustain the cell Fermentation used in muscle cells to convert pyruvate to lactate in the reduction process Slide 36 Glycolysis

What 3 glycolytic enzymes are regulated? How are they regulated & what regulates them?

1) Pyruvate kinase---regulated by allosteric regulators, glucose levels and glucagon, insulin Low levels of glucose will cause glucagon to be released & will cause pyruvate kinase to be phosphorylated & become much less active = favors gluconeogenesis Insulin promotes the transcription of pyruvate kinase = favors glycolysis Positive allosteric regulator: Fructose 1,6-bisphosphate Negative allosteric regulators: ATP and Alanine 2) Hexokinase---inhibited by glucose 6-phosphate by negative feedback inhibition. If too much glucose 6-phosphate being produced---will shut off hexokinase in allosteric manner 3) Phosphofructokinase: allosteric regulators, insulin Positive allosteric regulators: Fructose 2,6-bisphosphate and AMP Negative allosteric regulators: ATP and Citrate Insulin promotes the transcription of phosphofructokinase

Gluconeogenesis

1) Synthesis of glucose from noncarbohydrate precursors a) Lactic acid---product of anaerobic glycolysis b) Amino acids c) Glycerol---comes from breakdown of triacylglycerols/fats Important process in body, because brain primarily relies on glucose for its fuel & RBCs can ONLY use glucose for their fuel Need 160 g of glucose daily----120 g needed by brain In body, have 20 g of glucose on average at any given time Storage form, glycogen, have about 190 g glucose Have enough glucose around to cover us for one day In times of starvation = body needs another mechanism by which it can produce glucose that can be used by brain, RBCs, other cells 2) Gluconeogenic pathway converts pyruvate to glucose 3) Sites of gluconeogenesis a) Liver---major site = helps to maintain blood glucose levels, liver is either picking up glucose from the systemic circulation when there is a lot around (to process it), or in case of low glucose levels = produces glucose so it can be distributed to other cells b) Kidney---minor site Other cells (heart muscle, skeletal muscle, brain cells) can perform gluconeogenesis, but not very prevalent 4) Gluconeogenesis is not simple reversal of glycolysis Because have 3 steps in glycolysis that are irreversible, only go in one direction To go in the other direction, would be problematic

What are the diverse fates of pyruvate?

1) Transamination = becomes alanine 2) Carboxylation = becomes oxaloacetate = important component in gluconeogenesis & citric acid cycle 3) Oxidative decarboxylation = becomes acetyl CoA (makes NAD+)----goes through citric acid cycle and oxidative phosphorylation which both encompass aerobic glycolysis 4) Reduction/fermentation = becomes lactate---by anaerobic glycolysis

Actions of GLP-1 and GIP

2 other hormones that are important: GIP & GLP-1 GIP = Gastric inhibitory polypeptide GLP-1 = Glucagon-like peptide-1 Have multiple effects Both made by intestinal cells, and released by certain stimulation GIP's effects are on: 1) Adipocytes: Increases lipolysis 2) Beta cells of pancreas: Increases insulin release & biosynthesis GLP-1 effects are on: 1) Beta cells of pancreas: Increases insulin release & biosynthesis 2) Alpha cells of pancreas: decreases glucagon secretion 3) Stomach: Decreases gastric emptying, decreases gastric acid secretion 4) Brain: Increases satiety (feeling full), decreases food intake Some of the drugs being used to treat diabetes mimic these hormones Slide 5 Glycolysis

Phosphoenolpyruvate carboxykinase: Negative allosteric modulator

ADP

Pyruvate carboxylase: Negative allosteric modulator

ADP

Pyruvate kinase: Negative allosteric regulators

ATP Alanine

Phosphofructokinase: Negative allosteric regulators

ATP Citrate

Activation of Phosphofructokinase by Fructose 2,6-bisphosphate

Activates by inhibiting the effects of ATP Facilitating the affinity of fructose 6-phosphate to the enzyme If look at 1 mM fructose 6-phosphate If have no fructose 2,6-bisphosphate around---get this reaction rate If increase the level of fructose 2,6-bisphosphate = much higher reaction rates because have activated the enzyme---increased the affinity of fructose 6-phosphate for the enzyme Much greater activity with lower concentrations of substrate present With ATP, what fructose 2,6-bisphosphate does is essentially inhibit the inhibitory effects of ATP If look at a case where have this amount of ATP present, & have no fructose 2,6-bisphosphate = essentially have very little enzymatic activity = ATP is effectively inhibiting the enzyme As increase the concentration of fructose 2,6-bisphosphate = override the inhibitory effect of ATP, because now have very high enzymatic activity with that activator present Slide 23 Glycolysis

Galactosemia

An inherited deficiency in galactose 1-phosphate uridyl transferase Individuals that lack that enzyme, do not have ability to process galactose in diet Manifestations: Diarrhea Liver enlargement and jaundice Retarded mental development Cataracts---because galactose within the eye is acted upon by enzyme aldose reductase that gives a polyol substance = polyol acts as osmotic, because doesn't get out---brings in water & causes cataracts to form in the eye By limiting galactose & lactose type products in diet = can minimize liver enlargement and jaundice, but no effect on mental development, because need galactose in the right form to galactosylate the proteins Slide 42 Glycolysis

Glycolysis Stage 2

At this point, first stage is completed Through all these reactions, have not formed any ATP, but have consumed it Now have two three carbon units that can go down this pathway It's in stage 2 where now we start to form ATP molecules First step = glyceraldehyde 3-phosphate acted upon by glyceraldehyde 3-phosphate dehydrogenase to form 1,3-bisphosphoglycerate This is a molecule that has a high potential to transfer a phosphate group Slide 27 Glycolysis

Family of glucose transporters

Be familiar with GLUT 1, GLUT 2, GLUT 3, and GLUT 4 GLUT 1 and 3 = found in most tissues---have function of basal glucose uptake. Km is 1 mM, and glucose concentrations are normally from either 4 to 8 mM---means that these transporters are continually operating, because the plasma concentrations are higher than the Km for the transporter system---way glucose can continually get into cells of body Whether GLUT 1 or 3 = tissue localization GLUT 1 = commonly seen in BBB tissues GLUT 3 = neurons themselves In order for glucose to get into the brain, has to cross BBB---GLUT 1 facilitates that Has to get into neuron---GLUT 3 facilitates that When glucose concentrations get below levels of 1 mM---can happen in diabetic patient that has taken too much insulin, or has taken insulin & has not ingested food after that Insulin facilitates movement of glucose into adipose cells and striatal muscle---that will lower the plasma concentrations of glucose If levels get below 1 mM---then transporters are not going to be operational Ramifications for diabetic: Issues with dizziness, confusion regarding lack of sugar being brought to brain tissue---does require GLUT 1 and 3---if gets severe = could go into a coma, eventually die GLUT 2 = found in liver and pancreatic beta cells Km is much higher---15 to 20 mM = means that these transporters are really only activated when plasma concentrations of glucose become very, very high---after a meal Role of GLUT 2 in pancreas---plays a role in insulin secretion & in liver serves a role to remove excess glucose from the blood Liver is main organ for controlling plasma glucose concentrations---it will produce glucose to be released when plasma concentrations are low, when plasma concentrations are high = GLUT 2 transporter brings the glucose to the liver where it can be acted on by glucokinase GLUT 4 = muscle and fat cells Dependent upon insulin---insulin stimulates the expression of this transporter = facilitates glucose movement into adipose and muscle cells

Carboxybiotin

Biotin is connected to enzyme by carboxy group, usually linked through lysine residue = forming an amide bond What that provides is a long arm in which the active group. biotin that carries CO2 group, can be shuttled from one site to another site on this particular enzyme (or other enzymes that perform similar reactions) Pyruvate carboxylase has multiple domains = each domain carries out a different function Biotin, because it has to interact with all 3 sites, has to be able to move form one site to the next site to the next site Long arm from the lysine side chain & side chain of biotin allows the process to take place Biotin is a CO2 carrier---adds CO2 to organic molecules In this case, adding it to pyruvate to form oxalacetate Slide 50 Glycolysis

Cancer and Glycolysis

Cancer cells, which are very rapidly growing, need a source of energy Their source of energy is like any other cell, glucose However, when cancer cell starts to develop into a tumor = it's very hypoxic state = not a lot of blood vessels going into it to provide oxygen Under that hypoxia, the tumor cells are going to release a transcription factor (HIF-1)---this alters the metabolism of tumor cells It facilitates glycolysis and fermentation process Glycolysis can be anaerobic or aerobic in nature Oxygen limited = anaerobic Cancer cells differ from normal cells in that they have to rely on fermentation process---formation of lactic acid to sustain itself = glycolytic pathway & the ATP that it needs, at least in the early stages Transcription factor will start to cause upregulation of various components of glycolytic pathway = glucose transporters, enzymes that are involved in many of the glycolytic pathway steps (regulatory/not regulatory) All to facilitate the glycolytic pathway and the anaerobic metabolism that takes place Also is a drug target for cancer cells in this type of condition, because things are different than with a normal cell Slide 44 Glycolysis

Fructose 1,6-bisphosphatase: Positive allosteric modulator

Citrate

Gluconeogenic pathway

Compare the 2 pathways: Have to get around 3 irreversible steps in glycolysis Do that by involving: 1) Pyruvate carboxylase 2) Phosphoenolpyruvate carboxykinase 3) Fructose 1,6 bisphosphatase 4) Glucose 6-phosphatase 4 unique, irreversible reactions for gluconeogenic pathway Other components of gluconeogenic pathway are same as for glycolysis Whether goes in one direction or another, depends on the concentrations of products or reactants These precursors come into the pathway: Lactate & some amino acids come in to form pyruvate at very beginning Other amino acids come in at oxaloacetate stage Glycerol (product of fat breakdown) coming into dihydroxyacetone phosphate stage Do not need to memorize every step, will look at the unique ones that relate to gluconeogenic pathway and how they are controlled Slide 47 Gluconeogenesis

Phosphoenolpyruvate Carboxykinase

Convert oxaloacetate to phosphoenolpyruvate (one of intermediates in gluconeogenic pathway), so can move up the pathway Decarboxylation, trapping the enol with GTP = decarboxylation plus the transfer of phosphate group that drives the reaction very favorably that gives the phosphoenolpyruvate At this point, now have formed phosphoenolpyruvate, and that can move up the gluconeogenic pathway using the same enzymes we saw in glycolysis until get to point where get to fructose 1,6-bisphosphate At that point have to use a different enzyme, instead of phosphofructokinase to generate fructose 6-phosphate That enzyme is fructose 1,6-bisphosphatase That gives rise to fructose 6-phosphate, which can move up to glucose 6-phosphate Here have to have unique enzyme to convert back to glucose = glucose 6-phosphatase Slide 47 and 51 Glycolysis

Liver tissue: Insulin actions

Decrease gluconeogenesis Increase glycogen synthesis Increase lipogenesis

Carbohydrate Digestion

Dietary carbohydrates (starch & saccharides) initially metabolized by alpha-Amylase in the saliva & subsequently by alpha-Amylase that is secreted by the pancreas in the small intestine---that breaks down larger carbohydrates into oligosaccharides & disaccharides (lactose, sucrose, etc) Then glycosidases breakdown the disaccharide sugars to galactose, glucose, and fructose Then it's the active transport of those sugars via intestinal epithelial cells that bring them into systemic circulation Slide 2 Glycolysis

Stages of Glycolysis

Do not need to remember every intermediate & enzyme Will focus on a few regulatory points in pathway---to remember Overall idea of what pathway does & its importance, and those points where it's regulated Can divide glycolysis into 2 stages: Stage 1 (pink) = Process where glucose is trapped in the cell---done by phosphorylation That process also starts to destabilize glucose molecule so it is amenable to further metabolism/breakdown Breakdown is to form phosphorylated 3 carbon units Stage 2 (yellow) = Generation of ATP Early on, actually consume ATP in metabolism of glucose Stage 2, generate ATP Overall process = net formation of ATP Slide 8 Glycolysis

Phosphofructokinase: Key Enzyme in the Control of Glycolysis

Factors that control phosphofructokinase: 1) Inhibited by ATP ATP acts as allosteric inhibitor of enzyme, structure of enzyme (slide 21) = tetramer, uses ATP as a substrate---in catalytic site there is an ATP binding site in each unit there are also allosteric sites for ATP = when ATP concentrations build up within the cell = indication that there is plenty of ATP around that can bind to allosteric sites = inhibit the process in a negative feedback fashion If whole pathway of glycolysis is to make ATP, and plenty of ATP around in the cell---do not need to go down glycolytic pathway ATP will help regulate that system 2) Activated by AMP---high AMP levels = have consumed a lot of ATP & cell needs more ATP----activates the process = acts at same allosteric sites, but does opposite 3) Inhibited by citrate---compound that occurs in citric acid cycle = is measure of sources for other intermediates (amino acids, etc) going to be used from the cell = reflects ability of potential molecules that can be used elsewhere----when plenty of citrate around, tells cell do not need to make more molecules that can be used to make amino acids, etc = inhibits this process Glycolytic pathway is not only for creating ATP, but for making other molecules that can be used throughout the cell 4) Activated by Fructose 2,6-bisphosphate = most important regulator. Isomer of product of reaction Slide 20 and 21 Glycolysis

Glycolysis Stage 1

First reaction when glucose gets into the cell = first regulatory step = actions of hexokinase on glucose to essentially phosphorylate glucose to give glucose 6-phosphate Consume a molecule of ATP in this process Kinases (are several types) are examples of enzymes that are substrate induced-cleft closing enzymes Slide 14 Glycolysis

Entry of Glucose into Cells--Glucose Transporters

For this system to work, glucose has to get into the cell Glucose is not permeable to the cell, is very polar with all its hydroxy groups---makes it difficult to get across cell membrane by passive diffusion Have transporters that are designed to facilitate this process 12 transporters that have been identified Will look at 4 that are important to glycolysis All have a general structure that look like picture on slide 10 Glycolysis---12 transmembrane helical segments---all come together to form macromolecular process that can move glucose across the membrane What looking at---glucose moving down a thermodynamically favorable concentration gradient = larger concentrations of glucose outside the cell than inside the cell These transporters do not require energy, they are facilitative in nature---facilitate the process of glucose moving down its concentration gradient Illustrates the process where transporter is open to glucose & it binds to glucose on exterior side = binding causes conformational change that inverts the system that then allows glucose to enter into the cellular surface Slide 10 Glycolysis

Lactic Acid Fermentation

Form pyruvate and lactate dehydrogenase will take NADH and essentially reduce pyruvate to give lactate, regenerating NAD+----which can then go back to be used in glycolytic pathway When we start exercising muscles, and have used what ATP is present & glycolytic pathway has been activated = if oxygen is limited, will go down fermentation pathway (slide 34 glycolysis) to sustain the glycolytic pathway and generate the ATP that muscles need Ultimately when oxygen becomes plentiful, will go down other pathway (oxidative phosphorylation, which requires oxygen) to generate more ATP Whether going down one pathway or another, depends on how much oxygen is available Someone that's been a runner, & developed running skills will go down the oxygen dependent pathway much sooner than someone who just started running Lactic acid fermentation can go in both directions---to the right is the thermodynamically favorable process Lactate is used as fuel by the heart, RBCs & to do that----has to be converted back to pyruvate Slide 34 and 37 Glycolysis

Polyol Pathway

Formation of polyols = also important in diabetes Have diabetic individual with hyperglycemia = not being acted upon by normal metabolic processes----enzyme aldose reductase, which doesn't have great affinity for glucose--but when around in large concentrations, can react with it---when it does = forms things like sorbitol This can create osmotic effects within the cells More importantly, when it carries out this reaction, it consumes NADPH NADPH is very important in terms of minimizing oxidative stress, keeping glutathione in its reduced state, minimizing reactive oxygen species from being formed When consuming NADPH in polyol pathway, NADPH is not around to minimize oxidative stress When that occurs & cells start undergoing oxidative stress = reactive oxygen species formed, glutathione not in reduced state = damage taking place within cells which creates problems That is what you can see in uncontrolled diabetes, when large amounts of glucose are around---can be acted upon by aldose reductase and polyol pathway Slide 43 Glycolysis

Fructose 1,6-bisphosphatase: Negative allosteric modulators

Fructose 2,6-bisphosphate AMP

Phosphofructokinase: Positive allosteric regulators

Fructose 2,6-bisphosphate AMP

Release of Insulin by Pancreatic Beta Cells

GLUT 2 plays a role in this process When glucose levels increase (after a meal)---GLUT 2 will become active & will bring glucose into the pancreatic Beta cell There a glucokinase will phosphorylate it, this will start the process of glucose being metabolized to give increased concentrations of ATP This increased concentrations of ATP act on a potassium ATP-dependent channel---inhibiting it By inhibiting the potassium channel, that gives rise to depolarization within the beta cell, which in turn activates the calcium channel (voltage gated) Calcium will move into the beta cell, increasing level of intracellular calcium, facilitates exocytosis of vesicles containing stored insulin = released out into systemic circulation Hormones GIP & GLP1 stimulate release/secretion of insulin from pancreatic beta cells, because they act on GPCR receptors on the beta cell & activation of those receptors gives rise to an increase in cAMP = which also facilitates the exocytosis of insulin containing vesicles Several anti-diabetic drugs: Sulfonylureas, Meglitinides, GLP-1 mimics and DPP4 inhibitors---that metabolize these hormones, used to treat some conditions of diabetes---these agents are helping to stimulate the release of insulin Individuals who are type II diabetics = problem with secretion of insulin Beta cells make enough insulin, but processes in which insulin is released is not operating properly OR Signal transduction mechanisms (insulin acting on its receptors in muscle/adipose cells) is not functioning properly Diabetes is a wide syndrome of different populations of the disorder depending upon the underlying mechanism of which the effects of insulin are being effected Slide 12 Glycolysis

What is the tissue location and general role that is played by each of the glucose transporters?

GLUT1 = located in all mammalian tissues Role: basal glucose uptake Will not be operational when blood glucose concentration is very low---leads to dizziness & confusion because of lack of sugar being brought to brain tissue GLUT2 = located in liver and pancreatic B cells Role in Liver: removes excess glucose from blood Role in Pancreas: helps to regulate insulin---only activated when glucose levels are very, very high. Liver is main organ for controlling plasma glucose levels---when low, will release it---when high, removes excess glucose from blood with GLUT2 GLUT3 = located in all mammalian tissues Role: Basal glucose uptake Will not be operational when blood glucose concentrations very low----leads to dizziness & confusion because of lack of sugar being brought to brain tissue GLUT4 = Located in muscle and fat cells Role: Transports glucose into muscle and fat cells. Dependent on insulin---stimulates the production of insulin = moves glucose into muscle and adipose cells

Compare the GLUT1 and GLUT4 glucose transporters in terms of their tissue distribution and their function

GLUT1 is found in all mammalian tissues & is involved in basal glucose uptake---function is to get glucose into neurons GLUT4 is primarily located in muscle and fat cells & its uptake of glucose is stimulated by insulin----function is to get glucose into muscle and fat cells

Galactose-Glucose Interconversion Pathway

Galactose enters into glycolytic pathway through the galactose-glucose interconversion pathway Galactose is acted upon by galactokinase which phosphorylates it to give galactose 1-phosphate Then enzyme called galactose 1-phosphate uridyl transferase which utilizes UDP-glucose (another carrier molecule)---carrier of glucose, able to transfer glucose to other molecules Here an exchange happens where UDP-glucose and galactose 1-phosphate are switching places, where glucose molecule is cleaved off to form glucose 1-phosphate & then UDP-galactose is then formed Then an epimerase converts UDP-galactose back to UDP-glucose Which can be used again Both UDP-galactose and UDP-glucose are important, because many of the proteins, carbohydrates in cells have galactose or glucose residues put on the surface---they are important for the functioning of the proteins, carbs GPCRs are galactosylated (galactose is put on them) or glucose is put on them---and those are important for the function, particularly in the brain when UDP-galactose is not available to galactosylate those proteins---nerve activity is greatly hampered = can lead to mental illness & problems with development Slide 41 Glycolysis

How does glucokinase differ from the other hexokinases and what specific role does it play?

Glucokinase acts as an allosteric enzyme, even though it is a monomer. It phosphorylates glucose only when glucose is abundant---affinity for glucose is about 50 fold lower than hexokinase. Low affinity for glucose gives brain & muscles fist call on glucose when it is limited & ensures glucose will not be wasted when it is abundant. Coincides with GLUT2---only activated when high concentrations of glucose. High Km for glucose. Not inhibited by glucose 6-phosphate. Located in pancreas beta cells (regulates secretion of insulin) and liver (regulates usage of glucose). Phosphorylates glucose that comes into pancreas or liver, to trap it in the tissue.

Describe general mechanism by which insulin is released from the beta cells of the pancreas

Glucokinase in the beta cells of the pancreas increases the formation of glucose 6-phosphate when blood-glucose levels are elevated, which leads to secretion of insulin. Insulin signals the need to remove glucose from the blood for storage as glycogen or conversion into fat.

Energy Yield in the Conversion of Glucose into Pyruvate

Glucose + 2 Pi + 2 ADP + 2 NAD+ ----- 2 Pyruvate + 2 ATP + 2 NADH + 2 H+ + 2 H2O In glycolytic pathway, if look at net processes that have taken place = see that glucose + 2 inorganic phosphates + 2 ADP molecules + 2 NAD+ molecules are taken to 2 Pyruvate + 2 ATP + 2 NADH + 2 protons + 2 H2O At this point, balance within ourselves is not maintained Reason for that is that we have consumed 2 molecules of NAD+ to form 2 NADHs NAD+ and NADH are in very small quantities So if do not have mechanism by which we can convert NADH back to NAD+, the process will not be sustainable---will shut down Slide 35 Glycolysis

What are the three major pathways of glucose metabolism?

Glycolysis Gluconeogenesis Pentose phosphate pathway

What are the general characteristics of glycolysis and what is the general role of the 2 stages?

Glycolysis is a process in which glucose is metabolized to 2 molecules of pyruvate (6 unit molecule into two 3 unit molecules). Outcome that is important for the cell is ATP production. Glycolysis is an anaerobic process, nearly universal, and has a highly controlled pathway. In eukaryotes, glycolysis takes place in cytoplasm. It is the conversion of glucose into energy. Stage 1 is trapping/stabilizing and preparation phase, getting glucose ready to be phosphorylated and broken down---no ATP is made in this stage. During stage 1, glucose is converted into fructose 1,6-bisphosphate in 3 steps: phosphorylation, isomerization and another phosphorylation. Strategy of these initial steps is to trap glucose in the cell & form a compound that can be readily cleaved into phosphorylated 3 carbon units. Stage 1 is completed with the cleavage of the fructose 1,6-bisphosphate into two 3 carbon fragments, which are readily interconvertible. Stage 2: ATP is harvested when the 3 carbon fragments are oxidized to pyruvate

Fragmentation of Fructose 1,6-bisphosphate

Happens by aldolase Enzyme forms two 3 carbon units: Glyceraldehyde 3-phosphate & Dihydroxyacetone phosphate Glyceraldehyde 3-phosphate is on the glycolytic pathway to further metabolism Dihydroxyacetone phosphate is not To conserve, the cell has developed a way to convert dihydroxyacetone phosphate to glyceraldehyde 3-phosphate That enzyme is triose phosphate isomerase = very efficient enzyme, operates under diffusion control limit = as soon as it sees a substrate, it reacts This is quite important because of the transformation that takes place = if this enzyme was not kinetically perfect = would have very severe problems in cells Reason for that = look at mechanism by which enzyme reacts (do not need to memorize mechanism, just to illustrate why it's important) Slide 25 Glycolysis

Glycolysis Stage 2, more

Have molecule that has a high potential to transfer its phosphate group---transfers it to ADP to form ATP Phosphoglycerate kinase is the enzyme that catalyzes that reaction At this stage, with the 2 molecules of glyceraldehyde 3-phosphate coming in = have net zero in terms of ATP formation/use Stage 1 = have used 2 ATPs Stage 2 = have formed 2 ATPs (so far) Overall reaction, so far = net zero ATP 3-phosphoglycerate that results from the transfer of phosphate, is isomerized by mutase to give 2-phosphoglycerate Then enolase performs an isomerization to form phosphoenolpyruvate This is now another high phosphoryl potential molecule that can transfer its phosphate to ADP to from ATP Enzyme that carries that out is pyruvate kinase That is the last step in glycolytic pathway = 3rd regulatory step---irreversible step Slide 31 Glycolysis

3 regulatory steps in glycolytic pathway:

Hexokinase Phosphofructokinase Pyruvate Kinase Need to understand how those steps are regulated

Irreversible reaction sites in glycolysis

Hexokinase Phosphofructokinase Pyruvate kinase

Glycolytic Pathway Control

Highly regulated process Regulated to meet 2 major cellular needs: 1) Production of ATP 2) Production of building blocks---components used for amino acid or fat production Potential control sites---irreversible reactions (only run in one direction): 1) Hexokinase 2) Phosphofructokinase 3) Pyruvate kinase One of the ways to control this process is to control the amount of enzyme present--involves transcription and some of hormones mentioned have an effect on the system Insulin---increases amount of phosphofructokinase and pyruvate kinase present---turns on transcription and elevates their levels Other way to regulate = negative feedback inhibition or allosteric regulation Also regulation by phosphorylation---reversible modification of the enzyme

Covalent Enzyme-Bound Intermediate as a Mechanism of Energy Coupling

If didn't have coupling going on, would have this type of energy profile (on left of slide 30 Glycolysis)---forming an acid at the bottom Notice have large energy of activation in order to undergo acyl-phosphate formation By capturing the energy in the thioester intermediate, have a much lower energy of activation = makes whole reaction very facile Remember general concept, not specific mechanisms Slide 30 Glycolysis

Gluconeogenesis and Reversal of Glycolysis Comparison

If reversed glycolysis, would be very thermodynamically unfavorable---d/t those 3 irreversible steps What is done in gluconeogenesis = bypass those three steps (or have other steps instead of those steps) Stoichiometry of gluconeogenesis: It overall is a thermodynamically stable process Notice are consuming energy in form of ATP, GTP and reducing equivalents This is an anabolic process---converting smaller molecules into a larger molecule Pyruvate into glucose Slide 46 Glycolysis

Cooperation Between Glycolysis and Gluconeogenesis During a Sprint

In the liver---do not want glycolysis and gluconeogenesis taking place at the same time = not productive for liver cell However, is productive if one organ is doing glycolysis (muscle) and another is doing gluconeogenesis (liver) There are scenarios where that takes place, and is beneficial Provides mechanism where one organ helps supply the energy needs of another organ Example: running Muscle cells will be very active in terms of utilizing glucose metabolism needed to maintain ATP needed to continue muscle contraction When get down to pyruvate, can go into aerobic glycolysis or when oxygen is limited = goes into anaerobic glycolysis & form lactate Lactate can be released out into systemic circulation---that can go out to cardiac muscle & be used by cardiac muscle where will convert back to pyruvate & go through oxidative phosphorylation pathway to provide energy for heart to work Other place lactate will go: liver---liver will take pyruvate & it will go through gluconeogenic pathway to provide glucose which can then be excreted out into systemic circulation & then picked up by muscle cells to be utilized to sustain muscle contractions & keep running This is mechanism by which utilize ability of one organ (liver) to provide metabolic needs of another organ (muscle) to sustain activity Will see similar scenario if looking at starvation type conditions, using gluconeogenic pathway to help maintain activity of other tissues/functions This process is called the Cori Cycle Slide 55 Glycolysis

Liver tissue: Glucagon actions

Increase gluconeogenesis Increase glycogenolysis

Adipose tissue: Insulin actions

Increase glucose uptake Increase lipogenesis Decrease lipolysis

Adipose tissue: Glucagon actions

Increase lipolysis

Entry of Fructose and Galactose into Glycolysis

Ingest a lot of other sugars, has to be a mechanism in which those sugars can be used as fuel Process of utilizing those type of sugars is to bring them into the glycolytic pathway in terms of one or more of the intermediates of the glycolytic pathway Galactose is brought in through glucose 6-phosphate Fructose, depending on which tissue looking at, is brought into either fructose 6-phosphate by hexokinase---in adipose tissue OR More significantly, in the liver, where utilizes fructose 1-phosphate pathway which means it's brought into dihydroxyacetone phosphate and glyceraldehyde 3-phosphate intermediates Important in terms of fructose entering the liver at this particular point Slide 38 Glycolysis

Pyruvate Carboxylase

Initial reaction starting the process is pyruvate carboxylase Reaction that takes place in mitochondria When looked at how can control reactions = one way is compartmentalization Seeing compartmentalization process, where initial stage of gluconeogenesis is taking place within the mitochondria This is where we're going to be adding a CO2 molecule to the methyl group of pyruvate Doing that in 3 stages This enzyme carries out 3 separate reactions in trying to carry out this transformation 1st stage: activation of bicarbonate (source of CO2) with ATP to form mixed anhydride, activating the carboxyl group 2nd stage: Carboxyl group reacts with biotin cofactor, which will pick up the activated carboxyl group (CO2) to form a complex 3rd stage: Activated species will transfer CO2 to pyruvate to form oxalacetate Slide 48 Glycolysis

Regulation of GLUT 4 by Insulin

Insulin stimulates GLUT 4 In this process, insulin acts at its receptor---activating the receptor = causing GLUT 4 transporters (contained in specialized vesicles in adipose/muscle cells) to move to surface of those cells & become expressed on the surface When expressed, can transport glucose within muscle/adipose cells With time, those receptors can be endocytosed back into the cell & go through the whole process again In some diabetics, insulin is being released, but it is not effectively doing what it normally does in terms of activating the GLUT 4 receptor If it doesn't activate the GLUT 4 receptor---then that will diminish the amount of glucose that can get into muscle/adipose tissue This leads to hyperglycemia Type I diabetics = total absence of insulin being produced by beta cells = not sufficient insulin around to activate these transporters to move them to the surface of the muscle/adipose tissue Slide 13 Glycolysis

What transformation does triose phosphate isomerase catalyze and why is it important that it is nearly a perfect enzyme?

It catalyzes the transformation of dihydroxyacetone to glyceraldehyde 3-phosphate. Reason this one enzyme is so important is if the enediol intermediate (circled on top left of slide) were to escape into the environment---the bottom process---forming the written intermediate on bottom of slide, would be 100x more likely to occur than forming glyceraldehyde 3-phosphate Problem if the other product is formed = this is a reactive compound---would react with proteins, DNA, etc & in the process would mess up the whole biochemical machinery If were to take methylglyoxal, you would have severe toxicities because of its ability to react with different substances This does not occur, under normal circumstances, because this enzyme is so kinetically perfect = why it's so important See slide 26 Glycolysis

GLUT 2

Liver and pancreatic beta cells Pancreas: plays role in regulation of insulin---only activated when glucose levels are very, very high Liver: removes excess glucose from blood---only activated when glucose levels are very, very high Km = 15 to 20 mM

GLUT 4

Located: Muscle and fat cells Amount in muscle plasma membrane increases with endurance training Km = 5 mM Dependent on insulin---stimulates the production of insulin, moves glucose into muscle and adipose cells

GLUT 3

Located: all tissues Basal glucose uptake Km = 1 mM Neurons

Actions of Insulin and Glucagon in Metabolism

Multiple hormones play a key role in glucose metabolism process 4 are highlighted 1) Insulin 2) Glucagon Insulin is produced by beta cells of the pancreas---primarily acts on 3 tissues: 1) Adipose: to increase glucose uptake & increase lipogenesis (generation of fatty acids) & decrease lipolysis (breakdown of fatty acids) 2) Striatal muscle: stimulates glucose uptake, increases glycogen synthesis, increases protein synthesis 3) Liver: Decreases gluconeogenesis (biosynthesis of glucose), increases glycogen synthesis, increases lipogenesis (increase of production of fat) Insulin is involved in promoting fuel storage and growth Glucagon can be viewed as opposing force to insulin. It primarily acts on 2 tissues: 1) Adipose tissue--Increases lipolysis (breakdown of fats) 2) Liver---increases gluconeogenesis, increases glycogenolysis (breakdown of glycogen) Illustrates another way metabolism can be regulated in our bodies Different than the 3 ways metabolism is regulated at the cellular level---more macro-level in terms of tissue specificity & how different organs have different functions---illustrated by effects of glucagon and insulin Another way we can regulate metabolism that certain organs/tissues will be involved doing certain things, and other organs/tissues will be involved in doing other things in terms of metabolism = specialization Slide 4 Glycolysis

Oxaloacetate Mitochondrial Cytosol Shuttle

Once oxaloacetate has formed = in mitochondria, a lot of reactions that subsequently follow: take place in cytosol Somehow, oxaloacetate has to get back out into cytosol---does that through conversion of it to malate Oxaloacetate does not move across mitochondrial membranes (cannot do that) Malate can be transported out What see happening = Malate dehydrogenase in the mitochondria will convert oxaloacetate to malate = malate is pumped out & is then converted back by malate dehydrogenase in the cytosol to oxaloacetate Now have oxaloacetate in cytosol where can undergo next step in gluconeogenic pathway This is the reaction that this malate dehydrogenase carries out = reducing oxaloacetate to malate and then oxidizing it back from malate to oxaloacetate Slide 50 Glycolysis

Triose Phosphate Isomerase Catalytic Mechanism

One of the things that is formed is enediol intermediate This intermediate, when acted upon by isomerase, flows electrons as shown on slide 26 Glycolysis, picking up proton & generate glyceraldehyde 3-phosphate Another way these electrons at this point could flow That would be instead of flowing in the direction shown, could move in other direction and kick out phosphate When it does that it would generate this type of intermediate (drawn on slide), which can isomerize to give this type of compound (drawn on slide), essentially methylglyoxal Reason this one enzyme is so important is if the enediol intermediate (circled on top left of slide) were to escape into the environment---the bottom process---forming the written intermediate on bottom of slide, would be 100x more likely to occur than forming glyceraldehyde 3-phosphate Problem if the other product is formed = this is a reactive compound---would react with proteins, DNA, etc & in the process would mess up the whole biochemical machinery If were to take methylglyoxal, you would have severe toxicities because of its ability to react with different substances This does not occur, under normal circumstances, because this enzyme is so kinetically perfect = why it's so important Slide 26 Glycolysis

Hexokinase

Open form on the left---glucose will bind in the open cleft area When it does bind the enzyme closes around the molecule & allows the ATP to be used to phosphorylate the sugar molecule When do that, have trapped glucose within the cell Now glucose 6-phosphate can't get out of the cell This is the trapping of glucose in the cell by this process Another feature of most of hexokinases = they are inhibited by the product (glucose 6-phosphate) = negative feedback inhibition Too much glucose 6-phosphate being produced---not processed after it is produced, will turn off hexokinase activity in an allosteric manner Slide 15 Glycolysis

Pyruvate Kinase

Phosphoenolpyruvate transfers its phosphate to ADP to form ATP Is a very favorable reaction, because have enol-ketone conversion taking place Pyruvate is initially formed as an enol, but will isomerize to the ketone form = favorable reaction that drives the whole process Slide 32 Glycolysis

Pyruvate kinase is controlled by reversible phosphorylation. Describe the effect phosphorylation has on the activity of pyruvate kinase and name the hormone that triggers this phosphorylation. In what tissue does reversible phosphorylation of pyruvate kinase occur?

Phosphorylation of pyruvate kinase results in less active form of enzyme Hormone that triggers this is glucagon Reversible phosphorylation of pyruvate kinase takes place in liver

Glycolysis

Process in which glucose is metabolized to 2 molecules of pyruvate (one 6 carbon molecule---glucose, converted to two 3 carbon molecules---pyruvate) Nearly universal pathway---all our cells in our body have capability of producing this pathway The outcome that is important for the cell is it produces ATP Particularly in the brain, where glucose (under normal conditions) is the only food source Under starvation conditions---can revert to other ketone bodies for the brain, but basically brain relies on glycolysis to produce ATP RBCs are totally dependent on this process for ATP Anaerobic process---doesn't require oxygen Highly controlled pathway Slide 7 Glycolysis

Control of Pyruvate Kinase

Pyruvate is the end of the whole process ---serves as building block for other molecules and as an entryway into citric acid cycle It is a control point, several things that control the process 1) Isozymes of pyruvate kinase: have L form = in liver, and M form = in muscle/brain Difference between these = form in liver is controlled by phosphorylation of enzyme Diagram shown is what would happen in liver Dephosphorylated pyruvate kinase is a very active from, but it will be phosphorylated to give much less active form This occurs in the liver, the hormone that controls this is glucagon Glucagon acts on its receptor, it activates protein kinase A & protein kinase carries out phosphorylation This occurs when we have low blood levels of glucose Glucagon is then released, that will act on liver to induce gluconeogenesis---formation of glucose If going to form glucose, do not want to metabolize glucose One way in which glucagon counteracts metabolism of glucose, by shutting down or diminishing ability of pyruvate kinase to carry out its effect Reason why liver has this ability/regulation and not muscle/brain = liver is metabolic hub/controller of blood glucose levels When have low blood glucose levels = overall need for body is to ensure brain in particular has adequate concentrations of glucose, because glucose is brain's primary energy source Liver would shut down its ability to metabolize glucose to conserve whatever glucose there is, to be sure that the brain and muscle have enough glucose to operate Also regulated by allosteric regulation---both M and L form 2) Allosteric regulators: a) Fructose 1,6-bisphosphate = positive allosteric regulator---activates the enzyme = product of the phosphofructokinase reaction, one of other regulatory steps in pathway When that pathway is active = forming fructose 1,6-bisphosphate---this is going to serve as signal to pyruvate kinase that glycolytic pathway is being activated b) ATP, Alanine = negative allosteric regulators ATP is a reflection of energy charge within cell Making ATP in glycolytic pathway If plenty of ATP in the cell, do not need glycolysis to be taking place---glucose could be used to conserve, build glycogen for use at another time Makes sense that it would shut off the process Alanine also does this = alanine is a building block & pyruvate can be transformed through transamination to alanine When lots of alanine around, indication that building blocks are plentiful & do not need to form more pyruvate for the building block process = negative regulator 3 regulatory steps in glycolytic pathway: Hexokinase Phosphofructokinase Pyruvate Kinase Need to understand how those steps are regulated (1-2 questions on exam)

Coupled Reactions

Reaction from previous slide takes place in 2 stages: 1) Oxidation where glyceraldehyde 3-phosphate (aldehyde portion) is oxidized to carboxylic acid Oxidation is a process that will release energy Free energy change is -12 kcal/mol = thermodynamically favorable process, as expected Need to capture that energy 2) Reaction of acid with inorganic phosphate to form acyl phosphate This is thermodynamically unfavorable reaction, need to couple both reactions Can use ATP to make an unfavorable reaction, favorable---here we're not using ATP---have to use some type of other mechanism to make coupling take place Slide 28 Glycolysis

Major Pathways of Glucose Metabolism

Several major pathways in terms of carbohydrate metabolism---revolves around glucose Common intermediate is Glucose 6-phosphate From there, can go down glycolytic pathway---gives rise to pyruvate Or can go to pentose phosphate pathway---gives rise to pentose (5 carbon instead of 6 carbon) sugars---also provides reducing equivalents---very important for reducing oxidative stress that occurs in cells Or can be converted to glucose 1-phosphate, then can synthesize glycogen (storage form of glucose) Glucose is universal fuel for cells---provides good source of ATP that cells need to survive Slide 3 Glycolysis

Glucokinase (Hexokinase IV)

Specialized hexokinase = has characteristics that are different than the hexokinase just talked about (previous slide) High Km for glucose Not inhibited by glucose 6-phosphate Pancreatic Beta cells---regulates secretion of insulin Liver---regulates usage of glucose Coincides with GLUT 2 = found in beta cells & liver GLUT 2 only activated when high concentrations of glucose Glucokinase only becomes activated when high levels of glucose GLUT 2 and Glucokinase operate in tandem Role of glucokinase is to phosphorylate the glucose that is coming into the pancreas or the liver cells to trap it in that tissue

Describe mechanism by which sulfonylureas & GLP-1 mimics promote the release of insulin from pancreatic Beta cells

Sulfonylureas promote the release of insulin from pancreatic beta-cells by blocking the ATP-dependent potassium channel. This leads to membrane depolarization, which in turn activates a voltage gated calcium channel. The increase in calcium levels stimulates the exocytosis of insulin from vesicles containing the hormone. GLP-1 mimics promote the release of insulin from pancreatic beta cells by stimulating its receptor on the pancreatic beta cell. This brings about an increase in cAMP levels, which ultimately stimulates the exocytosis of insulin from vesicles containing the hormone.

Catalytic Mechanism of Glyceraldehyde 3-Phosphate Dehydrogenase

The coupling occurs by an enzyme intermediate that has a high energy type, that retains the energy until it needs to be transferred to second phase Do not need to memorize mechanism Have aldehyde that is reacted upon by enzyme with a sulfhydryl group in its active site, that reacts with the aldehyde group of glyceraldehyde 3-phosphate Form a hemithioacetal molecule, which is oxidized by NAD+ in active site Now form thioester intermediate Thioesters have high energy potential, because have ability to transfer acyl group to other molecules with the release of free energy What we've done is captured the free energy & oxidation in thioester intermediate NADH leaves, and another NAD+ comes in & now the thioester intermediate can react with inorganic phosphate to form the 1,3-bis-glyceraldehyde molecule Captured the energy released on oxidation in the thioester Slide 29 Glycolysis

Glucokinase Allosteric Activators as Potential Antidiabetic Agents

This enzyme is also a target for developing anti-diabetic drugs Several are in clinical trials---none have reached the market yet Example on slide 17 Glycolysis What looking at: molecules that will serve as allosteric activators of glucokinase Potential drugs are doing is binding to glucokinase in pancreatic beta or liver cells to an allosteric site = activating the enzyme = increase the affinity of glucose (normally the affinity is low, but if increase the affinity---will be active at much lower concentrations of glucose), and increase the Vmax = increases the rate at which the reaction takes place Reason potentially useful at treating diabetes = if activate glucokinase within the pancreatic beta cells by allosteric activators (GKA = glucokinase allosteric activator)---activate the glucokinase = facilitate the production of glucose 6-phosphate, which facilitates the production of ATP = facilitates insulin release Hepatic cells = where glucokinase is tied to uptake of glucose = glucose going through GLUT 2 transporter, gets into liver & if activate glucokinase = will more readily convert glucose to glucose 6-phosphate, trap it there = lower the amount of glucose found in plasma This type of agent would treat the 2 hallmarks of diabetes: hyperglycemia & insulin resistance---not enough insulin being produced/released to create an effect Slide 17 Glycolysis

Glucose 6-phosphatase

This is done in the ER Glucose 6-phosphate in the cytosol has to be transported across ER membrane Specific transporter (T1) that takes it across Then the glucose 6-phosphatase acts on it to release phosphate and glucose Those in turn have to be pumped out of the ER, glucose can then be pumped out of cell of the liver So can get into systemic circulation Do not need to know specific components, just that this unique last step to gluconeogenesis takes place in ER Important step, because if remained as glucose 6-phosphate would be trapped in the cell Liver's goal in terms of utilizing gluconeogenesis is not only to proved glucose for itself, but to provide glucose to the brain/muscles, other tissues in body Has to be a mechanism to that glucose being released, and this is the key to that being released & the subsequent transport of glucose out of liver cells Slide 52 Glycolysis

Hepatic Utilization of Fructose

Utilization fructose is important because we as consumers are ingesting large amounts of products that contain high fructose corn syrup---in those products more than 50% of the carbohydrates is fructose So this has ramifications in terms of metabolism in cells, because of where fructose enters glycolytic pathway On right of slide 39 Glucose, have glycolytic pathway Fructose on left, going through fructose 1-phosphate pathway---forming glyceraldehyde & dihydroxyacetone phosphate in the liver by the fructose The importance of this, is bringing in fructose into glycolytic pathway at a point beyond the key regulatory step = phosphofructokinase Bypassing the regulatory step What that means is then these products (glyceraldehyde & dihydroxyacetone phosphate) can be continually processed Even if phosphofructokinase may be shut off---still forming products in glycolytic pathway, down the pathway from where phosphofructokinase exists Ramifications: See acetyl CoA being formed in large quantities---leads to fatty acids Glyceraldehyde 3-phosphate being formed = form acyl glycerols = fats Significant effect on liver, because of this feature of bypassing regulatory steps & causing increases in fat Slide 39 Glycolysis

Glucose 6-phosphate Metabolic Fates

When form glucose 6-phosphates---can go on into several different directions Middle = glycolysis OR Can also go into 6-phosphogluconate = pentose phosphate pathway to give pentose phosphates OR Can go (in liver) to form glucose 1-phosphate which then goes into glycogen storage OR Can be formed into UDP-glucuronic acid to form glucuronides in liver metabolism of various drugs Formation of glucose 6-phosphate does not commit glucose to glycolytic pathway, is an intermediate that can go in several directions Slide 18 Glycolysis

Allosteric Regulation of Phosphofructokinase by ATP

When have low ATP present---hyperbolic curve High ATP = sigmoidal curve Certain level of reaction velocity = see that when have low ATP---can get reaction velocity with low levels of fructose 6-phosphate When high ATP around----need much higher levels of fructose 6-phosphate Have lowered the affinity of fructose 6-phosphate for the enzyme = won't be as active in terms of phosphorylation Slide 22 Glycolysis

Control of Synthesis and Degradation of Fructose 2,6-bisphosphate

When have the kinase active and phosphate inactive = fructose 6-phosphate will be converted to fructose 2,6-bisphosphate Stimulates glycolysis at phosphofructokinase step Inhibits gluconeogenesis at fructose 1,6-bisphosphatase step Factors that promote kinase activity to give rise to fructose 2,6-bisphosphate compound: 1) Large amount of fructose 6-phosphate around = has stimulatory effect on phosphatase = that converts the phosphorylated (fructose 1,6 bisphosphate) form to unphosphorylated (phosphofructokinase) form When in unphosphorylated form = kinase is active Phosphorylated form = phosphatase is active When fructose 1,6 bisphosphatase is active there, will dephosphorylate/convert fructose 2,6-bisphosphate back to fructose 6-phosphate, diminishing the amounts of the allosteric modulator (fructose 2,6-bisphosphate) The phosphorylation process of the enzyme is done by protein kinase A = controlled by glucagon Glucagon, when is around, activating that receptor = increases cAMP formation, which activates protein kinase A & that activates the fructose 1,6-bisphosphatase activity of system & will metabolize fructose 2,6-bisphosphate back to fructose 6-phosphate That augments gluconeogenesis and inhibits glycolysis Glucagon is promoting gluconeogenesis = this is one of ways does that When fructose 6-phosphate is plentiful, will see the Phosphoprotein phosphatase activated = converts phosphorylated enzyme species (fructose 1,6-bisphosphatase) back to dephosphorylated form (phosphofructokinase) At that point, kinase activity is now active = converts fructose 6-phosphate to fructose 2,6-bisphosphate (allosteric modulator) Other hormone that has effect = insulin Insulin also stimulates that Phosphoprotein phosphatase to cause formation of fructose 2,6-bisphosphate Insulin promotes glycolysis & storage of glucose Glucagon = promotes biosynthesis of glucose Hormones also have effect on various enzymes Insulin promotes phosphofructokinase transcription and pyruvate kinase transcription Glucagon promotes transcription of phosphatase and pyruvate carboxylase enzymes Need to be familiar with this---question on the exam Slide 54 Glycolysis

Predict the effect (increases or decreases) of each of the following mutations on the pace of glycolysis in liver cells and provide a brief explanation for the effect you predict: a) Loss of allosteric site for ATP in phosphofructokinase b) Loss of binding site for citrate in phosphofructokinase c) Loss of phosphate domain of the bifunctional enzyme that controls the level of fructose 2,6-bisphosphate d) Loss of binding site for fructose 1,6-bisphosphate in pyruvate kinase

a) Glycolysis increases because ATP can no longer inhibit phosphofructokinase b) Glycolysis increases because citrate can no longer inhibit phosphofructokinase c) Glycolysis will increase because in the absence of fructose 2,6-bisphosphatase, the level of fructose 2,6-bisphosphate will increase, resulting in activation of phosphofructokinase d) Glycolysis will decrease because fructose 1,6-bisphosphate can no longer activate pyruvate kinase

The concentration of lactate in blood plasma before, during, and after 400 meter sprint are shown in graph (Problem 1---Group In-class Exercise 1) a) What causes the rapid rise in lactate concentration? b) What causes the decline in lactate concentration after completion of the sprint? Why does the decline occur more slowly than the increase? c) Why is the concentration of lactate not zero during resting state?

a) Rapid depletion of ATP during strenuous exercise causes the rate of glycolysis to increase dramatically, producing higher cytosolic concentrations of pyruvate and NADH Lactate dehydrogenase converts these to lactate and NAD+ (lactic acid fermentation) especially when O2 demands outpace availability to muscle cells b) When energy demands are reduced, the oxidative capacity of the mitochondria is again adequate, and lactate is transformed to pyruvate by lactate dehydrogenase, and the pyruvate is converted to glucose. The rate of the dehydrogenase reaction is slower in this direction because of the limited availability of NAD+ and because the equilibrium of the reaction is strongly in favor of lactate (conversion of lactate to pyruvate is energy requiring) c) The equilibrium of the lactate dehydrogenase reaction is strongly in favor of lactate. Thus, even at very low concentrations of NADH and pyruvate, there is a significant concentration of lactate.