Biochemistry Exam 2 - Spring 2023

Biochem Exam 2 - Protein

Why are proteins important?

Proteins mediate every process in the cell

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Most abundant biomolecule in the body

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Thousands of different proteins in every cell

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Proteins from every organism are made from same 20 building blocks

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Building blocks are called amino acids

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The order of the amino acids gives diversity to proteins

Amino Acids Overview

20 Total

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Asparagine was first discovered in 1806

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Threonine was last to be identified in 1938

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All have common names, usually dependent on the source it was identified in

All amino acids have chiral centers except which one?

Glycine

What does Aliphatic mean?

Contains a hydrocarbon

Where are branched chain AA metabolized?

Muscle

What is Sickle Cell Anemia

Genetic mutation that occurred over time to protect population from Maleria

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Super painful

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Valine(nonpolar) replaces Glutamate (polar) in hemoglobin

*This causes the improper folding*

What is Maple Syrup Urine Disease

Genetic deficiency of enzymes required to metabolize branched chain amino acids (Valine, Leucine, Isoleucine)

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Symptoms: Urine with color, odor, and texture of maple syrup

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If untreated: Can cause brain damage and death

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Treatment": Avoid high protein food for life

What is Celiac Disease

Prevalence - 1.4% globally

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Etiology (Cause): Genetic susceptibility → exposure to gluten → environmental trigger and other risk factors → autoimmune response

What is phenylketonuria (PKU)

Genetic disorder that lacks enzyme (phenylalanine hydroxylase) necessary to metabolize phenylalanine

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Causes brain damage

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Treatment:Avoid phenylalanine in diet (low protein)

What is Cystic Fibrosis

Genetic disorder affecting 1/2,000 humans

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Missing 1 phenylalanine in protein that regulates transport of chloride ions across cell membranes

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Protein is not folded correctly and this degraded

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Causes: mucus to build up in lungs and digestive issues

Alzheimer’s Disease

Proteins:

Amyloids → normal cellular proteins that unravel and stick together

Lysine is key for holding it together through hydrophobic and electrostatic interactions

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Tangles → Microtubules become phosphorylated and behave unnaturally

Accumulation can cause cell to rupture

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Potential Treatments: CLR01 binds lysine and arginine

Lots of research going into treating this.

What is Aspartame

Artificial sweetener made of Aspartate and Phenylalanine

Glucogenic

Used to make glucose, if necessary

Product = Pyruvate

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Ketogenic

Turned into Kreb’s cycle intermediate, ketone bodies, or fatty acids, if in excess

Product = Acetyl CoA

Know AA Chart

*See Image*

How do AA act as acids and bases?

Amphoteric = dual acid-base nature

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All have at least 2 charges at physiologic pH, some have 3

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They are least soluble at their isoelectric point

When pH is high, what happens to AA?

AA can donate a H+ and become a proton donor - become an acid

When pH is low, what happens to AA?

AA can accept H+, becoming a proton acceptor - become a base

pKa General Rules

pKa is a lab determined number that is relatively constant

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pH will change substantially throughout body fluids

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If pH is below pKa = H+ is still attached

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If pH is above pKa = H+ is lost

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What is a Zwitterion?

The point where an AA or protein is neutral

Protein Overview

They are huge

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In theory could form infinite conformational structures

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Each protein has a specific 3D structure

Structure Themes

3D structure of a protein is determined by its amino acid sequence

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The function of a protein is dependent on its structure

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Most proteins exist in 1 or a few stable forms

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Most important stabilizing force for protein structures are noncovalent interactions

Hydrophobic Interactions → drives folding of nonpolar AA to interior of protein

Carbonyls & Amines → H bond with water

Charged R groups → Participate in electrostatic interactions

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Common structural motifs are found in most proteins

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Protein structures are not static, they are dynamic

Protein Structure: Conformation

Spatial Arrangement of atoms in a protein

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Rotation around a bond changes in conformation

Protein Structure: Native

Proteins in their functional, folded conformation

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Most Stable

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Has the lowest Gibbs free energy

Protein Structure: Stability

Protein’s ability to maintain the native confirmation

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Held together by disulfide bonds (covalent bonds between cysteines) and weak (noncovalent) interactions - H bonds, electrostatic interactions, hydrophobic interactions

What are the 4 levels of Protein Structure?

Primary → AA sequence

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Secondary → Initial Folding

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Tertiary → 3D folding of 1 strand

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Quaternary → 3D folding of multiple strands

Primary Structure → Peptides & Proteins

Peptide bonds - Formed via condensation/dehydration reactions

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Trans in nature

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Not broken by denaturation

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Peptide bond is rigid → Not allowed to rotate = stuck → Puts constraints on the overall protein structure

Synthesis of ALL macronutrients are what reactions?

Dehydration

Secondary Structure

Alpha helix, Beta Sheets, B turns

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Alpha Helices →

Tightly wound ‘spring’ like structure

Each turn contains 3.6AA

Structure held together by H-bonding

C=O on one AA is H-bonded to N-H 4AA apart

R groups point outward

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Stability of Helix dependent on:

Likelihood of a-helix formation → Interactions of R groups 3 or 4AA away → Size of R group → Occurrence of proline and glycine → Interactions of AA at ends of helix

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Types of Proteins

Fibrous →

Peptides found in long strands or sheets

Contain single secondary structure

Function as providers of support, shape, and external protection

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Globular →

Peptides folded in a spherical structure

Contain several secondary structures

Function as enzymes, regulatory proteins, and transport molecules

Keratin Proteins

Found only in mammals

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2 Alpha-helix coiled together to make a coiled coil

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Crosslinks between coiled coils held together by disulfide bonds

Collagen

Provides strength for connective tissue (bone, tendons, cartilage)

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30 different types of collagen in mammals

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Contains a unique secondary structure (also a coiled coil, but contains 3 polypeptide chains wound together)

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AA Sequence is generally a repeating tripeptide unit

Glycine - X-Y

X = often proline

Y = often hydroxyproline

Scurvy

Due to lack of Vitamin C (ascorbic acid)

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Required for hydroxylation of proline and lysine in collagen synthesis

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Deficiency leads to degeneration of connective tissue

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S/S - Corkscrew hairs, bleeding gums

Collagen Crosslinking

Lysyl Oxidase

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Copper (Cu) - dependent enzyme

Deaminates lysine forming reactive aldehyde

Aldehyde condenses with lysine or hydroxylysine on neighboring collagen

Collagen Degradation

Half-life of years

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Constant remodeling occurs due to growth and/or injury

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Degraded by MMPs

What are MMPs?

Matrix metalloproteinases

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Require metals → Ca and Zn

Main class of proteases in the body

Required for tissue remodeling

Which protein is mostly triple helical and virtually insoluble in water?

Collagen

Which posttranslational modification is extremely important for collagen structure?

Hydroxylation

Which is NOT found in high concentrations in collagen?

Valine

Types of Proteins

Fibrous →

Peptides found in long strands or sheets

Contain single secondary structure

Function as providers of support, shape, and external protection

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Globular →

Peptides folded in a spherical structure

Contain several secondary structures

Function as enzymes, regulatory proteins, and transport molecules

What is Motif?

Recognizable folding pattern containing 2 or more secondary structures

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Not inherently stable

Domain

Part of polypeptide chain that is independently stable

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If removed from polypeptide, the 3D structure remains the same

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Different domains can have different functions

Unstructured Proteins

Intrinsically Unstructured Proteins →

At least part of protein does not have a specific structure

50% of proteins have at least 1 unstructured region

This portion changes into a specific structure when interacting with another protein - gives flexibility with shape

Important in signaling pathways

Protein Folding

Folded proteins are in their native confirmation

*in it’s functioning state*

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Determined by the primary structure (sequence of AA)

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Not Random

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Some proteins required assisted folding through use of *chaperones*

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Steps:

Secondary structures fold first guided by electrostatic interactions → Local IMF form → Long-range IMF form between the different secondary structures form

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Defects in Protein Folding

Protein Misfolding → 25% of all proteins fold incorrectly

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When misfolded, there are 2 options:

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Degraded →

Which is what’s happening with Cystic Fibrosis

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Accumulation →

What’s happening with Sickle Cell Anemia and Amyloids in Alzheimer’s

Which of the following Amino Acids is normally found in the interior of globular proteins?

*asking about polarity, which one is a non polar*

Valine

Protein Unfolding Through →

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Proteostasis

*Protein Homeostasis*

Coordinated cellular processes that control protein synthesis, folding, refolding, and degradation of proteins

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Autophagy - Cell eating

Protein Unfolding Through →

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Denaturation

Protein no longer functions as it should

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Increases surface area for digestion

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*Does NOT change primary structure*

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Increases digestibility

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Globular proteins are easier to denature than fibrous proteins

Other Protein Unfolding Mechanisms

Heat or Excessive Cold →

Alters H-bonds & hydrophobicity

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Strong Acids/Bases →

Alters net charge, disrupting H-bonds & causing repulsive electrostatic interactions

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Mechanical →

Unfolding protein, exposing hydrophobic groups

Protein Function Overview

Most functions involve reversible binding of a ligand

*ligand can be anything that can bind*

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Proteins are dynamic and flexible

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Reversible Binding

Molecule reversibly bound by a protein is called a ligand

*ligands can be any type of compound, including another protein*

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Interactions are transient

*able to respond quickly and reversibly to changing environment*

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Ligand binds to binding site

*Complementary site on protein that corresponds to size, shape, hydrophobicity of ligand*

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Able to selectively bind

*Proteins may contain multiple binding sites for different molecules*

Protein Binding

Binding of a ligand often is coupled with a confirmational change making it easier for the ligand to bind the binding site

*this structural adaptation is called induced fit*

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Often, binding of a ligand can also cause a confirmational change

*sometimes 1 ligand will bind, the protein changes structure and now a 2nd ligand can bind*

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Reversible Protein Binding

Ka = Association constant

Measure of the affinity for the ligand to bind a protein

Bigger # = more it likes the ligand

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Kd = Dissociation constant

Used more in chemistry as a measure of affinity

Kd = 1/Ka

A measure of how much of a ligand must be in solution for 1/2 of the protein to be bound

Smaller he Kd, the tighter the protein will bind the ligand = Greater affinity!

Oxygen-Binding Proteins

Specialized proteins with a heme ring are called hemeproteins →

Fe can form 6 bonds:

4 N, 1 Histidine, 1 O2

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Examples of Hemeproteins:

Myoglobin - Oxygen Carrier in Muscles

Hemoglobin - Oxygen carrier in blood

Neuroglobin - Oxygen and nitric oxide carrier in neurons

Cytoglobin - Oxygen carrier in many tissues *thought to protect tissues from oxidative stress*

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Myoglobin

Hemeprotein in heart and skeletal muscle

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Oxygen carrier for aerobic metabolism (glycolysis, Kreb’s cycle, electron transport chain)

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Number of polypeptides - 1

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Interior stabilized by hydrophobic interactions

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1 heme = 1 Fe = 1 O2

Red Blood Cells (RBCs)

Aka Erythrocytes (aka hemoglobin)

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Produced from stem cells, called hemocytoblasts

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During maturation, daughter cells produced large amounts of hemoglobin and then cellular organelles lose their function

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RBCs are incomplete and unable to reproduce

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Derive energy from glycolysis (since they lack mitochondria, they can’t form ATP through the electron transport chain and can’t burn fat)

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Lifespan of 120 days

Hemoglobin

Transports O2 from lungs to tissues

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Number of polypeptides = 4

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Can transport CO2 and H+ from tissues to lungs

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4 heme = 4 Fe = 4 O2

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2 Types:

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Oxyhemoglobin →

R state = relaxed

Less electrostatic interactions (ion pairs)

High affinity for binding oxygen

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Deoxyhemoglobin →

T state = Tense (aka taut state)

More electrostatic interactions (ion pairs)

Low affinity for binding oxygen

Oxygen Binding

Degree of saturation of oxygen-binding sites varies from 0% - 100%

Myoglobin can only bind 1, so it is either 0% or 100%

Hemoglobin can cary from 0-100% bound

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Depends on partial pressure of oxygen (pO2)

Partial pressure is a measure of concentration

Oxygen Dissociation Curve

Myoglobin →

Binds O2 at low concentrations found in muscle

Releases O2 in response to oxygen demand

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Hemoglobin →

Cooperative binding = binding O2 at one heme increases affinity for binding O2 at other heme sites

Cooperative Binding of Hemoglobin

Each hemoglobin can bind a max of 4 oxygens → first oxygen binds T site weakly → causes a conformational change in adjacent subunits → Transitions subunit from T to R state -. Increases affinity to bind next oxygen → Causes a conformational change in adjacent subunits → Transitions from T to R state → Increases affinity to bind 3rd oxygen and so on.

Cooperative Binding

Allows Hb to respond to small changes in oxygen concentrations

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Lungs = Oxygen saturates Hb (loads)

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Peripheral Tissues = oxyHb releases oxygen (unloads)

Allosteric Proteins

Allosteric proteins have several possible structural confirmations induced by binding of a type of ligand called a *modulator*

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Conformational changes may make the protein more or less active

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Types of Interactions:

Homotropic → When ligand and modulator are identical

Heterotropic → When ligand and modulator are different compounds

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Hemoglobin as an Allosteric Protein

The binding of 1 ligand (oxygen) affects the affinities of unfilled binding sites

*oxygen is both a ligand and an activating homotropic modulator*

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Hemoglobin has other *inhibiting heterotropic modulators*

CO

CO2

H+

2,3 bisphosphoglycerate

Hemoglobin Transport

CO2 and H+ are both inversely proportional to O2 binding

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Decrease in pH shifts toward deoxyhemoglobin (T)

Increase in pH shifts toward oxyhemoglobin (R)

Bohr Effect

Effect of H+ and CO2 concentration on oxygen binding and releasing → *inversely proportional*

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Peripheral Tissues →

high H+, low pH, and high CO2 concentrations

Hb affinity for oxygen decreases

R to T state

Facilitates O2 unloading

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Lungs →

low H+, higher pH, low CO2 concentrations

Hb affinity for oxygen increases

T to R state

Facilitates oxygen loading

Heterotropic Allosteric Effector (Inhibitor)

2, 3 - bisphosphoglycerate (BPG) →

Intermediate in glycolysis

Found in high concentrations in RBC

Binds to deoxyHb, decreasing affinity for O2 by stabilizing T structure

Inversely related to O2 binding

Important for physiologic adaptation to less oxygen at high altitudes

Carbon Monoxide

Heterorophic! - Activator

Enhances loading

Binds to heme - preferential to O2

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Colorless, odorless, heme has a 250 times greater affinity for CO than for O2

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When CO is bound, it shifts the O2 dissociation curve, not allowing O2 to be released, basically suffocated from the inside out.

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at 50% bound = coma and death.

Hemoglobin Degradation

Cell membrane of RBC becomes fragile and bursts → Hb is released and phagocytized by macrophages in Kupffer cells of liver, spleen, and bone marrow → Iron released and transported in blood by transferrin → Porphyrin ring converted to bilirubin by macrophages

Jaundice

Caused by large concentrations of bilirubin in extracellular fluid

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Causes:

Hemolytic → Excessive destruction of RBC

high levels of free unconjugated bilirubin

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Obstructive → Obstruction of bile duct or liver damage

high levels of conjugated bilirubin

Proteins and Ligands in the Immune System

Most ligands bind to a specific amino acid sequence in an area on the protein called a binding site

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Immune system is important for recognizing foreign compounds

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Immune response to protein are very specific and extremely sensitive

Enzymes - Biological Catalysts

Globular proteins

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Speed up reactions without being consumed

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Do not shift equilibrium, just reach it faster

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Very specific

Apoenzyme

Protein part of enzyme only

Holoenzyme

Complete (active) enzyme

Protein + cofactor/coenzyme

Cofactor vs Coenzyme

Cofactor →

Minerals

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Coenzyme →

Vitamins

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*see chart for more details, some on exam*

Zymogen

Enzyme precursor (inactive) - must be converted to active form

Name Enzymes

Name is based on:

What it reacts with

How it reacts

Add -ase to ending

Not always, but most of the time

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Ex. Lactose is milk sugar, digested by lactase

Alcohol dehygrogenase - enzyme that removed hydrogen from alcohol to form an aldehyde

What are the 7 classes of enzymes?

Oxidoreductase

Transferase

Isomerase

Hydrolase

Lyase

Ligase

Translocase

Oxidoreductase

Addition or removal of a H

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Requires coenzymes (NAD+ and FAD)

Dehydrogenase - removes H

Reductase - adds H

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Ex. Enzymes found in electron transport chain in mitochondria, Kreb’s cycle, AA metabolism

Transferase

2R and 2P

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A-B + C < - > A + B-C

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Transfer a functional group from one molecule to another

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Ex. Krebs cycle, AA metabolism

Isomerase

1R and 1 P

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A-B-C < - > A-C-B

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Rearrange functional groups on one molecule

Hydrolase

A-B + H2O < - > A-H + B-OH

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Causes hydrolysis reactions

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Water breaks bond

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Lipase, protease, phosphatase, amylase

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Ex. Digestive enzymes (enzymes to digest lipids, carbohydrates, porteins, etc.) AA metabolism

Lyase

Cleaves C-C, C-S, or C-N bonds (excluding peptide bonds)

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Ligase

A+B+ATP < - > A-B+ADP+Pi

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Energy released from removing phosphate drives reaction

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Generally need energy from ATP to form bond

Translocase

Movement of molecules or ions across membranes or their separation within membranes

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Ex. cations, anions, amino acids, small peptides, carbs, other compounds

Oxidoreductase

Removal or addition of H or O (NAD+ or FAD)

Transferase

Transfer of group from one molecule to another

Isomerase

Structural rearrangement

Hydrolase

Water breaks bonds

Lyase

Break bond without water or redox

Ligase

Make bond (uses ATP)

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Energy is USED for the reaction, phosphate is NOT transfered

Translocase

Transport across membranes

How enzymes work

Enzymes contain an active site

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React with specific substrate

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Enzymes speed up reaction rates

will not determine how fast the reaction will go

will reach equilibrium faster

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Negative change in free energy (delta G) will determine if the reaction will occur spontaneously - exergonic

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Positive change in free energy requires energy to be added to the system for the reaction to happen - endergonic

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Delta G = 0 at equilibrium

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Catalysts speed up reactions by lowering the activation energy

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In a pathway with multiple steps: Overall rate is determined by the step with the highest activation energy - called the *rate limiting step*

Enzyme Active Sites

Much of the catalytic power of enzymes comes from the free energy released through formation of weak bonds in active sites (IMFs) - called Binding Energy

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Binding Energy also gives the enzyme it’s specificity - the ability to discriminate between substrates

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Weak interactions are optimized at the transition state

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How Substrates Bind

Decrease in entropy to binding an enzyme (keeps substrate in the correct orientation)

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Weak bonds form between E and S, disrupting water layer (desolvation)

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Binding energy from IMF in transition state drives the reaction

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Enzyme undergoes conformational change caused by IMF with S (induced fit)

Enzyme Kinetics

Vo = reaction velocity

Number of reactions catalyzed by enzyme per second

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Vmax = maximum velocity

Enzyme is completely saturated with substrate (100% bound)

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Km = Michaelis constant

affinity for binding

amount of substrate required for 50% of enzyme to be bound (1/2 vmax)

Smaller number = better at binding

\[S\]

1-49% of substrate is bound

\[S\] = Km

50% bound

\[S\]>Km

51-99% bound

\[S\]>>Km

100% bound

How does pH affect enzyme activity?

Each enzyme has optimum pH, depends on pK or R groups

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Extreme pH can lead to denaturation