BIOC 440 Exam 2

Lectures 11-18

Glycolysis Pathway Steps

1. Glucose to Glucose 6-phosphate
2. Glucose 6-phosphate to Fructose 6-phosphate
3. Fructose 6-phosphate Glyceraldehyde 3-phosphate
4. Glyceraldehyde 3-phosphate to Pyruvate (NADH, ATP byproducts)

PPP Oxidative Pathway Steps

Irreversible

1. Oxidation of Glucose 6-phosphate + NADP+ to 6-Phosphogluconate by G6P dehydrogenase
2. Hydrolysis of 6-Phosphogluconate + NADP+ to NADPH, CO2, Ribulose 5-phosphate (for nucleotide biosynthesis) by 6-Phosphogluconolactonase
2. Oxidation decarboxylation of Ribulose 5-phosphate to Xylulose 5-phosphate via 6-Phosphogluconate dehydrogenase

PPP Non-Oxidative Pathway Steps

Reversible; Utilizes transketolase or transaldolase

1. Xylulose 5-phosphate to either Ribulose 5-phosphate or Fructose 6-phosphate (for glycolysis) or Glyceraldehyde 3-phosphate (for glycolysis)

Glucose 6-phosphate Dehydrogenase Deficiency

Fatal in cases of high oxidative stress

Isomers

Same molecular formula, different chemical structure

Epimers

Isomers that different in configuration at a single carbon

Transketolase

2 carbon transfers, Uses TPP

Transaldolase

3 carbon transfers

PPP: Need more R5P than NADPH

1. Glucose 6-phosphate to Fructose 6-phosphate
2. Fructose 6-phosphate to ribose 5-phosphate

Allows some carbons to go through glycolysis

PPP: Need BOTH R5P and NADPH

1. Glucose 6-phosphate to + NADP+ to Ribulose 5-phosphate and NADPH
2. Ribulose 5-phosphate to R5P

Generates equal amounts of both

PPP: Need more NADPH than R5P

1. Glucose 6-phosphate to + NADP+ to Ribulose 5-phosphate and NADPH
2. Ribulose 5-phosphate to R5P
3. R5P to Fructose 6-phosphate OR glyceraldehyde 3-phosphate
4. glyceraldehyde 3-phosphate to dihydroxyacetone phosphate OR fructose 1,6-biphosphate
5. fructose 1,6-biphosphate to fructose 6-phosphate
6. fructose 6-phosphate tp G6P

Recycles carbons for further oxidation

PPP: Need NADPH, pyruvate, ATP

1. Glucose 6-phosphate to + NADP+ to Ribulose 5-phosphate and NADPH
2. Ribulose 5-phosphate to R5P
3. R5P to Fructose 6-phosphate OR glyceraldehyde 3-phosphate (step 5)
4. Fructose 6-phosphate to fructose 1,6-biphosphate
5. fructose 1,6-biphosphate to dihydroxyacetone phosphate or glyceraldehyde 3-phosphate
6. glyceraldehyde 3-phosphate to ATP and Pyruvate

Inhibition of G6P dehydrogenase

NADPH inhibits PPP (allows for increase in glycolysis)

Glucagon

Responds to low blood glucose by releasing more glucose, stimulates insulin secretion

Epinephrine

Fight or flight response by releasing more glucose

Insulin

Responds to high blood glucose by storing glucose, stimulates glucagon secretion

Insulin formation

Proteolytic cleavage of preproinsulin and disulfide formation of proinsulin leads to mature insulin

Tissues that Synthesize Glucose

Liver & Kidney

Gluconeogenesis

Generation of glucose using NON-carbohydrate precursors; Primarily cystolic

Cori-Cycle

M-Glycogen to M-Lactate (and ATP) to blood lactate to L-lactate (and ATP) to L-glucose to blood glucose back to M-glycogen

Relationship between Glycolysis and Gluconeogenesis

NOT the reverse of each other; Gluconeogenesis is anabolic and energetically expensive

Steady State

Rate of input = Rate of output (does NOT mean that rate of forward reaction = rate of backward reaction)

Flux (rate)

Regulated by changing enzyme amount or activity via transcriptional/translational regulation, allosteric control, covalent PTMs, or compartmentalization

Allosteric Control

Muscle isozyme inhibited by G6P

Sequestration

Liver enzyme regulated by increased in F6P

Inhibition of PFK-1

ATP, citrate

Activation of PFK-1

ADP, AMP, Fructose 2,6 bisphosphate

Leads to glycolysis

Increase of Fructose 2,6 bisphosphate

PFK-2 active, FBPase-2 inactive, stimulates glycolysis, inhibits gluconeogenesis

Decrease of Fructose 2,6 bisphosphate

PFK-2 inactive, FBPase-2 active, inhibits glycolysis, stimulates gluconeogenesis

Regulation of Pyruvate Kinase

Glucagon makes it inactive

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Regulation of Pyruvate Carboxylase

Acetyl-CoA inhibits pyruvate dehydrogenase complex and activates pyruvate carboxylase to form oxaloacetate

Glycolysis

Used by aerobic and anaerobic organisms; Oxidation of 1 molecule of glucose to 2 molecules of pyruvate; 10 enzyme catalyzed reactions

GLUT2

Found in liver and B-pancreatic cells

Low affinity and high capacity for glucose

GLUT3

Found in brain

High affinity for glucose

GLUT4

Found in skeletal muscle and fat

Responsive to insulin

Glycolysis: Preparatory Phase

1. Glucose --hexokinase--> G6P (needs ATP)
2. G6P --phosphohexose isomerase--> F6P
3. F6P --PFK-1--> F 1,6bisphophate (needs ATP)
4. F 1,6bisphophate --aldolase--> glyceraldehyde 3-phosphate & Dihydroxyacetone phosphate

Glycolysis Payoff Phase

5. glyceraldehyde 3-phosphate & Dihydroxyacetone phosphate --triose phosphate isomerase--> glyceraldehyde 3-phosphate
6. glyceraldehyde 3-phosphate --glyceraldehyde 3-phosphate dehydrogenase--> 1,3 biphosphoglycerate
7. 1,3 biphosphoglycerate --phosphoglycerate kinase--> 3-phosphoglycerate
8. 3-phosphoglycerate --phosphoglycerate mutase--> 2-phosphoglycerate
9. 2-phosphoglycerate --enolase--> phosphoenolpyruvate
10. phosphoenolpyruvate --pyruvate kinase--> pyruvate

Fates of Pyruvate

Anaerobic: Ethanol, Lactate
Aerobic: acetyl CoA --> citric acid cycle --> CO2, H2O

Lactic Acid Fermentation

Pyruvate --lactate dehydrogenase--> Lactate

Ethanol Fermentation

Pyruvate --pyruvate decarboxylase--> Acetaldehyde --alcohol dehydrogenase--> ethanol

Pyruvate decarboxylase coenzyme

Thiamine pyrophosphate (TPP)

Involved in decarboxylation of of alpha-keto acids and transfers of 2 carbon units

-ΔG

Spontaneous

+ΔG

Non-spontaneous

Standard State

1 Molar, pH = 7, 55M H2O, 25C

ΔG = RTlnQ - RTlnKeq

R = 8.314 kj/mol
T = temp in K
Q = [P]/[R]
Keq = constant

ATP Hydrolysis

ATP + H2O --> ADP

Phosphoryl group has high transfer potential; Favored to relieve charge repulsion, for resonance stabilization, and ionization of ADP; High activation energy for hydrolysis

ATP provides energy by ...

Group transfers

Oxidation

Loss of electrons

Reduction

Gain of electrons

Oxidation Number Trend

The higher the number, the more oxidized

Oxidation #: Hydrogen Bond

-1

Oxidation #: Carbon bond

0

Oxidation #: O, N, S bond

+1

Transcriptomics

RNA sequencing: Relies on new technology, sensitive with broad dynamic range

Proteomics

Large scale studying of proteins: Limited to characteristics of protein

Metabolomics

GC-MS: studies low MW molecules

NMR

Ranges are similar for different groups, low MW molecules

Pulse-Chase Experiments

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Protease

Catalyzes hydrolysis of peptide bonds

Enolase

Catalysis my metal ion and acid/base

Enzyme function

lower activation energy

Km

Michaelis Constant = 1/2 Vmax

Michaelis-Menten Equation: low [S]

[S] << Km, Vo = Vmax [S]/Km

Michaelis-Menten Equation: high [S]

[S] > Km, V0 = Vmax

Catalytic efficiency

kcat/Km --> higher = more efficient enzyme

kcat = vmax/[E]