Medical Biochemistry

Glycolysis/TCA/ETS

Robert F. Waters, PhD

Glycolysis(Introduction)Glucose + 2 ADP + 2 NAD+ + 2 Pi -----> 2 Pyruvate + 2 ATP + 2 NADH + 2 H+

Cofactors needed Mg++ for ATP

Ca++, Zn++, Cd++ for absorption of glucose

Glycolysis

Anaerobic (Hypoxia) Aerobic (Inhibits Glycolysis)

Tries to make pyruvate Cytosol Glucose Absorption and Transport

Glucose General Information Glucose turnover of 70kg (154 lb) person

~2mg/kg/min or 200g/24hrs.

Hypoglycemic substances Insulin

Binds on cells to IRS-1 (Insulin receptor substrate) Stimulates TG synthesis Blocks Lipolysis (Increases LDLs)

Hyperglycemic substances Glucagon, epinephrine, HGH, cortisol

Stimulatory Substances for Insulin Production GIP-glucose dependent insulinotropic peptide CCK-cholecystokinin (pancreozymin) GLP-1 glucagon like peptide VIP-vasoactive intestinal peptide

NOTE: This is the reason the body has a better insulin response orally rather than IV.

Digestion Cont:

Glucose Absorption Glucose Transporters (GLUTs)

Glut-1 = erythrocytes Glut-2 = liver and pancreas Glut-3 = brain Glut-4 = skeletal muscle and adipose tissue Glut-5 = small intestine (Fructose Transport)

Cotransported with Na+ Na+ dependent glucose transport

Active Transport Most monosaccharides can cross brush border but extremely slow

(diffusion) Fructose is absorbed by Na+ independent facilitated transport (ATP

consumed as well)

Glycolysis Glucose to Glucose-6-phosphate

Hexokinaseglucose-6-phosphate (G6P)is the first reaction of glycolysis,

and is catalyzed by tissue-specific isoenzymes known as hexokinases

Glucokinase• Four mammalian isozymes of hexokinase are known (Types I - IV),

with the Type IV isozyme often referred to as glucokinase. Glucokinase is the form of the enzyme found in hepatocytes. The high Km of glucokinase for glucose means that this enzyme is saturated only at very high concentrations of substrate.

Glycolysis

The Km for hexokinase is significantly lower (0.1mM) than that of

glucokinase (10mM).

Glycolysis

G-6-P inhibits hexokinase Glucose stimulates hexokinase production Concept of Km? Definition of a Kinase Mg++ or Mn++ Inhibited by Fluorine

Glycolysis

Glucose-6-Phosphate to Fructose-6-Phosphate Isomerase

-D-Fructose-6-phosphate

Glycolysis Fructose-6-phosphate to Fructose-1,6-

bisphosphatePFK-1PFK-2 Insulin to Glucagon ratioCitrate InhibitsATP inhibitsMn++ or Mg++ Inhibited by fluorine

-D-Fructose-1,6-bisphosphate

Glycolysis

Split of F-1,6-bisphosphate into dihydroxyacetone phosphate and phosphoglyceraldehyde (DHAP and PGAL)

Triose phosphate isomerase Aldolase A (Isoenzyme) isomerase

Glycolysis

PGAL to 1,3 bisphosphoglycerate Substrate level Phosphorylation Inhibited by Arsenate Add Pi

Glycolysis

Erythrocytes1,3-bisphosphoglycerate to 2,3-bisphosphoglycerate to

3-phosphoglycerateMutasephosphatase

All cells metabolizing1,3-bisphosphoglycerate to 3-phosphoglyceratephosphoglycerokinase

Glycolysis

3-phosphoglycerate to 2-phosphoglyceratePhosphoglyceromutase

Glycolysis

2-phosphoglycerate to phosphoenolpyruvateEnolase Inhibited by fluorine (halogens)

Glycolysis

PEP to PyruvatePyruvate kinase

Glycolysis

Anaerobic conditionsPyruvate to lactate

Lactate dehydrogenase

Aerobic conditionsPyruvate to mitochondrial TCA cycle

Pyruvate-keto acid form of alanine

Glycolytic Control Mechanisms

Hexokinase Insulin StimulatesGlucose Stimulates Inhibited by G-6-P

G-6-P does not inhibit glucokinase

Inhibited by Glucagon

Glycolytic Control—PFK-1

Stimulated by AMP Stimulated by Fructose 2,6-bisphosphate Inhibited by ATP Inhibited by Citrate Inhibited by Glucagon

Glycolysis Control Mechanisms

PFK-1 and PFK-2

Insulin to Glucagon Ratio

Glycolytic Control-Pyruvate Kinase

Stimulated by Fructose 1,6-bisphosphate Inhibited by Glucagon

Glycolytic Metabolic Lesions Hexokinase Deficiency

Hemolytic Anemia Lactic Acidosis

Normal Blood Levels-1.2 mM High Levels 5 mM or more May be due to high lactate production or utilization

Hypoxia Reduces Mitochondrial ATP Production Activates PFK-1 Stimulates Glycolysis Increased Lactate Production May be caused [hypoxia] by reduced blood flow in tissue (shock), respiratory disorders, etc.

Pyruvate Kinase Deficiency

Pyruvate Kinase exists as isoenzymes One or more subunits may be affected Ionic imbalance causing erythrocytes to swell Hemolytic anemia-excessive RBC destruction

Pyruvate Kinase Deficiency-Cont. Presentation

Newborn anemic and jaunticed Hematology

Variability in cell morphology Above normal reticulocytes/total RBC ratio

Pyruvate Kinase activity is ~20% of normal Treatment

None for the most part Splenectomy under high anemic conditions Probably splenomegaly and hepatomegaly

The Mitochondria

mtDNA mtRNA Inorganic Phosphate Carrier Molecule ATP Transport Molecules

Pyruvate Dehydrogenase Complex

Decarboxylation Vitamins and pseudo-vitamins

Niacin, Riboflavin, -lipoic acid, Thiamine, CoA (Pantothenate)

Enzyme Complex (E1,E2,E3)Pyruvate Decarboxylase (CO2) 3C to 2C,

dihydrolipoyltransacetylase, dihydrolipoyldehydrogenase

PDH Regulation

Inhibited by;Acetyl CoANADH

Stimulated by;CoASHNAD+Pyruvate

PDH Stimulation Through Inhibition For example NAD+ inhibits Protein Kinase which does

NOT phosphorylate PDH enzymes and keeps them active. ATP and Mg++ are necessary for Protein Kinase Phosphoprotein Phosphatase removes phosphates from

phosphorylated enzymes. This enzyme is stimulated by Ca++

NOTE: [Ca] concentration and [ATP] concentration in mitochondria are inversely related.

Further PDH Regulation

Insulin Stimulates PDH in Adipose Tissue Catecholamines in cardiac muscle stimulates PDH

DopamineNorepinephrineepinephrine

Example of Cortisol Activating PDH in Cardiac Muscle Adrenal Cortex Secretes Cortisol

A glucocorticoid (Also anti-inflammatory)

Cortisol moves into adrenal medulla Stimulates Phenylethanolamine N-methyltransferase

Converts norepinephrine to epinephrine

Epinephrine secreted into blood and activates PDH complex Genetic inhibition by epinephrine

Epinephrine Production

Defects in PDH Complex

Severe cases are fatal Symptoms

Lactic AcidosisNeurological DisordersHigh Serum [Pyruvate]High Serum [Alanine]

PDH Defect Treatments

Large doses of thiaminHelps with E1 defect

Large doses of lipoic acidHelps with E2 defect

Ketogenic Diet rather than GlucogenicKetogenic Amino Acids

Isoleucine, leucine, tryptophan, lysine, phenylalanine, tyrosine

TCA Cycle

Tricarboxylic Acid Cycle Krebs Cycle Citric Acid Cycle Mitochondrial Matrix

TCA Cycle

OxaloacetateKeto Acid form of AspartateRegenerating Substrate (4-carbons)

Acetyl-CoAStoichiometric Substrate (2-carbons)

Citrate Synthase (Irreversible)Produce Citrate (6-carbons)

TCA Cycle Citrate to Isocitrate

Citrate less water cis-Aconitate Cis-Aconitate plus water isocitrate Prochiral carbon

Carbon with three different groups therefore distinguish between which COO- and stereospecificity with enzyme

Cis-aconitase Bidirectional (isoergonic)

Reactants to products favored (exergonic) Products to reactants favored (endergonic)

TCA Cycle

Isocitrate to -ketoglutarate Isocitrate dehydrogenase

Regulatory enzymeNADHCO2

Oxidative decarboxylationCoupled with reduced NAD and oxidative phosphorylation

TCA Cycle

-ketoglutarate to Succinyl-CoA -ketoglutarate dehydrogenase

Niacin, Riboflavin, Thiamine, -lipoic acidMulti-subunit enzyme structure

CO2Keto acid form of Glutamate

TCA Cycle

Succinyl CoA to SuccinateSuccinate thiokinase (kinase)Coupled Reaction

GDP + Pi GTPADP ATPSubstrate level Phosphorylation NOT Oxidative

Phosphorylation in the production of ATP

TCA Cycle

Succinate to FumarateSuccinate Dehydrogenase

Oxidation of succinate to fumarateReduction of FAD+ to FADH

TCA Cycle

Fumarate to MalateFumarase

Hydration of fumarate to malateisoergonic

TCA Cycle

Malate to OxaloacetateMalate dehydrogenase

Reduced NAD (NADH)IsoergonicSlighty endergonic (Slightly favors malate formation)

ATP Production

8 ATP - Glycolysis 30 ATP - PDH and TCA Cycle Theoretical Number of ATP (38) Actual ~36 ATP per mole of glucose

TCA Cycle Control

Citrate Synthase (Synthetase)Condensing Enzyme Inhibited By:

ATPNADHSuccinyl CoA

TCA Cycle Control-Cont:

Isocitrate DehydrogenaseActivated By:

ADP

Inhibited By:ATPNADH

TCA Cycle Control-Cont:

-Ketoglutarate Dehydrogenase Inhibited by:

Succinyl CoANADHATP

Contains tightly bound Tpp, lipoamide, FADSimilar to PDH complex

E3 subunit the same

TCA Cycle Control-Cont:

Succinyl CoA SynthetaseCoupled reaction with GTPEnzyme that catalyses coupled reaction is called

Nucleotidediphosphate Kinase

TCA Cycle Control-Cont:

Succinate DehydrogenaseHas Iron-Sulfur CentersCovalently Bound with FAD

General TCA Information Amphibolic

Involved in catabolic and anabolic processes Anaplerotic Reactions

Increase concentrations of TCA cycle intermediates Example: Amino Acid Metabolism

Aminotransferase: Glutamate and -ketoglutarate Aminotransferase: Aspartate and Oxaloacetate Pyruvate Carboxylase: Gluconeogenesis

Pyruvate + CO2 Oxaloacetate

The aminotransferase associated with alanine and pyruvate is not anaplerotic because pyruvate is not TCA intermediate.

Specifics of Pyruvate Dehydrogenase Complex E1

Pyruvate Decarboxylase Liberates CO2

Thiamine (TPP) E2

Dihydrolipoyltransacetylase Produces Acetyl-CoA -lipoic Acid CoA-Pantothenic Acid

E3 Dihydrolipoyldehydrogenase

Produces FADH to NADH Riboflavin and Niacin

Malate Aspartate Shuttle

Glycerophosphate Shuttle

Cytochromes

Cytochrome c oxidase contains two haem a groups, one binuclear copper site (CuA), one mononuclear copper site (CuB) and one bound Mg2+ per

monomer plus FeII and FeIII iron associated with sulfur (S).

Cytochrome c Oxidase (COX)

Do NOT get confused with cyclooxygenase also called COX!

Cytochrome c Oxidase Cycle

Co-enzyme Qx

1. Impaired coenzyme Q10 synthesis due to nutritional deficiencies. (Ubiquinone)

2. Genetic or acquired defect in coenzyme Q10 synthesis.

3. Increased tissue needs resulting from a particular medical condition.

Co-enzyme Q10

Interconversion Isoprenoid Units

Electron Transport System

Oxidative Phosphorylation

Electron Transport Continued;

Enzyme Information

   kDa   

Complex INADH dehydrogenase (or)NADH-coenzyme Q reductase

800 25

Complex IISuccinate dehydrogenase (or)Succinate-coenzyme Q reductase

140 4

Complex III Cytochrome C - coenzyme Q oxidoreductase 250 9-10

Complex IV Cytochrome oxidase (Copper) 170 13

Complex V ATP synthase 380 12-14

Electron Transport Continued;

Complex I

NADH + H+

         FMN

         Fe2+S

         CoQ

NAD+ FMNH2 Fe3+S CoQH2

Electron Transport Continued;

Complex II

Succinate          

FAD          

Fe2+S          

CoQ

Fumarate FADH2 Fe3+S CoQH2

Electron Transport Continued;

Complex III

CoQH2          

cyt b ox          

Fe2+S          

cyt c1 ox          

cyt c red

CoQ cyt b red Fe3+S cyt c1 red cyt c ox

Electron Transport Continued;

Complex IV

cyt c red          

cyt a ox          

cyt a3 red          

O2

cyt c ox cyt a red cyt a3 ox 2 H2O

Electron Transport Continued;

Chemistry of Complex V

ETS-Cont:

   kDa   

Complex INADH dehydrogenase (or)NADH-coenzyme Q reductase

800 25

Complex IISuccinate dehydrogenase (or)Succinate-coenzyme Q reductase

140 4

Complex III Cytochrome C - coenzyme Q oxidoreductase 250 9-10

Complex IV Cytochrome oxidase (Copper) 170 13

Complex V ATP synthase 380 12-14

Electron Transport Inhibitors

Complex IMany insecticidesBarbiturates

AmobarbitalSecobarbital

Some antibiotics

Electron Transport Inhibitors-Cont:

Complex IIISome antibiotics

Complex IVH2S

CyanideAzideCO

Electron Transport Inhibitors-Cont: Cyanide Poisoning (Example)

Almond smell of gaseous CN (breath)Severe Acidosis

A cancer treatment in Mexico is called Amygdalin (extracted from almonds) may be harmful due to CN is metabolite.

TreatmentInfusion of thiosulfate (binds CN)Ventilation with 100% O2

Administer Sodium Bicarbonate