Biochem Unit 2 before quiz

functions of globular proteins

1. storage of ions and molecules- myoglobin, ferritin

2. transport of ions and moelcules- hemoglobin, seratoinni transporter

3. defense against pathogens- antibodies, cytokines

4. muscle contraction- acid and myosin

5. biological catlysis- lysosome and chymotrypsin

protein function and binding

relies on interactions with other moelcuels

binding is reversible- aka transient because it is not helpful if things are allays covalently linked- ALL REACTIONS ARE REVERSIBLE

ligand

molecule that a protein can bind to in a reversible manner- small molecule

binding site

site on protein where ligand binds- specific

binds via noncovalent forces which are thae same as the ones that dictate protein structure which is what allows interactions to be transient

2 potential methods for ligand binding

1. lock and key model- high specificity explained by complementary of the binding site and the lignad

complimentary in size, shape, charge, and hydrophobicity

• less likely is what happens because that likely would have a rigid interaction and would not be able ot interact with each other for very long

likely true model:

2. induced fit mode- proteins are flexible where both the ligand and protein can change their conformations upon invading, makes binding site more complementary to ligand

• bind tighter because they conformationally change to bind together

myoglobin function and structure

compact globular protein composed of a single polypeptide chain- momoner- 153 amino acids in length

carries and stores oxygen in msucle

contained a heme prostethic group- a perphoryin ring completed with an iron ion (fe2+)

oxygen binds to Mb via the heme

myoglobin structure

has eight alpha-helical regions which are designated A though H which surround the Heme group N-terminus is the A group

the Mb polypeptide chain golds to form a cradle that nestles the heme prosthetic group

• protects the heme iron atom from oxidation

• provides pocket into which the oxygen molecule can fit

two polar His residues are found in the interiior'; they are involved in interactions with the heme group and bound oxygen

heme

perphoryrin ring and iron

myoglobins prosttic group which is tetra dentate with a heme in the middle. without this group, myoglobin cant bind to oxygen

the Fe binds to two more things other than the amine groups on the ring which include a histidine residue-called the proximal histidine residue, and oxygen

the distal histidine

statically inhibits oxygen from binding perpendicularly to the heme plane

oxygen/carbon monoxide binding to heme effect of distal histidine

o2 has a 25,000x weaker affinity for iron but because the distal histidine is interacting with it, it bends the o2. this prevents the carbon oxygen triple bond from binding because it does not want to interact with the distal histidine

• not flexible triple bond

binding affinity

affinity is a measure of how tight a protein P binds to a ligand L. This is also expressed as a dissociation constant, Kd, which is simply an equilibrium constant for the dissociation reacition. Kd has units of M, the lower the value of Kd, the tighter the binding

the value of Kd is always the same for a specific protein-ligand interaction

how to find Kd

when the concentration fo the protein bound to ligand equals the concentration of free protein then P/PL= 1 and the concentration of free ligand equals the Kd. therefore Kd=L at 50% saturaiton

• concentration of ligand when the protein is half saturated

L=kd

L>kd

L<kd

=, half of P will be bound to L and half will be free

L>kd, P will mostly be bound to L

L<kd, P will mostly be bound to L

the ligand binding equation

what is the graphical trend as the Kd gets smaller

the binding curve becomes steeper

what type of grpah is it

hyperbolic

ligand binding equation

myoglobin vs hemoglobin

myoglobin- monomer

o2 storage

1 heme group so It can only old one o2

hemoglobin- heterodimer 2 alpha and 2 beta subints

function- o2 transport

4 heme groups- therefore can carry 4 oxygens

hemoglobin quaternary structure- tense and relaxed

tense state has a huge hole in the middle- T (tense) state deoxyHb- no O2 bound- is more stable (which is how o2 is able to dissociate)- lower affinity for o2- more interactins, more stable

relaxed state- R state- oxyHb- o2 bound- less stable- also almost no hole in the middle- higher affinity for 02- fewer interactions, more flexible

o2 binding triggers what conformational change

from T→R

conformational change from teh T state to the R state involved breaking ion pairs between teh Alpha 1 and beta 2 interface

as soon as hemoglobin binds to one oxygen, it wants to bind to al. of them

T versus R state conformation perforin ring

t state, perforiyrin ring is puckered. it is not in the R state so it pulls the histadine towards it which pulls the alpha helix its connected to closer which causes an overall structural change which is called cooperativity because it increases the interaction now between the rest of the hemes and oxygen

cooperatively

• if the binding of one ligand molecule affects the affinity of another ligand molecule on teh same rotein

• multiple subunits usually required

• positive cooperatively occurs when the binding of a ligand increases the binding affinity of subsequent ligands

• negative cooperatively occurs when the binding of a ligand decreases the bidning affinity of subsequent igand

• Hb subunits exhibit positive cooperatively upon binding oxygen

two cooperatively models

concerted vs sequentiall

concerted is that all the subunits are either all tense or all relaxed but ligand bonding still occurs one by one

sequential- more likely- turns into a relaxed state as ligands bind

Mb binding curve- one-site binding

fraction saturation on teh side

Mb0 hypersonic binding curve for o2- insensitive to small chanfges in oxygen concentration. Mb cannot bind to O2 in a cooperative manner why? Bec cues theres only one binding site and one subunit (monomer)

the binding curves for hemoglobin and myoglobin global are consideredly more complaated due to multiple binding sites

Mb vs Hb binding curbe

Mb- hyperbolic- binding curve for O2

Hb- sigmoidal binding curve for O2- reflects a transition from low to high affinity binding this makes Hb highly sensitive to changes in oxygen concentration

sigmoidal curve purpose

has to have a low affinity in tissues and a high affinity in lungs so it can pick up the O2 at lungs (13 kpa) and dispers it in tissues(4kpa)

ligand interactions can be regulated

reversible binding of ligands is essentail for regulation

binding of ligands other than oxygen affects oxygen-binding properties of hemoglobin

protons, carbon monoxide, carbon dioxide, chloride ions, and BPG all affect the binding of oxygen by Hb

allosteric proteins w multiple binding site son the same protein

• umbrella term which includes cooperativity

• how binding of one ligand effects binding of another ligand to a different inning site on the same protein

• does not always require multiple subunits

poistive- binding of one ligand increases affinity for the other

negative- binding of one ligand decreases the affinity for the other

bohr effect

shows how pH difference affects affinity

can be a negative or positive effector of an enzyme

for Hb, It is an antagonisht- negative effector and it decreases oxygens ability to hold on to hemoglobin in tissues- makes it give up the oxygen in tissues because you produce lactic acid in the body when you work out so you need more oxygen

2,3-bisphosphogycerate (BPG) is a negative allosteric effector of O2 binding

BPG binds to Hb and promotes oxygen release

• does this by cross linking the two beta subunits which stabilizes it in the deoxygenated form (T-state)

• in oxyHb, the central cavity is too small for BPG to fit

• BPG shifts the oxygen saturation curve of Hb to the right, allow it to release oxygen to tissues

carbon monoxide is a positive effector

CO-colorless, odorless gas

250-fold greater affinity for Hb than O2

healthy person- 1% CO-Hb

smoker- 3-8%

china smocker- 15%

greater risk- smoker, heart/lung/blood disease/ fetus

sickle cell anemia

replacement of Glu residue at position 6 in the beta chain by Val marks the only chemical difference between Hb A and Hb S

a hydrophobic pocket forms in teh corner of each beta chain when it is in the deoxy state

these pockets nicely accommodate the val side chain of neighboring Hb S molecules leading ot aggregation into long chain polymeric structures

Hb S is less soluble than Hb A

insoluble Hb S distort red cell into elongated sickle shape

cooperatively- Kd

cooperative proteins have multiplee ligand bindings sites so the Kd becomes

the hill plot of cooperatively

key topics in protein function

reversible binding of ligands is essential

specificity of ligands and binding sites- high specificity- only certain ligands bind

ligand binding is often coupled to conformational changes, sometimes quite dramatic- induced fit

in multi-subunit proteins, conformational changes in one subunit can affect the others- cooperatitvity

interactions can be regulated

the bodies immune defenses

1. innate defense

• what you are born with, do not have to be exposed to a pathogen to gain

• external and internal innate defenses

  • external- skin, secretions, mucuou

  • internal- phagocytic cells

  • naturak killer cells

  • defensiveproteins

  • inflammatory response

2. adaptive defense

• antibodies

• lyphocytes

  • b cells

  • t cells

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immune response

macrophage will endocytose a pathogen with a specific antigen, it will then show the antigen on its surface, and the B cell will interact with it and produce antibodies for it. the T cell then interacts with the macrophage and B cell with said antigen and targets those cells for death

memory cells

recognize different antigens through antigen receptor on cell surface.

these cells will either become memory cells which maintain antigen receptor or they will become plasma cells where they release the antibody

antigens and antibodies

antigens- foreign molecule that stimulate production fo antibodies- proteins or large polysaccharides on the surfaces of biruses or freight cells

antibodies- protiens produced by B cells that bind specifically to antigens

• binding of antibodies to antigens will make the antigen for destruction or interfere with its function

• a given antibody will bind to a small region of the antigen

• one antigen can have several epitopes

• epitopes recognized by antibody- unique shape- 5-10 amino acid sequences

• one again may have more than one antibody against it, etch antibody regonizeing a unique amino acid sequence

antibodies structure and function

y shaped structure

made of four peptide cains

• two identical light chains, and two identical heavy chaiins

• help to gather by disulfide bonds

n- termini of light and heavy come together to form antgen binding site (unique antino amongds/epitope bind here)

structural bases of antibody diversity

immunoglobulins are divided into 5 isotopes: IgA, IgD, IgE, IgG, IgM. they differentiate in structure in the constant region and have different effector functions

IgG is the most abundant antibody in blood and lymph and I used as the prototype in illustrating antibody structure

typical immune response

following exposure to a foreign antigen, the immune sytem produces antibodies directed against the diseased antigens

body first produced antibody called immunoglobulin M (IgM)

• within a week of ideates exposure IgM can be detected in blood- approximately three weeks later, IgM levels drop and IgG levels rise and then fall within a few days

the second time teh body is exposed to the same disease, the immune response is stronger and faster than the first time

this is the principal behind vaccinationand booster shops

antibgens bind via induced fit

antigen binding auses significat structural changes to the antibody

Uses of antibodies in biochemistry

applications:

• westernblotting protein identification following SDS-PAGE

• immunoprecipitation- isolation of specific protiens and binding partners

• immunoflouresence microscopy- localizatin of specific proteins in cells

• ELIZA- detection of protiens in a sample

muscle proteins

msucle fiber0 large, single, elongated multinuclear cell

each fiber contains about 1,00 myofibrils which is one cell

actomyosin cycle

enzymes

systems cannot increase temperature without risking damaging structures so they use catalyssits- cah[nge the rate of a reaction without net change of catalyst, increase reationr ates without being consumed and without altering equalbrium

catalysts increase reactionaries without… (2 things)

without being consuemed

without altering equalbrium

enzyme

a substance produced by a living organism that acts as a catalyst to bring about a specie biochemical reaction; present at low concentrations

vast majority of biological catalysts

globular proteins

some RNA also catalyze reactions- ribbonymes

definition of : substratecte, product, active site

substrate- substance acted upon

product- resulting species

active site- location in enzyme where reaction occurs (extremely specific for substrates- but nonspecific for product usually)

enzymes increase reaction rate

can increase up to 10^19 times faster than the uncataluzed reaction

enzyme activity

many enzymes can actin the forward and reverse reactions

specific interactions in active site determine binding affinity- shape, charge, hydrophobic

enzyme activity is regulated by

• pH- can change structure by changing charges- likely in active site

• temperature- can increase reactionr ate

• concnetration of enzyme

• concentration of sybstrate

• concnettartion of cofactors or coenzymes

last 3 re more likely to regulate in vitro

cofactors

include coenzymes - organic molecules

• cosubstrate- bound more loosely- consumed within reaction

• proseteic groups- tightly bound, not consumed

Inorganic moleucles

• 1/3 of all known enzymes require metal ions- metalloenzymes

• usually positively charged

app vs holo enzyme

apo is inactive

holo is active and is an app enzyme with a cofactor or coenzyme

activation energy (free energy of activation)

chemical reaction occurs when colliding molecules possess a minimum amount of energy caled the activation energy

activation energy is the difference between energy levels of the ground state and the transition state

the rate of a reaction reflects this activation energy

• a higher free energy of activation corresponds to a slower paction

free energy change

the amoutn of energy available to do work

each compound in a chemical reaction contains a certain amount of potential energy related to the kind and number of its bonds, in reactions that occur spontaneously, the products have less free energy than teh reactions. thus the reaction releases free energy, which is then available to do work

many reactions that are spontaneous (neg. dG) will proceed at slow rates because they do not have the energy or correct orientation

• the rate at which a spontaneous reaction occurs iimporives with increasing the temperature or using a ctalyst

spontaneous if free energy changes is negative

free energy

dG predicts the direction that a reaction will proceed

• negatie dG indicates spontenaity and exergonic - rxn will go forward

• positive d G indicates nonsponteanous nad endergonic- rxn will go backward

• when dG is zero, at equilibrium

Is dG changed by an enzyme

not the Gibbs free energy, but the free energy of activation is changed where the activation decreases but the still total amoutn of energy release will still be the same

dg is the difference in energy between substrate and product it is not changed by an enzyme

what does change ins the free energy difference between substrate and the transition state

how? enzymes are flexible

how do enzymes increase rates

a catalystt reduces the activation energy of a reaction

the free energy of activation is the amoutn of energy needed to convert 1 molecule fo substrate from the ground state to the transition state

complementarity of active site- stickase

no enzyme- reaction will eventually happne

enzyme complementary to substtate- lock and key method

enzyme complementary to transition state- induced fit model

general acid-base catalysis

general acid- partial proton transfer from an acid lowers free energy of the reactions transition state

general base- partial proton abstraction by a base lowers free energy of a reactions transition state

covalent catalysis

covalent bond forms between enzymeand substreate

• a transient covalent bond between enzyme and substrate

• rchanges reaction pathway- reaction mechaism

requires a nucleophile on the enzyme

• can be a reactive hydroyl, thiol, amine, or carboxyl

metal ion catalysis

involved a metal ion bound to the enzyme- metal ion interacts with substrate to facilitate binding

what metal ions can do:

• help oritein substrate for reaction

• stabilize negative chanrges

• medicate oxidation-reduction eactions

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chymotrypsin

small intestine enzyme which breaks down free protiens

protease (protein-ases)

during digestion, dietary proteins must be broken into small peptides by proteases

chymotrypsin is one of several proteases that cuts peptides at specific locations ont he ppttide backbones

• likely has aromatic binding site because only cleaves next to aromatics

• this protease cleave the peptide bond adjacent to (c-terminal to) aromatic amino acids

chymotrypsin is a ____ protease and requires a ____ _____

serine protease; catalytic triad

what amino acids are apart of the catalytic triad

histidine, aspartic acid, and serine

oligeomerization

heterotrimer; alpha beta and c chain

asp, his, see, and gly make up internal binding pocket

covalent catalysis using chymotrypsin; steps

1. bind subtrate

2. form covalent bond

3. cleave peptide bond

4. release 1s product (c-terminal)

5. break covalent bond

6. release product 2 (n-terminus)

enzyme is unchanged when we finish it

chymotrypsin mechanism

serine residue hydrogen bound to the histidine residue with a nonbonding but able to hydrogen bond glycine residue.

substrate then enters and aromatic ring will bind to hydrophobic pocket

when bound to pocket, c-terminal =o and hydrogen on serine is h-bonding with the nitrogen on histidine

step 2

histidine takes serines proton, serines now negatively charged oxygen is now a nucleophile and attacks the carboxyl carbon which causes the =o to become a —o-

this —o- can now go back to being a =o which kicks off teh c-terminal peptide, giving the extra electrons to hydrogen

step 3

c terminus exits and n terminus is still hydrogen bound to to serine residue still

step 4

water comes in and the nitrogen on the histidine attacks the hydrogen on the water and then the hydroxide attacks the carboxyl carbon which then forms a —o- bond which can now h-bond with the glycine’s backbone. t

step 5

distance between nitrogen and hydrogen is now too far, their bond breaks because serine wants it back. serine’s O- bond with carbon also breaks because the —o- can go back to forming the =o

step 6

the product can now dissociate

six classes of enzymes

1. oxidoreductases

2. transferases

3. hdyrolases

4. lyases “synthases”

5. isomerases

6. ligases “synthetases”

oxidoreductases

catalyze electron transfer in redox reactions

transferases

transfer a funcitonal group from one molecule to another

hydrolases

cause cleavage of a bond using water- water receives the functional group being transferred- known as hydrolysis

lyases “synthases”

cleave or form a bond between two molecules

isomerases

move groups within a molecule (intramolecular rearrangement)

what types of co-substrates are commonly present in enzymatic reactions

water, ATP, and NADh (e- accept or donator)

enzymes are used in cellular pathways but why isnt the rection compelted in jsut one step? why so many intermediates?

each reaction releases energy becaues it is exothermic and favorable. therefore, cannot happen all at once because cells cannot tolerate large amounts of energy or heat being released. they cannot generate a large energy burst to drive energy-requiring processes.

instead, transformations take place via sequential series of chemical reactions whose overall affect achieves dramatic energy changes, even though any given reaction in teh series proceeds with only modest input or output of energy

enzyme clinical relevance

enzymes are used clinically in three principal ways

1. diagnosis and prognosis- measure enzyme activity

2. measurement of substances (cholesterol, glucose, hormones)

3. therepautics