Biochemistry

Enzymes

Biological catalyst that: Increases the reaction rate by decreasing the activation energy, are reaction specific, and are not consumed or produced during a reaction

Michaelis Menten Equation

E+S → (k,on) ← (k,off) ES → (k,cat) E+P

Michaelis Menten Equation (V)

V = Vmax[S]/Km+[S]

Michaelis Menten Equation (Km)

K(off)+K(cat)/K(on)

Michaelis Menten Kinetics Assumption: Only used to describe the initial reaction velocity

[S] > [P]

Michaelis Menten Kinetics Assumption: Steady-state approximation

[ES] = constant

Michaelis Menten Kinetics Assumption: Free Ligand Approximation

[S] > [E]

Michaelis Menten Kinetics Assumption: Rapid Equilibrium Assumption

k(off)>k(cat), km=k(off)/k(on)= k(D)

Catalytic Efficiency

k(cat)/k(m)

Michaelis-menten saturation curve

This is a hyperbolic curve, the y-axis represents reaction rate and the x-axis represents substrate concentration. At low substrate concentrations, the reaction rate increases sharply.

Competitive Inhibition

E+S → ← ES → E+P, inhibitor binds to E (Active Site), increases effect on Km, and has no effect on the Vmax

Uncompetitive Inhibition

E+S → ← ES → ← E+P, inhibitor binds to ES (Allosteric Site), decreases effect on Km, and decreases the Vmax

Mixed Inhibition

E+S → ← ES → E+P, binds to E + ES (Allosteric Site/Unequal Affinity) increases if inhibitor has a greater affinity for E or decreases effect on Km if inhibitor has a greater affinity for ES, and decreases the Vmax

Noncompetitive Inhibition

Mixed inhibition where the inhibitor has equal affinity for E and ES, binds to E + ES (Allosteric Site), has no effect on Km, and decreases Vmax

Lineweaver-Burk Plots

1/v = (Km/Vmax)(1/s) + 1/Vmax

Lineweaver-Burk Plots x-intercept

-1/Km

Lineweaver-Burk Plots Slope

Km/Vmax

Lineweaver-Burk Plots y-intercept

1/Vmax

LIneweaver-Burk Plots Competitive Inhibitor

Competes for the enzyme but can be beaten if overtaken with enough substrate. Km higher (more competition) Vmax same. So same y intercept and (higher Km = 1/Km = smaller number) smaller x intercept.

LIneweaver-Burk Plots Uncompetitive Inhibitor

Unfairly competes by only binding to enzyme substrate complex. Since it's bound to the complex however, it looks like the enzyme is interacting a lot with the substrate (so Km is lower). Vmax decreased since the enzyme is stuck to the complex. Since both values decrease, 1/smaller value > 1/bigger value so both intercepts are bigger on the plot.

LIneweaver-Burk Plots Noncompetitive Inhibitor

Too lazy to compete (no competition) so it binds whatever it sees, complexed or not. Km is same and Vmax is decreased. So y intercept is higher and x intercept is same,

Cellular Respiration

Glucose is a high energy molecule that can be oxidized to release energy

Cellular Respiration Overall Reaction

C(6)H(12)O(6) + 6O(2) → 6CO(2) + 6H(2)O

Glycolysis

An evolutionary conserved biochemical pathway that breaks glucose down into two molecules of pyruvate and the energy that is released in this process is used in the net production of two ATP and two NADH molecules

Preparatory Phase of Glycolysis

Steps 1-5

Step 1. Phosphorylation of Glucose (Glycolysis)

Irreversible (∆G < 0), glucose is phosphorylated to glucose 6-Phosphate, and is catalyzed by hexokinase

Kinases

Transferase enzymes that transfer a phosphate group from one molecule to another

Phosphorylases

Add an inorganic phosphate to a molecule

Step 2. Isomerization of Glucose-6 Phosphate to Fructose 6-Phosphate (Glycolysis)

The aldehyde in glucose 6-phosphate is is converted to a ketone in fructose 6-phosphate and is catalyzed by phosphoglucose isomerase

Step 3. Phosphorylation of Fructose 6-Phosphate (Glycolysis)

Irreversible (∆G < 0), fructose-6-phosphate is phosphorylated to fructose 1, 6-bisphosphate, catalyzed by phosphofructokinase-1 (PFK-1), and is the committed step (First irreversible step unique to glycolysis)

Bisphosphates

Molecules with two phosphate groups at different positions (Ex: Fructose 1, 6-Bisphosphate)

Diphosphates

Molecules with two phosphate groups linked together (Ex: ADP)

Step 4. Cleavage of Fructose 1, 6-Bisphosphate (Glycolysis)

Fructose 1, 6-bisphosphate is broken down into glyceralde 3-phosphate and dihydroxyacetone phosphate (DHAP), and is catalyzed by aldolase

Step 5. Conversion of Triose Phosphates (Glycolysis)

Dihydroxyacetone phosphate is converted to glyceraldehyde 3-phosphate and is catalyzed by triose phosphate isomerase

Payoff Phase

Steps 6-10

Step 6. Oxidation of Glyceraldehyde 3-Phosphate (Glycolysis)

Glyceraldehyde 3-Phosphate is oxidized and an inorganic phosphate is added to form 1, 3 bisphosphoglycerate, NAD+ is reduced to NADH, and is catalyzed by glyceralde 3-phosphate dehydrogenase

Step 7. Transfer of Phosphate Group from 1, 3-Bisphosphoglycerate (Glycolysis)

A phosphate group is transferred from 1, 3-bisphosphoglycerate to ADP to form ATP and 3-phosphoglycerate (This form of ATP formation is called substrate-level phosphorylation) and is catalyzed by phosphoglycerate kinase

Step 8. Conversion of 3-Phosphoglycerate to 2-Phosphoglycerate (Glycolysis)

The phosphate group on 3-phosphoglycerate is moved from the C-3 to the C-2 position to form 2-phosphoglycerate and is catalyzed by phosphoglycerate mutase

Step 9. Dehydration of 2-Phosphoglycerate (Glycolysis)

Water is removed from 2-phosphoglycerate to form phosphoenolpyruvate and is catalyzed by enolase

Step 10. Phosphate Group Transfer from Phosphoenolpyruvate

Irreversible (G∆ < 0), the phosphate group from phosphoenolpyruvate is transferred to ADP to form ATP and pyruvate (substrate-level phosphorylation) and is catalyzed by pyruvate kinase

Glycolysis Preparatory Phase Results

Steps (1-5), glucose is broken down into two molecules of glyceraldehyde 3-phosphate and two molecules of ATP are invested

Glycolysis Payoff Phase Results

Steps (6-10), the two glyceraldehyde 3-phosphate molecules are converted to two pyruvate molecules, four molecules of ATP are produced, and two molecules of NADH are produces

Glycolysis Overall Reaction

Glucose + 2NAD+ + 2ADP + 2P(i) → 2 Pyruvate + @NADH + 2H+ + 2ATP + 2H(2)O

Glycolysis Energy Results

Only a small amount of energy is released in glycolysis; pyruvate is a high energy molecule that can be further degraded to release energy

Fermentation

Anaerobic degradation of glucose to produce ATP, oxygen cannot be used as the final electron acceptor in the electron chain, and another electron acceptor is required to regenerate NAD+ from NADH

Lactic Acid Fermentation Example

Active skeletal muscles under hypoxic conditions

Lactic Acid Fermentation

Pyruvate is the electron acceptor and is reduced to lactate, NADH is oxidized to NAD+, and is catalyzed by lactate dehydrogenase

Lactate in Lactic Acid Fermentation

Is released into the blood and is eventually converted back to glucose by liver

Ethanol Fermentation

Pyruvate is first broken down to acetaldehyde and carbon dioxide, acetaldehyde is reduced to ethanol, and NADH is oxidized to NAD+ (Ex: Yeast Cells)

Pyruvate Dehydrogenase Complex

Located in the mitochondrial matrix, a complex of enzymes that decarboxylates and oxidizes pyruvate to form acetyl-CoA and carbon dioxide, and NAD+ is reduced to NADH

Glycolysis Location

Glycolysis occurs in the cytosol, so pyruvate is first transported to the mitochondrial matrix

Acetyl-CoA

Is the starting material of citric acid cycle

Citric Acid Cycle Location

Mitochondrial Matrix

Citric Acid Cycle

Oxidation of the acetyl group in Acetyl-CoA to two molecules of carbon dioxide and the two carbons in the acetyl group of acetyl-CoA are converted to CO(2)

Net Result of One Round of the Citric Acid Cycle

acetyl-Coa + 3NAD+ + FAD + GDP +2H(2)O + P(i) → CoA + 2CO(2) + 3NADH + 3H+ + FADH(2) + GTP

Step 1. Formation of Citrate (Citric Acid Cycle)

Acetyl-CoA donates its acetyl group (2C) to oxaloacetate (4C) to form citrate (6C), catalyzed by citrate synthase, and hydrolysis of the thioester bond in acetyl-CoA is highly exergonic ( ∆G < 0 )

Step 2. Formation of Isocitrate (Citric Acid Cycle)

Citrate is isomerized to isocitrate, catalyzed by aconitase, and cis-aconitase is an intermediate

Step 3. Oxidative Decarboxylation of Isocitrate (Citric Acid Cycle)

Isocitrate is oxidized and decarboxylated to form alpha-ketoglutarate (5C) and carbon dioxide, NAD+ is reduced to NADH, and is catalyzed by isocitrate dehydrogenase

Step 4. Oxidative Decarboxylation of Alpha-Ketoglutarate (Citric Acid Cycle)

Alpha-ketoglutarate is oxidized and decarboxylated to form succinyl-CoA (4C) and carbon dioxide, NAD+ is reduced to NADH, and is catalyzed by alpha-ketoglutarate dehydrogenase complex

Step 5. Formation of Succinate (Citric Acid Cycle)

Succinyl-CoA is hydrolyzed to succinate (4C), hydrolysis of thioester bond in succinyl-CoA is highly exergonic ( ∆ G < 0 ), GTP is formed by substrate-level phosphorylation, and is catalyzed by succinyl-CoA synthetase

Step 6. Oxidation of Succinate (Citric Acid Cycle)

Succinate is oxidized to fumarate (4C), FAD is reduced to FADH(2), and is catalyzed by succinate dehydrogenase

Succinate Dehydrogenase

The only enzyme that participates in both the citric acid cycle and the electron transport chain

Step 7. Hydration of Fumarate (Citric Acid Cycle)

Water is added to fumarate to form malate (4C) and is catalyzed by fumarase

Step 8. Oxidation of Malate (Citric Acid Cycle)

Malate is oxidized to oxaloacetate (4C), NAD+ is reduced to NADH, and oxaloacetate is regenerated so the citric acid cycle can continue to run

Oxidative Phosphorylation

Energy from the oxidation of NADH and FADH(2) is used to produce ATP

Electron Transport Chain

Electrons from NADH and FADH(2) are passed through a series of electron carriers to oxygen to form water and H+ are pumped from the mitochondrial matrix to the intermembrane space

ETC Complex I: NADH Dehydrogenase

NADH is oxidized to NAD+ and ubiquinone (Q) is reduced to ubiquinol (QH(2)), two electrons are transferred from NADH to ubiquinone, and four H+ are transferred to the intermembrane space

Ubiquinone (Q)

The oxidized form of coenzyme Q

Ubiquinol (QH(2))

The reduced form of coenzyme Q

ETC Complex II: Succinate Dehydrogenase

FADH(2) is oxidized to FAD+, ubiquinone (Q) is reduced to ubiquinol (QH(2)), two electrons are transferred from FADH(2) to ubiquinone, and no H+ are transferred to the intermembrane space

ETC Complex III: Coenzyme Q: Cytochrome C Oxidoreductase

Ubiquinol is oxidized to ubiquinone, two molecules of cytochrome c(Fe3+) is reduced to cytochrome c(Fe2+), two electrons are transferred from ubiquinol to two molecules of cytochrome c(Fe3+), and four H+ is pumped into the intermembrane space

Cytochrome c

A one electron carrier

ETC Complex IV: Cytochrome C Oxidase

Two molecules of cytochrome c(Fe2+) is oxidized to cytochrome c(Fe3+), oxygen is the final electron accepter and is reduced to water, two electrons are transferred from two molecules of cytochrome c (Fe2+) to oxygen, and two h+ are transferred into the intermembrane space

Chemiosmotic Theory

States that the energy produced from oxidizing NADH and FADH(2) is stored as a H+ concentration difference across the inner mitochondrial membrane

High [H+]

In the intermembrane space

Low [H+]

In the mitochondrial matrix

Proton Motive Force (pmf)

The energy stored in the H+ gradient produced by the electron transport chain, has both chemical potential energy and electrical potential energy

ATP Synthase

H+ can flow down their concentration gradient through protein pores (________) to produce ATP

Gluconeogenesis

Synthesis of glucose from pyruvate and related compounds, 7 of the 10 steps in glycolysis are reversed and use the same enzymes, and the three irreversible steps ( ∆G < 0 ) require a separate set of enzymes

Gluconeogenesis Location

Liver

Gluconeogenesis Overall Reaction

2 Pyruvate + 4 ATP + 2 GTP + 2 NADH + 2 H+ + 2H(2)O → Glucose + 4 ADP + 2 GDP + 6P(i) + 2 NAD+

Reaction coupling to ATP/GTP hydrolysis

Allows gluconeogenesis to be irreversible ( ∆G < 0 )

Irreversible Step 1. Conversion of Pyruvate to Phosphoenolpyruvate (Gluconeogenesis)

Requires two steps and two enzymes: pyruvate is converted to oxaloacetate by pyruvate carboxylase, oxaloacetate is reduced to phosphoenolpyruvate by phosphoenolpyruvate carboxykinase

Irreversible Step 1. Conversion of Pyruvate to Phosphoenolpyruvate Requirements (Gluconeogenesis)

A high energy phosphate group (One from ATP and GTP)

Irreversible Step 2. Conversion of Fructose 1, 6-Bisphosphate to Fructose-6-Phosphate

Fructose 1, 6-bisphosphate is hydrolyzed to fructose-6-phosphate by fructose 1, 6-bisphosphotase

Irreversible Step 3. Conversion of Glucose 6-Phosphate to Glucose

Glucose 6-phosphate is hydrolyzed to glucose by glucose 6-phosphotase

Glycogen

Glucose can be stored as this polymer and released as needed to produce ATP; an effective way of storing large quantities of glucose without affecting cellular osmolarity

Glucose concentration without glycogen

0.4 M

Glucose concentration as glycogen

0.01 (micro)M

Location where glycogen is stored

Liver and Skeletal Muscle

Glycogen Branched Structure

Allows for rapid breakdown

Glycogen Straight Chains

Held together by alpha-1,4 linkages

Glycogen Branches

Formed from alpha-1, 6 linkages

Glycogenolysis

Glycogen phosphorylase uses an inorganic phosphate to break alpha-1, 4 linkages to form glucose 1-phosphate

At four glucose residues from a branch point (Glycogenolysis)

Debranching enzyme transfers three of the glucose residues to another branch and cleaves the alpha-1, 6 linkage to produces glucose

Phosphoglucomutase (Glycogenolysis)

Converts glucose 1-phosphate to glucose 6-phosphate

Glucose 6-Phosphatase (Glycogenolysis)

Removes the phosphate group from glucose-6-phosphate to produce glucose that can be released into the blood

Skeletal Muscle Cells (Glycogenolysis)

Do not express glucose 6-phosphatase

Phosphoglucose Mutase (Glycogenolysis)

Converts glucose 6-phosphate to glucose 1-phosphate

UDP-Glucose Pyrophosphorylase (Glycogenolysis)

Converts glucose 1-phosphate to UDP-Glucose : glucose 1-phosphate + UTP → UDP-glucose + PPi