1.1.1 Pre-Midterm Biochem - Glycolysis Lecture

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Front 2

Define glycolysis and explain its role in the generation of metabolic energy

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Glycolysis (sugar-splitting) is a cytosolic specific pathway by which cells get energy from glucose. Glucose can either be phosphorylated into G6P, stored as glycogen, or passed through glycolysis in the cytosol. The PPP pathway uses glucose to make reducing equivalents for biosynthetic reactions and ribose for nucleotide synthesis. There are two phases associated with glycolysis: phase 1 involves the investment of E (2 ATP) to form F-1,6 BP while phase 2 involves the generation of E (4ATP) to form two molecules of pyruvate. Thus the net yield of glycolysis is 2ATP. The second phase of glycolysis involves two high E intermediates: 1,3 BPG and PEP.

Front 3

Define substrate level phosphorylation and intendiry those reaction in the glycolitic pathway.

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Substrate level P produces ATP by direct transfer of a P to ADP; oxidation and phosphorylation are not coupled; in oxidative phosphorylation, electrons are transferred from a donor to an acceptor (oxygen) in a redox reaction. The two enzymes that produce substrate level phosphorylation in glycolysis are: glyceraldehyde kinase and pyruvate kinase.

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Explain the significance of lactate production in anaerobic glycolysis. Indicate the further fate of lactate formed in muscle (Cori Cycle)

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Pyruvate is converted to lactate via lactate dehydrogenase (E1, E2, E3, TPP, FAD, lipoic acid, coenzyme A, NAD+) under anaerobic conditions in order to re-oxidize NADH and provide additional NAD+ to glycolysis. Lactate enters the blood stream where it is transported to aerobic tissues (liver and heart, Cori cycle). In these tissues, pyruvate is regenerated and changed into acetyl-CoA for the TCA cycle. As lactate concentration increases, the H+ levels rise, and lactic academia can occur.

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Outline the regulation of glycolysis indicating the regulatory enzymes. Appraise the role of AMP, ATP and fructose 2,6 bisphosphate on glycolysis

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There are three irreversible steps in glycolysis that serve as the points for regulation: hexokinase/glucokinase, PFK-1 (RLS), and pyruvate kinase. Hormonally, all three of these enzymes are activated by insulin and inhibited by glucagon.
Glucokinase has a lower affinity for glucose in the liver (fed state, high Km), than does hexokinase in the muscle (lower Km). The phosphorylation of glucose traps it into the cell; glucose can enter cell (heart, muscle, adipose) via GLUT 4 insulin dependent receptors (extracellular glucose is at a higher cellular concentration than intracellular glucose, too hydrophilic to enter cell by means of passive diffusion). Hexokinase can be allosterically inhibited by G6P (product), but this isn’t an important regulatory site for glycolyis.
PFK-1 is composd of a tetramer of L and M subunits (rich in L4 in liver, M4 in muscle). It is inhibited by ATP and activated by AMP and fructose 2,6 bisphosphate (signaling molecule).
Pyruvate kinase is allosterically regulated by being inhibited by high ATP concentrations and activated by fructose 1,6 bisphosphate.

Front 6

Outline the function of glycolysis in specific tissues (RBC, liver, brain, muscle, eye, tumor cells)

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In cells without mitochondria (RENT KSS: RBCs, eyes, neonates, tumors, kidney medulla, skin, skeletal muscle), glycolysis is the only means to generate ATP. Glycolysis provides G6P to the PPP and connects glycogen degradation in myocytes and cardiocytes to the TCA cycle under aerobic conditions. Anaerobic glycolysis provides ATP in muscle tissues during anaerobic exercise and produces NAD+ which can be reused in glycolysis. Glycolysis provides anaplerotic intermediates for synthesis of AA (G,A,S). Glycerol can be derived from glycolysis and used as a precursor for acylgylcerol (lipid) synthesis.
Hepatic glycolysis functions to buffer the high blood glucose levels following a carbohydrate rich meal. Glucose is converted into TAG via acetyl CoA and is exported from the liver to be stored in adipose tissues as a FA. Hepatic PFK-2 phosphorylates F6P into F2,6 bisP (signaling molecule, not in glycolysis that activates PFK1 and inhibits F16 bisphosphatase in gluconeogenesis) and is active when unphosphorylated and inactive when phosphorylated. Insulin stimulates hepatic glycolysis; glucagon stimulates cAMP which inhibits glycolysis and stimulates gluconeogenesis.

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Analyze the effect of pentavalent arsenateon glycolytic enzymes

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Arsenate is an anion analogous to phosphate. Thus in the presence of arsenate, when glyceraldehde 3P dehydrogenase transfers a P to glyceraldehydes 3 P it forms 1-arseno-3-phosphogylcerate rather than 1,3 BPG. This is unstable and breaks down to 3-phosphoglycerate, thus the phosphorylation reaction catalyzed by phosphoglycerate kinase is by-passed. This means that glycolysis continues in the presence of arsenate but at the expense of one less ATP being formed. Both Hg and arsenite bind to dihydrolipoyl groups on pyruvate dehydrogenase complex and leads to CNS pathologies.

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Analyze the effect of fluoride ions on glycolytic enzymes

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Fluroride is a strong competitive inhibitor of enolase, but it bears no resemblance to 2-phosphoglycerate. Fluoride binds to the Mg2+ ions and phosphate forming a complex at the active site.

Front 9

Identify the biochemical mechanisms involved in the occurrence of lactic acidosis in states of poor tissue perfusion/oxygenation

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Pyruvate is converted to lactate via lactate dehydrogenase (E1, E2, E3, TPP, FAD, lipoic acid, coenzyme A, NAD+) under anaerobic conditions in order to re-oxidize NADH and provide additional NAD+ to glycolysis. Lactate enters the blood stream where it is transported to aerobic tissues (liver and heart, Cori cycle). In these tissues, pyruvate is regenerated and changed into acetyl-CoA for the TCA cycle. As lactate concentration increases, the H+ levels rise, and lactic academia can occur.

Front 10

Explain biochemical mechanism for the occurance of hemolysis in inherited pyruvate kinase deficiency

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Erythrocytes are totally dependent on glycolysis
ATP is required for the maintenance of Na+/K+ transporting-
ATPases
Cells swell and lyse without ATP
Reticulocytes lack pyruvate kinase (PK), but possess mitochondria that generate ATP by oxidative phosphorylation

Front 11

Differentiate between pyruvate kinase deficiency and glucose 6-phosphate dehydrogenase deficiency

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Pyruvate Kinase (PK) deficiency
Severity of anaemia depends on the % of PK deficiency
Less than 5% activity is fatal.
Affected isoform found only in erythrocytes.

Glucose 6 Phosphate deficiency
Genetic disease
Leads to NADPH dificeincy in erythroctyes resulting in haemolytic anemia
Over 400 mutation but only a few are symptomatic
Asymptomatic individuals may develop haemolytic deficiency caused by precipitating factors such as oxidants, drugs, favism, infection, neonatal jaundice.

Front 12

Describe the effects of methanol and ethylene glycol poisoning

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Initial effects of MeOH poisoning include impaired vision. MeOH slowly metabolizes to formaldehyde by alcohol dehydrogenase. Formaldehyde is then rapidly converted into formic acid causing metabolic acidosis.
Ethylene glycol poisoning is used as an antifreeze that is sweet to taste but odorless. It is absorbed via the GI tract and primarily metabolized in the liver. Alcohol dehydrogenase converts ethylene glycol into glycoaldehyde which eventually results in glycolate which interferes with some hepatic enzymes and is responsible for metabolic acidosis.