Biochemistry

What are the 9 reactions of the TCA cycle?

1. Oxidative decarboxylation of pyruvate

2. Synthesis of citrate from acetyl CoA and oxaloacetate

3. Isomerization of citrate

4. Oxidation and decarboxylation of isocitrate

5. Oxidative decarboxylation of α-ketoglutarate

6. Cleavage of succinyl CoA

7. Oxidation of succinate

8. Hydration of fumarate

9. Oxidation of malate

How is pyruvte transported to mitochondria before entering TCA?

Special pyruvate transporter cross the mitochondrial membrane

Once in the matrix, the pyruvate is converted to _____ by _______

Acetyl CoA by the pyruvate dehydrogenase complex

What are the 3 stages of catabolism?

1. Hydrolysis of complex molecules: Complex molecules are broken down to simpler molecules (e.g. proteins to amino acids)

2. Conversion of building blocks to simple intermediates: These building blocks are further degraded to Acetyl CoA and other simple molecules. A small amount of ATP captured

3. Oxidation of acetyl CoA: Oxidation of acetyl CoA generates large amounts of ATP via oxidative phosphorylation as electrons flow from NADH and FADH2 to oxygen

Differentiate between anabolic and catabolic reactions.

Anabolic (synthetic):

• form complex products from simple molecules

• divergent (Few products become many)

Catabolic (degradative):

• break down complex products into simpler products

• convergent (Many molecules become small end products)

What are second messenger systems?

They intervene between the original messenger (the neurotransmitter or hormone) and the ultimate effect on the cell—are part of the cascade of events that translates hormone or neurotransmitter binding into a cellular response

Example: calcium/phosphatidylinositol and the adenylyl cyclase system

They also greatly amplify the strength of the signal

What are the 3 major classes of second messenger signals?

1. cyclic nucleotides (cAMP and cGMP)

2. inositol trisphosphate (IP₃) and diacylglycerol (DAG)

3. calcium ions

What is the overall equation of glycolysis?

C6H12O6 + 2ADP + 2Pi + 2NAD+   →   2C3H4O3 + 2H2O + 2ATP + 2NADH + 2H+

or

Glucose + Adenosine diphosphate + Phosphate  +  Nicotinamide adenine dinucleotide

↓

Pyruvate + Water + Adenosine triphosphate + Nicotinamide adenine dinucleotide + Hydrogen ions

Where are the enzymes of most glycolytic reactions?

In the extra-mitochondrial fraction of the cell in the cytosol.

Name the 10 steps of glycolysis

1. Phosphorylation of glucose

2. Isomerization of Glucose-6-Phosphate

3. Phosphorylation of fructose-6-phosphate

4. cleavage of fructose-1, 6-diphosphate

5. Isomerization of dihydroxy acetone phosphate

6. Oxidative phosphorylation of glyceraldehyde 3-phosphate

7. Transfer of phosphate from 1, 3 diphosphoglycerate to ADP

8. Isomerization of -phosphoglycerate

9. Dehydration of 2-phosphoglycerate

10. Transfer of phosphate from phosphoenolpyruvate

Glycolysis: Phosphorylation of glucose (Step 1)

• The glucose is initiated or primed for the subsequent steps by phosphorylation at the C₆ carbon

• Transfer of phosphate from ATP to glucose making Glucose-6-phosphate

• Loss of energy as heat

• enzymes hexokinase and glucokinase

Glycolysis: Isomerization of Glucose-6-Phosphate (step 2)

• Glucose-6-phosphate is reversibly isomerized to fructose-6-phosphate

• phosphoglucoisomerase the enzyme

Glycolysis: phosphorylation of fructose-6-phosphate (step 3)

• fructose-6-phosphate is converted into fructose-1,6-bisphosphate

• enzyme: phosphofructokinase (PFK1)

• Phosphate is transferred from ATP while energy is lost

Glycolysis: Cleavage of fructose 1, 6-diphosphate (step 4)

Aldolase cleaves fructose 1,6-bisphosphate to:

• dihydroxy acetone phosphate

• glyceraldehyde 3-phosphate

It is reversible and not regulated

Aldolase B, the isoform in the liver and kidney, also cleaves fructose 1-phosphate and functions in the metabolism of dietary fructose

Glycolysis: Isomerization of dihydroxyacetone phosphate (step 5)

• dihydroxyacetone phosphate can be isomerized into glyceraldehyde 3-phosphate

• enzyme: triose phosphate isomerase

• net production of two molecules of glyceraldehyde 3-phosphate

Glycolysis: Oxidative Phosphorylation of Glyceraldehyde 3-phosphate (step 6)

• One of three energy-conserving or forming steps

• glyceraldehyde 3-phosphate is converted into 1,3-bisphosphoglycerate

• enzyme: glyceraldehyde 3-phosphate dehydrogenase

• NAD⁺ is reduced to coenzyme NADH by the H^– from glyceraldehydes 3-phosphate

• two moles of glyceraldehyde 3-phosphate are formed from one mole of glucose,

• two NADH are generated in this step

Glycolysis: Transfer of phosphate from 1, 3-diphosphoglycerate to ADP (step 7)

• ATP-generating step of glycolysis

• Transfer of phosphate group from 1, 3-bisphosphoglycerate to ADP

• enzyme: phosphoglycerate kinase

• produces: ATP and 3-phosphoglycerate

• two moles of 1, 3-bisphosphoglycerate are formed from one mole of glucose

• 2 ATPs are generated

Glycolysis: Isomerization of 3-phosphoglycerate (step 8)

• 3-phosphoglycerate is converted into 2-phosphoglycerate

• caused by shift of phosphoryl group from C3 to C2

• enzyme: phosphoglycerate mutase

• reversible

Glycolysis: Dehydration of 2-phosphoglycerate (step 9)

• 2-phosphoglycerate is dehydrated

• enzyme: enolase

• reversible, 2 water moles are lost

• results in phosphoenolpyruvate

Glycolysis: Transfer of phosphate from phosphoenolpyruvate (step 10)

• second energy-generating step of glycolysis

• Phosphoenolpyruvate is converted into an enol form of pyruvate

• enzyme: pyruvate kinase

• enol pyruvate rearranges to become ketopyruvate

• the enzyme catalyzes the transfer of a phosphoryl group from phosphoenolpyruvate to ADP, thus forming ATP

Adenylyl cyclase

• Recognition of a chemical signal by membrane receptors like β- and α2-adrenergic receptors

• triggers increase/decrease in adenylyl cyclase

• membrane-bound enzyme

• converts ATP to 3',5'-adenosine monophosphate (cAMP)

What are G-proteins?

• heterotrimeric (alpha, beta, gamma subunits)

• They bind Guanosine nucleotides (GTP and GDP), form a link in the chain of communication between a receptor and adenylyl cyclase

• Inactive state: GDP bound to alpha subunit of G-protein

• Once a ligand binds, GDP replaced with GTP

• a subunit disassociates from BY subunit and moves to adenylyl cyclase, which is activated

What are G-protein coupled receptors characterized by?

• extracellular ligand binding region

• seven transmembrane helices

• intracellular domain that interacts with G-proteins

Describe Vibrio chlorae and Bordetella pertussis

• Inappropriate activation of adenylyl cyclase through covalent modification (ADP ribosylation) of different G proteins

• Cholera: GTPase activity of Gas inhibited

• Whooping cough: Gai is inactivated

What is the second link in the cAMP second messenger system?

Protein kinases:

• activation by cAMP of enzymes called cAMP-dependent protein kinases (eg protein kinase A)

• cAMP activates protein kinase A by binding to 2 regulatory subunits causing release of active catalytic subunits

• The active subunits catalyse the transfer of phosphate from ATP to serine/threonine residues of protein substrates

• The phosphorylated proteins may act directly on cells ion channels or, if enzymes, become activated/

• Protein kinase A can also phosphorylate proteins that bind to DNA, causing changes in gene expression

Third step of cAMP second messenger system

Dephosphorylation of proteins

• Phosphate groups added to proteins by protein kinases are removed by protein phosphatases

• hydrolytically cleave phosphate esters

• This ensures changes in protein activity induced by phosphorylation are not permanent

Fourth step of cAMP second messenger system

Hydrolysis of cAMP:

• cAMP is rapidly hydrolyzed to 5'-AMP

• enzyme: phospho diesterase

• 5'-AMP is not an intracellular signaling molecule, so effects of hormone/neurotransmitter mediated increases of cAMP are terminated when extracellular signal removed

What are the 2 types of transport into cells (glycolysis)?

1. a Na+-independent, facilitated diffusion transport system

2. Na+-monosaccharide cotransporter system

What are Na+-independent facilitated diffusion transporters? (glycolysis)

• GLUT-1 to GLUT-14

• Extra cellular glucose binds to the transporter, which then alters its conformation, transporting glucose across the cell membrane

• They exist in two conformational states

What are the two characteristics of Na+ independent facilitated diffusion?

1. Tissue specificity:

   • Tissue specific pattern of expression

   • GLUT-3 is primary glucose transporter in neurons

   • GLUT-1 abundant in erythrocytes/blood brain barrier, low in adult muscle

   • GLUT-4 abundant in adipose tissues/skeletal muscle (active number is increased by insulin)

2. Specialized function of GLUT isoforms:

   • follows conc gradient, from high to low

   • GLUT-1,3,4 are involved in glucose uptake from blood

   • GLUT-2, mostly in liver cells, either transports glucose into or out of cells, where glucose conc can be high or low

Glycolysis: Why don’t phosphorylated sugar molecules penetrate cell membranes? (2 reasons)

1. No specific transmembrane carriers for these compounds

2. They are too polar to diffuse through the lipid core of membranes

Glycolysis: What is the Na+-dependent monosaccharide cotransporter system?

• energy-requiring process that transports glucose against concentration gradient

• from low glucose outside to high glucose inside cell

• carrier-mediated process

• movement of glucose coupled with concentration gradient of Na+

• SGLT: Sodium dependent GLucose Transporter

• Occurs in intestinal epithelial cells, renal tubules, choroid plexus

What does hexokinase do?

What is it inhibited by?

What are it’s Km and Vmax?

What does it do in the hypothalamus?

• phosphorylation of glucose catalyzed by hexokinase

• inhibited by reaction product, glucose-6-phosphate

• It has low Km, therefore high affinity

• permits the phosphorylation and metabolism of glucose even when tissue concentrations are low

• Low Vmax, thus cannot trap cellular phosphate more than cell can use

• serves as a glucose sensor in neurons of the hypothalamus, playing a key role in the adrenergic response to hypoglycemia

Where is glucokinase?

What does it do?

What regulated glucokinase?

How is it reactivated again?

• It is in liver cells and B cells of pancreas

• β cells: glucokinase functions as the glucose sensor, determining the threshold for insulin secretion

• liver cells: facilitates glucose phosphorylation during hyperglycemia

• Glucokinase regulatory protein (GKRP) regulates activity of glucokinase through reversible binding

  • GK is translocated to the nucleus in the presence of F6P, binding tightly to regulatory protein

  • renders enzyme inactive

  • When blood glucose levels increase, GK is released from the regulatory protein and the enzyme re-enters cytosol where it phosphorylates Glucose to G6P

How does glucokinase differ from hexokinase? (4 reasons)

1. Glucokinase has higher Km, requiring higher glucose concentration for half-saturation

2. It functions only when the intracellular concentration of glucose in the hepatocyte is elevated, like a sugar-rich meal

3. Glucokinase has high Vmax, allowing the liver to effectively remove the flood of glucose delivered by the portal blood

   • This prevents large amounts of carbs entering systemic circulation after a sugary meal, minimizing hyperglycemia

4. Glucokinase activity is not directly inhibited by Glucose-6-phosphate, but fructose-6-phosphate (it is in equilibrium with fructose-6-phosphate)

Glycolysis: What is PFK-1 controlled by?

• Available concentrations of the substrates ATP and fructose 6- 6-phosphate

• Fructose 2,6 biphosphate:

  • activator of PFK-1

  • activates enzyme even with high ATP level

  • formed by PFK-2

• Energy levels within cell:

  • allosterically by elevated levels of ATP

  • Elevated levels of citrate

  • (Conversely, PFK-1 is activated allosterically by high concentrations of AMP, which signal that the cell’s energy stores are depleted)

What does PFK-2 do?

• bifunctional protein

• kinase activity that produces fructose 2,6-bisphosphate

• phosphatase activity that dephosphorylates fructose 2,6-bisphosphate back to fructose 6-phosphate

Fructose 2,6-bisphosphate is an inhibitor of _______

fructose 1,6-bisphosphatase, an enzyme of gluconeogenesis

Describe the activity of Fructose 2,6-bisphosphate in the well-fed and starved state

Well-fed state:

• Decreased levels of glucagon and elevated levels of insulin

• cause an increase in fructose 2,6-bisphosphate

• thus, in the rate of glycolysis in the liver

• Fructose 2,6-bisphosphate acts as an intracellular signal, indicating that glucose is abundant.

Starved state:

• Elevated levels of glucagon and low levels of insulin

• decrease the intracellular concentration of hepatic fructose 2,6-bisphosphate.

• This results in a decrease in the overall rate of glycolysis and an increase in gluconeogenesis.

How is 1,3-bisphosphoglycerate synthesized?

• The glyceraldehyde 3-phosphate’s aldehyde group is oxidized to a carboxyl group

• coupled to Pi of the carboxyl group

Describe the mechanism of arsenic poisoning

Method 1:

• inhibition of enzymes that require lipoic acid as a coenzyme

• like including E2 of the PDH complex, or α-ketoglutarate dehydrogenase

• Arsenite forms a stable complex with -thiol groups

• when it binds to lipoic acid in the PDH complex, pyruvate accumulates

  Method 2:

• competing with inorganic phosphate as a substrate

• enzyme: glyceraldehyde 3-phosphate dehydrogenase

• forms a complex that spontaneously hydrolyzes to form 3-phosphoglycerate

• By bypassing the synthesis of and phosphate transfer from 1,3- Biphosphoglycerate, the cell is deprived of energy usually obtained from the glycolytic pathway

• Arsenic also replaces Pi on the F1 domain of ATP synthase, resulting in the formation of ADP-arsenate that is rapidly hydrolyzed.

How is 2,3-bisphosphoglycerate synthesized?

• 1,3-BPG is converted to 2,3-BPG by the action of bisphosphoglycerate mutase

• found mostly in red blood cells

• 2,3-BPG is hydrolyzed by a phosphatase to 3-phosphoglycerate

What is feed-forward regulation? (step 10 of glycolysis, step 1)

• In liver, pyruvate kinase is activated by fructose 1,6-biphosphate

• which is the product of PFK-1 reaction

• increased phosphofructokinase activity results in elevated levels of fructose 1,6-bisphosphate, which activates pyruvate kinase

What is covalent modulation of pyruvate kinase? (step 10 of glycolysis, step 2)

• Phosphorylation by a cAMP-dependent protein kinase leads to inactivation of pyruvate kinase in the liver

• When blood glucose levels are low, elevated glucagon increases the intracellular level of cAMP

• causes the phosphorylation and inactivation of pyruvate kinase.

• patic glycolysis and stimulation of gluconeogenesis by glucagon. Dephosphorylation of pyruvate kinase by a phosphoprotein phosphatase results in re activation of the enzyme

How is lactate formed in anaerobic glycolysis in eukaryotic cells?

It is formed by lactate dehydrogenase,

Lactate formation in muscle:

• exercising skeletal muscle, NADH production

• exceeds the oxidative capacity of the respiratory chain

• results in an elevated NADH/NAD+ ratio, favoring reduction of pyruvate to lactate

• during exercise, lactate accumulates in muscle, causing a drop in the intracellular pH, potentially resulting in cramps

• lactate diffuses into the bloodstream, and can be used by the liver to make glucose

  Lactate consumption:

• direction of the lactate dehydrogenase reaction depends on the relative intracellular concentrations of pyruvate and lactate, and on the ratio of NADH/NAD+ in the cell.

• liver and heart, the ratio of NADH/NAD+ is lower than in exercising muscle. These tissues oxidize lactate (obtained from the blood) to pyruvate

• the liver, pyruvate is either converted to glucose by gluconeogenesis or oxidized in the TCA cycle.

• Heart muscle exclusively oxidizes lactate to CO2 and H2O via the citric acid cycle.

What is lactate acidiosis?

• Elevated concentrations of lactate in the plasma

• The failure to bring adequate amounts of oxygen to the tissues results in impaired oxidative phosphorylation and decreased ATP synthesis

• To survive, the cells use anaerobic glycolysis as a backup system for generating ATP, producing lactic acid as the endproduct

What is the energy yield of anaerobic and aerobic glycolysis?

Anaerobic:

• Two molecules of ATP are generated for each molecule of glucose converted to two molecules of lactate.

• There is no net production or consumption of NADH.

Aerobic:

• a net gain of two ATP per molecule of glucose.

• Two molecules of NADH are also produced per molecule of glucose

What are the 3 alternate fates of pyruvate in glycolysis?

1. Oxidative decarboxylation of pyruvate

   • Oxidative decarboxylation of pyruvate by pyruvate dehydrogenase complex

   • Pyruvate dehydrogenase irreversibly converts pyruvate, the end product of glycolysis, into acetyl CoA, a major fuel for the TCA cycle

2. Carboxylation of pyruvate to oxaloacetate

   • Carboxylation of pyruvate to oxaloacetate (OAA) by pyruvate carboxylase

   • replenishes the citric acid cycle intermediates

3. Reduction of pyruvate to ethanol (microorganisms)

   • conversion of pyruvate to ethanol

   • decarboxylation of pyruvate by pyruvate decarboxylase occurs in yeast and certain other micro - organisms, but not in humans

   • enzyme requires thiamine pyro - phosphate as a coenzyme,

Step 1 of TCA cycle: Oxidative decarboxylation of pyruvate

• pyruvate, endproduct of glycolysis, must be transported to mitochondria for TCA cycle

• a specific pyruvate transporter that helps pyruvate cross the inner mitochondrial membrane

• pyruvate is converted to acetyl CoA by the pyruvate dehydrogenase complex,

What is the pyruvate dehydrogenase complex composed of?

E₁: decarboxylase

E₂: dihydro lipoyl transacetylase

E₃: dihydro lipoyl dehydrogenase

What are the coenzymes of PDH complex?

E₁: decarboxylase

• thiamine pyro phosphate

E₂: dihydro lipoyl transacetylase

• lipoic acid

• CoA

E₃: dihydro lipoyl dehydrogenase

• FAD

• NAD+.

How is the PDH complex regulated?

• cAMP-independent PDH kinase phosphorylates/inhibits E₁

• PDH phosphatase activates/phosphorylates E₁

Step 2 of TCA cycle: Synthesis of citrate from acetyl CoA and oxaloacetate

• Acetyl CoA and oxaloacetate are condensed

• product: citrate

• enzyme: citrate synthase (not allosteric)

• citrate synthase is inhibited by citrate

• equilibrium far in the direction of citrate synthesis

• binding of oxaloacetate causes a conformational change in the enzyme that generates a binding site for acetyl CoA

Step 3 of TCA cycle: Isomerization of citrate

• Citrate becomes isocitrate

• enzyme: aconitase, Fe-S protein

Step 4 of TCA cycle: Oxidation and decarboxylation of isocitrate

• oxidative decarboxylation of isocitrate

• enzyme: Isocitrate dehydrogenase

• produces NADH, CO₂

What regulates isocitrate dehydrogenase?

Activate: Allosterically by ADP and Ca₂

Deactivate: inhibited by ATP and NADH,

Step 5 of TCA cycle: Oxidative decarboxylation of α-ketoglutarate

• α-ketoglutarate to succinyl CoA

• enzyme: α-ketoglutarate dehydrogenase complex

• oxidative decarboxylation

• equilibrium of the reaction is far in the direction of succinyl CoA

Step 6 of TCA cycle: Cleavage of succinyl CoA

• Enzyme: Succinate thiokinase

• GTP + ADP →←GDP + ATP

Name the 3 substrates for gluconeogenesis

1. Glycerol:

   • released during hydrolysis of triacylglycerols in adipose tissue

   • delivered by blood to liver

   • phosphorylated by glycerol kinase

   • glycerol phosphate,

   • oxidized by glycerol phosphate dehydrogenase

   • to dihydroxy acetone phosphate

2. Lactate:

   • released into the blood by exercising skeletal muscle

   • by cells that lack mitochondria like RBC

   • lactate is taken up by the liver and reconverted to glucose

3. Amino Acids:

   • derived from hydrolysis of tissue proteins

Name the 5 reactions unique to gluconeogenesis

1. Carboxylation of pyruvate

2. Transport of oxaloacetate to blood

3. Decarboxylation of cytosolic oxaloacetate

4. Dephosphorylation of Fructose 1,6-biphosphate

5. Dephosphorylation of Glucose-6-phosphae

Gluconeogenesis: Carboxylation of pyruvate: (step 1)

• block to overcome: Pyruvate Kinase: PEP to pyruvate

• pyruvate is first carboxylate by pyruvate carboxylase to Oxaloacetate

• converted to PEP

• enzyme: PEP- carboxykinase

What is the co-enzyme of Pyruvate Carboxylase?

What forms the complex?

• Biotin

• covalently bound to the ε-amino group

• p of a lysine residue in enzyme

• Hydrolysis of ATP drives the formation of an enzyme–biotin–CO2 intermediate.

• reaction in mitochondria of liver + kidney

How is pyruvate carboxylase activated?

• allosterically activated by Acetyl CoA

• Elevated levels of Acetyl CoA in mitochondria mean increased OAA synthesis

• Low levels of Acetyl CoA pyruvate carboxylase is inactive,

Gluconeogenesis: Transport of oxaloacetate to the cytosol (step 2)

• OAA must be converted to PEP

• PEP from mitochondria transported to cytosol

• OAA cannot cross inner mitochondrial membrane

• reduced to malate

• enzyme: malate dehydrogenase

• reoxidized to OAA by malate dehydrogenase. NAD+ reduced

Gluconeogenesis: Decarboxylation of cytosolic oxaloacetate (step 3)

• Oxaloacetate is decarboxylated

• phosphorylated to PEP in cytosol

• enzyme: PEP-carboxykinase

• hydrolysis of guanosine triphosphate

• provide an energetically favorable pathway from pyruvate to PEP

• Then, PEP is acted on by Glycolysis reverse reactions until it becomes fructose 1,6 biphosphate

Gluconeogenesis: Dephosphorylation of fructose 1,6-bisphosphate (step 4)

Regulation by energy and fructose 2, 6 biphosphate

• Hydrolysis of fructose 1,6-bisphosphate

• enzyme: fructose 1,6-bisphosphatase

• bypasses: irreversible phosphofructokinase-1

• Regulation by energy:

  • fructose 1,6 biphosphate inhibited by AMP

• Regulation by fructose 2-6 biphosphate

  • fructose 1,6 biphosphate, inhibited by fructose 2,6- bisphosphate

Gluconeogenesis: Dephosphorylation of glucose 6-phosphate (Step 5)

• Hydrolysis of Glucose 6 phosphate by glucose 6-phosphatase

• bypasses hexokinase reaction

• Liver/Kidney release free Glucose from G6P

• enzymes: glucose 6-phosphate translocase

• transports glucose 6-phosphate across the endoplasmic reticulum (ER) membrane, and the ER enzyme, glucose 6-phosphatase (found only in gluconeogenic cells), which removes the phosphate, producing free glucose