Biochemistry exam 2 glycolytic pathway/gluconeogenesis/ pyruvate dehydrogenase complex/

NAD+ regenerated in glycolysis

Activated electron carrier

• accepts Glyceraldehyde 3 phosphate electrons

• NAD reduced to NADH

Alcohol Fermentation

Pyruvate decarboxylase converts pyruvate into acetaldehyde

• Dehydrogenase converts acetaldehyde into ethanol and ox NADH to NAD+ for glycolysis

Lactic Acid fermentation

Lactate dehydrogenase converts pyruvate into lactate and ox NADH to NAD+ for glycolysis 

Fructose enters glycolytic pathway: Liver

Step 1:Fructokinase phospho. fructose by ATP hydrolysis, produce ADP and F1P

Step 2: F1P aldolase splits it into glyceraldehyde and dihydroxyacetone phosphate 

• Dihydroxyacetone phosphate enters at step 5

Step 3: Triose kinase uses atp to create ADP and Glyceraldehyde 3 phosphate

• Glyceraldehyde 3 phosphate enters at step

Fructose enters glycolytic pathway: Tissue

Phosphorylated by hexokinase by ATP hydrolysis, forming ADP and F6P

Galactose enters glycolytic pathway

At G6P

Step 1: galactokinase phospho galactose by using atp, produce galactose 1 phosphate

Step 2: Galactose 1 phosphate and UDP-glucose by (galactose 1 phosphate uridyl transferase) converted to UDP galactose and glucose 1 phosphate 

Step 3: UDP galactose 4 epimerase regenerates UDP glucose from udp galactose 

Step 4: Phosphoglucomutase convert G1P to G6P to enter step 2

Muscle cell regulates glycolysis

To meet ATP needs

glycolysis inhibited at rest and activated when moving

Hexokinase

• inhibited by G6P

Phosphofructokinase (rate determining step)

• Inhibited by atp, lower affinity for F6P

• Stimulated by AMP

Pyruvate kinase

• Inhibited by ATP, stimulated by F1,6-bisphosphate 

• inhibited b phospho to glucagon when blood glucose is low (prevent liver from using it)

Hypoxic conditions

Yeast and eukaryotes increase glycolytic bodies

• Glycolytic bodies: fermentation granules

Tumours displace enhanced rates of glucose uptake/golycolysis

Warburg effect

Cancer cells metabolize glucose to lactate even with oxygen

• Lactate secretion lowers pH

• increasing glycolytic intermediates stimulates PPP

• cancer cell growth faster than blood vessels 

Cancer cells experience hypoxia

Hypoxia induced transcription factor (HIF-1a)

• increases gene expression of glycolytic enzymes, GLUT transporters and growth factors for angiogenesis

Endurance training affect muscle glycolysis

Anaerobic exercise forces cell to rely on lactic acid fermentation for ATP production, stimulating HIF-1A

Oxygen levels rise, HIF-1a degraded by Q-proteasome pathway 

Gluconeogenesis

Synthesis of glucose from pyruvate and noncarbohydrate precursors

• LACTATE: enters as pyruvate

• GLUCOGENIC amino acids: enter as pyruvate or oxaloacetate

• GLYCEROL: enters as dihydroxyacetone phosphate

Gluconeogenesis occurs

In liver and kidneys

• Important for starvation/fasting

• Glucose: red blood cells/brain fuel only

3 irreversible steps in glycolysis bypassed in glyconeogenesis

Hexokinase, phosphofructokinase, pyruvate kinase

• have large negative delta G

4 Gluconeogenesis reactions not in glycolysis 

Step 1: Pyruvate carboxylase
Step 2: Phosphyenolyase carboxykinase
Step 9: Fructose 1,6-Bisphosphate
Step 11: Glucose 6 Phosphatase

Gluconeogenesis step 1

Pyruvate carboxylase-ligase

• Converts pyruvate into oxaloacetate by ATP hydrolysis

• Biotin coenzyme, carrier of CO2

• 4 step reaction

Gluconeogenesis step 2

Phosphoenolpyruvate carboxykinase

• convert oxaloacetate into phosphoenolpyruvate by GTP hydrolysis

• Reactions 1 and 2 bypass pyruvate kinase

Gluconeogenesis step 9

Fructose 1,6-bisphosphate

• Convert F1,6-bis. into F6P

• produce Pi

• inhibited by F2,6Bis

• Bypasses phosphofructokinase

Gluconeogenesis step 11

Glucose 6 phosphatase-hydrolase 

• Converts G6P into glucose into bloodstream

• Occurs in liver

• Enzyme integrated into inner ER membrane

2 Pyruvate molecules make 1 glucose molecule 

Requires 6 ATP

• 1 ATP pyruvate carboxylas

• 1 GTP phosphoenolpyruvate carboxykinase

• 1 ATP phosphoglycerate kinase 

2 Glycerol molecules make 1 glucose molecule 

Enters GNG as dihydroxyacetone phosphate

Required 2 ATP

• 1 ATP convert glycerol to dihydroacetone phosphate 

2 lactate and 2 alanine molecules make 1 glucose molecule

Both enter GNG as pyruvate, requires 6 ATP 

Metabolism in liver: Eating

High insulin to glucagon ratio

• insulin stimulate Fatty acid synthesis

• Glucose goes in liver, releasing insulin for storage, oxidation, and FAS

Metabolism in liver: fasting

Low insulin to glucagon ratio

• Glucagon stimulates degradation of “stores: and new glucose synthesis

• glucagon and fatty acids go on, glucose, oxidation, and ketone bodies go out 

Fructose 2,6 Bisphosphate activate phosphofructokinase

Liver: F6P rises when blood-glucose high

• F6P: synthesis of F-2,6-BP

Binding F6P increases affinity of phosphofructokinase for F6P

Stops inhibitory effects of ATP 

Substrate cycle

Reactions not fully active at same time cuz of allosteric controls

• F6P to F1,6-P and hydrolysis back to F6P= undesirable futile cycle

• Cycles in opposite directions cancel each other out 

Small changes= impact net flux 

Cori cycle and glucose alanine cycle transfer free energy

Lactate released into blood after muscle contraction (Cori)

Alanine released into bloodstream after breakdown of proteins in muscle tissue (glucose alanine)

Liver removes lactate and alanine, converts to glucose, release back to blood

Triose phosphate Isomerase Deficiency

Affects Glycolysis and GNG

• increases of advanced glycosylation end products and reactive oxygen species

  • Increased methyl group  

leads to hemolytic anemia/ neurological disorders 

Pyruvate Kinase deficiency

Causes hemolytic anemia due to reduced pyruvate and ATP production

• affects glycolysis outflow

Genetic: recessive

Phosphoenopyruvate to pyruvate by kinase 

Pyruvate Carboxylase Deficiency

Affects GNG and metabolism due to oxaloacetate intermediate

Causes hypoglycemia (low blood sugar) and lactic acidosis (high lactate in blood) 

Pyruvate Oxidation mammalian cells

In mitochrondrion

Mitochrondrial pyruvate carrier transports pyruvate into mitochon. matrix

Pyruvate dehydrogenase complex converts pyruvate to acetyl CoA 

Pyruvate dehydrogenase enzmes

E1: Pyruvate dehydrogenase

• Cofactor: TPP

• Oxidative decarbox. of pyruvate

E2: Dihydrolipoyl transacetylase

• Cofactor: lipamide

• Tranfer acetyl group to CoA

E3: Dihydrolipoyl dehydrogenase

• Cofactor: FAD

• Regenerate ox form of lipoamide 

Pyruvate dehydrogenase Coenzymes

Catalytic coenzymes: TPP (B1), lipoamide, and FAD (B2)

Stoichiometric coenzymes: CoA(B5), NAD+(B3)

Pyruvate dehydrogenase cycle step 1

Decarboxylation

• Catalyzed by pyruvate dehydrogenase (E1)

• Pyruvate + TPP then decarboxylate into hydroxyethyl TPP

Pyruvate dehydrogenase cycle step 2

Oxidation and transfer to Lipoamide

• Catalyzed by E1

• Hydroxyethyl-TPP oxidized to form acetyl group while transferred to lipoamide

• makes Acetyl lipoamide

Pyruvate dehydrogenase cycle step 3

Formation of Acetyl Coa

• Catalyzed by dihydrolipoyl transacetylase (E2)

• aceytl group transfeered from acetyl lipoamide to COA

• forming acetyl coa

Pyruvate dehydrogenase cycle step 4

Regeneration of Oxidized Lipoamide

• Catalyzed by dihydrolipoyl dehydrogenase (E3)

• E3 uses disulfide bond and FAD to reoxidize dihydrolipoamide 

Pyruvate dehydrogenase cycle step 5

Regeneration of Oxidized E3

• Catalyzed by E3

• electron move from FAD to form FADH2

• Move from FADH2 to NAD+

• E3 back to oxidative state 

PDH regulation

Pyruvate to acetyl CoA: irreversible

• Feedback inhibition

• Covalent modification of E1

PDH Feedback inhibition

A lot of NADH and acetyl COA

• only make acetyl CoA when NADH and acetyl CoA present

PDH Covalent Modification of E1

Kinase: phosphosry. and inactivates E1

Phosphatase: removes phosphate and activates E1

PDH phosphatase deficiency

PDH complex inactive

• too much lactic acid in blood, damage CNS

• treatment: Dichloroacetate/ketogenic diet

Diabetic Neuropathy

Overactive PDH kinase

• treatment: inhibit PDH kinase/LDH enzyme (gene editing)

Beriberi

Lack of thiamine (B1)

• TPP from thiamine

• treatment: B1 vitamin

Mercury and Arsenic Poisoning

Arsenitie inhibits PDH by inactivating dihydrolipoamide comp. of transacetylase

• treatment: 2,3 dimercaptopropanol

PDH kinase developing cancer

Leads to PDH inactivity

• only lactate produced by pyruvate

Aerobic glycolysis: more glucose than actate promoted, leading to ATP produced at faster rate

• Allows cancer cell rapid generation (Warburg effect)