BMB 401- PART II PROTEINS AND ENZYMES

2.1 AMINO ACIDS

a. Nomenclature
Proteins are polymers made up of amino acids.
20 aa- standard aa or alpha aa
standard aa share structure but have different side chains- R group
linked together to generate a polypeptide chain via peptide or amino bonds
some aa side chains have ionizable groups.
Ionizable- can lose or gain a proton.
General structure
R group is variable. Rest in constant
Alpha carbon
Proline- has a secondary amino group and a cooh.
3 letter code. Proteins is single letter. Know 3 letter, single letter.
3 major classes: apolar R, polar R. charged residues.
N terminal nh2 is free, C terminal- cooh free.
1.
-

Apolar residues
Is non polar.
Glycine- H
Alanine- methyl group
Valine- isopropyl
Leucine- isobutyl (iso is where butyl, etc is attached)
Isoleucine- sec-butyl
Methionine s-methylethyl
Proline- pyrrolidine
Phenylalanine- benzyl (phenyl)
Trypophan- indolylmethyl (indole)

2.
-

Polar residues
Molecules that have a polarized bond.
Charge separation- dipole in their R group.
Serine- hydroxymethyl
Threonine- hydroxyethyl
Asparagine- amidomethyl
Glutamine- amidoethyl
Tyrosine- hydroxybenzyl
Cysteine- sulfhydrylmethyl

Pkr- refers to the side chain pka.

Above a pka above 10.5 these will be hydrolyzed.

Understand to be given a value and see where the reaction will go.
Above 10.5 will ionize bc at ph 10.5 will be 50/50 percent. 50% oh and
50% o- . above ph 10.5, more will be o-. will have more protons.
at ph 10 (go down) then will have more protons, will push rxn backwardswill push o- to neutral form.
3. Charged residues
- Lysine
- Arginine- has guanidine group attached. Only aa with guanidine. Bridge
is propyl.
- Histidine- has imidazole functional group. Is attached to the amino acid
backbone.
- Aspartic acid- carboxyl r group
- Glutamic acid. carboxyl r group.
- Given pka is 6 or 8. Which way rxn goes.
- Basic residues- accept a proton. At 10.5 50% protonated 50% neutral.
Ph 7.4/7.5 is predominantly one state- is protonated because is going
down. Now have enough protons will push it to the right.
- Acidic acid go to acetate plus a proton. Ph4- is 50/50, at ph 2 will be
protonated all acid. At ph7 will be predominately in the unionic form.
- Histidine- only basic or neutral- never acidic. Ph 6- 50/50 percent
protonated and neutral. Phh 7.4- will be neutral, below ph 6 is
protonated.
- Ph<pka- r group is protonated.
Dipolar amino acids- Zwitterions
- Pka of amino and carboxylic groups are 9 and 2.
Nomenclature- Alpha amino, alpha carboxyl.
- Greek alphabet extends.
- Ex. Epsilon amino group was replaced.
- Two bonds- peptide and disulfide bonds.
Peptide linkage- condensation
- When you conjugate things the nomenclature changes.
b. Stereochemistry
- All amino acids are L
- Most sugars are D
Polarized light
- Is perpendicular and is in phase.
- Light has 2 electromagnetic wave. B is always perpendicular
- E can oscillate in all direction- is nonpolarized.
- E can be made to oscillate vertically or horizontally or elliptically.
- Polarization of light refers to the direction of oscillation of E.

c. Derivatives
- Altering PTM can cause an entire protein to change. Can make it active
inactive, etc.
- PTMs- phosphorylation, methylation, acetylation.
- Are reversible.
- Are molecular switches to alter and modulate protein function.
- Free aa can also be modified.
- 1. Serine- phosphorylated derivative
- 2. Glutamate- carboxylated derivative.
- 3. Proline- hydroxylated
- 4. Histidine- methylated. After methylated the histidine is neutralized,
side chain cant be protonated anymore.
- 5. Lysine- is acetylated. Acetylation neutralizes or reduces the charge.
-

Proteins are colorless- made up of white light.

Green fluorescent protein- stick a carboxyl group to amino, kick water

out, cyclizes. Delocalized, conjugated pi bond. Electrical properties
conjugate visible light. All lights are absorbed except for green (510)

Neurotransmitters
Decarboxylated glutamate
Histamine- carboxylate missing
Dopamine
Tyrosine

2.2 Protein sequence

a. Protein Diversity
-

Protein is a biopolymer that is comprised of aa residues convalently

linked via repetitive peptide bonds
Size and composition of polypeptide chain is unlimited
Diversity is limited by
Primary structure
o Proteins can be unitary- one chain that can fold together.
o Many proteins are multi-polypeptides.
o Protein can be comprised of more than one polypepide.
o Insulin- two chain protein. Is disulfide linkages or bridges that
hold it together.
o Inter chains- hold chains together
o Intra- within the same chain
o Multisubunit proteins dont have to be covalently linked.
o Alpha and beta chains

2.2b
-

Diversity of protein size

o Size can vary.
o Are many peptides that regulate metabolism etc.
o Most proteins are 100-1000 amino acids
o Cyt c is a small proteins.
o 100 small
o 200-500 medium
o 500 plus is large
o above 1000 mega
o by looking at size can estimate quickly.
o Aa average molar mass is 110g/mol
o Proteins are usually in KD.
o Larger residue chain gets the more accurate the guess gets.

Protein purification
work with proteins in isolation
want to purify it and isolate it so no other proteins are with it
need to purify proteins in order to understand their biophysical and
biochem properties- thermos, kinetics, catalysis, structure.
- Want to know the structure of proteins- if we knew what the protein
looked like then can infer its function- not true.
- Look at protein unique structure and chemistry in order to separate it
from other molecules
- Ph temp and pressure is important. These affect protein function and
stability
- Isoelectric point- pI is simply the pH at which a given protein carries
no net charge. Need to know this or else protein will precipitate out. If
no net charge then protein will precipitate out and not solubilize.
Isoelectric point
- Dont need to know pIs
- Ex. Myoglobin or hemoglobin wont have charge at neutral pH 7. Equal
number means they have a equal number of positive and negative
residues.
- Net charge is sensitive to pH.
- Proton concentration changes- the equilibrium shifts. Change pH =
change its state.
- pI is the pKi (only refers to the charged aa- ionizable ones)
- N= number of charged residues.
- Add up pKis and divide by N.
- Protein purification should be done away from the pI or else it will
precipitate protein- enzymes will become inactive. Have to know pI of
protein from its aa sequence.
- If purifying myoglobin or hemoglobin want to work away from 7. Want
to work ideally above 8 or below 6.

Salting out
- Outdated method
- Precipitates proteins
- Generally dont want to do this.
- Salt ions compete with protein molecules for bulk solvent.
- Add salt and make protein come out.
- Salt interact with water and will compete with the protein for the water.
- Crash- coming out of solution.
- Protein is colorless liquid- that means protein is soluble. Proteins can
be membrane or water soluble. All about water soluble. This section.
- Water sol- proteins in the cell, hemoglobin, myoglobin etc. not
membrane proteins like gpcrs.
- Increase salt concentration- salt competes with protein for water- will
therefore have a high concentration- not enough solute available to
compete. Salt will come in and push protein out.
- Want to be right at the spot of pI- is where the protein is least soluble.
Proteins least soluble at their pI.
- Most common used salt is ammonium sulfate- is KOSMOTROPHIC.
(dont need to know) has a high affinity for water and can take away a
lot of water.
- Makes a hydration shell- bigger hydration shell means it can take away
more water.
- Protein in solution- yellow protein is the one we want.
- Different proteins have different solubilities.
- Green more soluble than purple.
- Solution has no salt. Will spin out, least soluble protein will sediment to
the bottom by centrifugal force.
- Increase the salt concentration, remove purple, next least soluble
protein will sediment. Will come out as precipitate. Then only yellow
protein remains.
- Exploiting out solubility
Ion exchange chromatography IEC
- Are exploiting the charge, proteins can be negative, neutral and
positive depending on pH.
- Is ion exchange
- Ion exchange column. Column is attached to exchangers. If protein is
negative- charge protein with DEAE. Protein is covalently bonded to
this. Anion exchanger.
- Protein is positive use CM- cation exchanger, will bind cations.
- Our protein is negative, have column with DEAE- anion exchanger.
Have solution with a bunch of proteins
- Apply to column, will run through. Proteins that are neutral or positive
will run through- charge isnt compatible so will not stick. Your protein
has neg charge so has high affinity and will bind and have electrostatic
interactions.

Size
-

Add salt (nacl) cl ions will bind to thing, na ions will bind to the proteintarget protein can now come off the column.
Exploiting net charge of protein and column is charged, your proteins
will be selective.

exclusion chromatography
Exploiting out size of protein as well as its conformation (shape)
Proteins can be round, globular
Apply protein to a column- use a gel with small tiny pores.
Is a filter- molecular sieve. Has a proe matrix with tiny pores. Protein
mixture has different sizes. Small proteins enter the pore, large
proteins dont enter.
Smaller they are- more likely to enter.
As it goes through- small molecules will enter the gell. Larger ones will
bypass.
Small- enter gel matrix, will slow down.
Larger molecules will run through faster because they werent impeded
Small- motion down the column isnt impeded.
Two proteins can be same number of residues- globular protein- may
enter column, rod may not enter column
Smaller impeded
Larger come out faster.

Affinity chromatography
- Have a ligand that has an affinity for your protein of interest.
- Ligands can bind to proteins- can engineer proteins.
- Clone the protein= express in bacteria outside of body.
- Clone the protein with a Gst tag onto the protein= are called fusion
proteins- your protein plus a tag. Marker can be a gst tag or a histidine
tag (6 residues)
- Gst- is a protein- if protein is not soluble will enhance the solubility of
the protein- gst is very soluble and stable and will keep the protein in
the solution. However protein is tagged so can affect its behavior.
- If you do gst tag- charge the column with glu.
- Immobilize ligand onto column
- Bacterioize column = apply impure protein onto column- protein will
bind to the gst. All other proteins will just go thru.
- If have histidine- charge column with nickle- his tag can coordinate
with nickle ions. Strong affinity rxn. All will go thru. After go thru make
sure protein come off column
- Gluthatione if use gst to get protein off column
- Imidazole- allows ur protein to come off column if use his.

Quality control
- Purity and yield
- Purity- visually analyze- use SDS page- gives you an idea if there are
any contaminants
- Sds denatures protein- use tiny bit of purified protein- denature it.
- Look at protein concentration
Sds concentration
- Electrophoresis- charged molecules migrate in a fluid medium like gel
under the influence of electric field.
- PAGE- trying to separate proteins on the basis of their mass.
- Charge and shape arent depended on molar mass of protein. Get no
resolution- two molecules can migrate the same if they are similar.
- Separating molecules on basis of size with PAGE.
- Electrophoresis- highly charged.
- Coat protein with sds- will denature them and make them all rods. All
proteins small and large will have identical q/m ratios. Since they can
bind more SDS- no matter size of protein. Cant separate proteins bc
have same q/m ratio in sds page.
Gel contribution
- All the proteins get same force and same weight.
- If bigger- get more drag- is impeded- more slower. Gel separates them.
- Separate out bc the bigger proteins cannot move. So big bc the gel
impedes their movement. Tiny proteins will run thru the gel.
Beer lambert law- measure protein concentration. Can calculate or have
epsilon value
a- Measure.
Can calculate concentration.
Units.
2.2c Protein sequencing
- First protein in 1955
- Make larger protein to smaller fragments- make them overlapping
fragments
- Step 3-5 for all proteins
1. Identify n terminal residues
a. Only for multisubunit protein
b. React polypeptide chain with dansyl cl
c. Breaks residue
d. N terminal is a derivative
e. Purifcy with hpl- is hydrophobic. Different molecules have
different rxns.
2. Separate subunits
a. Ex. Insulin , disulfide bonds- most common lab agent is
reducing agent.

b. Reduces disulfide- get two individual peptides

c. Make sure that they dont link again by adding iodoacetate.
3. Overlap fragments
a. Cut the protein into small individual fragments.
b. Use other peptidases
c. Trypsin- bond that will be cut is scissle bond- ly or arg bond.
Trypsin will cut this. Proline- causes a kink and sterically
hinders- prevents enzyme action.
d. Elastase- small residues- cant be proline.
e. Cut at different points. Get overlapping fragments.
f. Cyanogen bromide- only works on met. Causes cyclization of
met. C terminus of met- cyanogen bromide will cleave it on c
terminus of met.
4. Sequencing fragments
a. Subject the protein. Treat with
Looking at fragments, can look at mass and tell what kind of
sequence it may be
Cant distinguish leu/ile or glyn/lys

2.4 Protein function

a. Oxygen transport
-

Hb is a oxygen
Mb serves as a reservoir- stores oxy.
M has a hyperbolic o binding curve.
H binds in a sigmoidal o2 binding curve and serves its function
Ph and bpg- these two affect the ability of h to bind oxygen.
Allosteric= change the conformation of hemoglobin.

O needed for respiration. Many other organisms can rely on direct

diffusion from the environment

Hemoglobin is a tetramer
Myoglobin is predominately in the muscle
Plays a role to store o2 and to facilitate the release of hemoglobin
Will snap o2 off hemoglobin and will help it relay to tissues
Function of both is related to a cofactor- heme.
Prosthetic group- a specific type of cofactor.

- myoglobin is a single chain

- hemoglobin is composed of two chains
- closely look at them- are evolutionarily related.

- share very little identity or similarity in amino acid sequence. Structures are
really similar
- proteins that have little in common can have same form
- structures look like alpha form with different helices- a through h.
- stretch them out- helices are a to 8.
- Bring back together and fold it- tertiary structure
- Ab is a customary can be called alpha or beta helix.
- Myoglobin is a single polypeptide- is monomeric
- Have 4 of them- 2 alpha and two beta- is important to hemoglobins
function.
- Macrocyclic ring- dont have extra moieties- have 4 pyrol groups.
- Proferin center of heme lies iron.
- Histidine from helix f.
- Helix is a-h in hemoglobin.
- Proximal face- side pointing away from where the oxygen binds.
Myoglobin
- In addition to oxygen , other gases can compete with oxygen and can
bind to myoglobin. Can deprive o2 from binding.
- Allosteric- when something binds to a protein- binding on one side
causes a conformational change on the other side.
- Allosteric- proteins when u look at their structures- are very flexible.
Ligand binding causes a conformational change.
- Is advantageous to proteins because dont have a pocket for binding
ligand.
- Dont have final shape fully formed until they see the ligand in order to
fit.
- Can go conformational change rather than before binding.
- Allostery key role in binding of o2 to myoglobin.
- K is concentration of two reactants divided by the products.
- Myoglobin bound to oxygen- is equal to the product.
- Concentration divided by k.
- At equilibrium- concentration of free myoglobin is equal to bound- is
concentration of the ligand where protein is concentrated.
- Less we need, the stronger.
- In equilibrium- the lower the value the better because need less
concentration.
- Y is the amount oxygen bound to the myoglobin.
- Myoglobin can only be free or bound.
- Oxygen is a gas- express in partial pressure.
- At higher concentrations the curve flattens out.
- Curve has typical hyperbolic response- no oxygen
Hyperbolic curve.
- Curve flattens at around 10 torr.

Equilibrium constant- value of ligand concentration at half

saturation
- Ex. 50% concentration- get 3 torr.
- Is a strong binder
- Lower the k, higher the binding affinity.
- 3 torr- is fully saturated at physiological conditions.
Hemoglobin
- is a TETRAMER
- Image is a monomer for simplicity- Heme in center
- Phi8 at the proximal face
- Epsilon 7 at the distal face
- Deoxy hb and oxy hb
- Deoxy conformation0 slightly nonplanar.
- fe 2 atom in pulled out of plane because of interaction and
repulsion due to valence electrons.
- Will absorb like in 500-600nm- gives red color.
Oxygenated conformation
- Oxygenated version absorbs light at 600. Arterial blood appear
bright red.
- As the o2 pulls the f2 atom to the center- this draws the histidine
residue upward, causes a conformational change of the protein
- Oxygen can sneak in. this triggers the neighboring molecules.
Will trigger the neighbors to adopt a conformation. When o2
binds to one monomer- it triggers the other two to open and be
ready to get o2. Is cooperative, binding to one monomer
facilitates binding to other ones. Get a sigmoidal curve.
- Deoxy conformation doesnt bind oxygen easily.
-

Oxy hb is bright red

Deoxy is dark red.
In oxy hb- largely absorbs below 600.

Venous blood is blue

White light has a bunch of colors
Light from the sun is scattered.
Rayleigh scattering- has to do with scattering between small
particles- proteins scattering visible light. Proteins are much
smaller than the wavelength of light. Used when particles are
much smaller than the wavelength.
White light from sun is scattered.
Scattering intensity is reversely proportional to 1/lambda4
Shorter wavelength is scattered more than longer wavelength.
Light from sun- sky is blue because blue scattered more than the
red. Same way venous blood is blue. Blue and red component is
scattered- blue component scatters more.

Tetramer- n molecules bind of oxy

Hill coefficient- gives degree of cooperativity.
Equilibrium dissociation constant- K is for the entire hemoglobin.
Different monomers have different binding affinities.
N becomes the exponent of the k.
Amount of oxygen bound to hemoglobin.
If n is greater than 1= then get a sigmoidal curve. Will flat out at
high concentrations of oxygen.
Binds rapidly and flattens out. If hemoglobin were to bind oxygen
in a cooperative manner.
3 torr- for myoglobin.
Carbon monoxide binds to hemoglobin more stronger- is toxic.
K is equal to the venous blood.
Sigmoidal- allows hemoglobin to offload more oxygen than is
needed- if deprived of oxygen- is 20% saturated. Can offload
80% of oxygen. Makes hemoglobin a better carrier.
Sigmoidal behavior- can pick up more oxy where its needed in
the lungs and drop off to the tissues - sigmoidal will do it better
than hyperbolic. Myoglobin is a reservoir- want to keep the
oxygen, dont want to offload so want a hyperbolic curve.
Phase a is below 1. Is equal to 10 torr.
N=1 there is no cooperativity. In the beginning, hemoglobin binds
the oxygen in a noncooperative manner- this is when the first
monomer binds- has no help in binding.
C is also non cooperative binding- last monomer binding. Is
struggling to bind oxygen.
In phase b- is cooperative. Is fast binding and bigger than 1. N is
around 3.
Center has the cooperativity
Last one has no cooperativity because other 3 are already
bound. No help in binding.
In proteins, binding of a ligand.
Square- is deoxy,
Circle is conformation hemoglobin adopts when introduce
oxygen- will drive the equilibrium this way- is called the
equilibrium shift model. Is preformed.
In the deoxy conformation- many residues engage in iron pairs
and h binding context.
Asp94- iron pairing or salt bridging with histidine.
Histidine will not be protonated, eq shift model. Tiny fraction is
protonated. If you can find a partner that stabilizes you, the
equilibrium can shift.
Oxygen binding will break this interaction and a proton is
released. Protonation of hb will mitigate or reduce its affinity for

oxygen. If introduce more protons then will reduce affinity for o2

called bohr effect.
Pushing hemoglobin and facilitating release of oxygen from
hemoglobin- lower ph.
Will shift equilibrium. Want to kick the protons out.

2.5 Enzyme catalysis

differences
Higher reaction rates
Enzymatic catalyzed reactions are under mild conditions, and close to
neutral ph.
Enzymes have a high substrate specificity.
Allosteric modulators- modulate enzyme activity. Like phosphorylation
activate or deactivate.
Oxidoreductase- redox,
Transferases- transfer of functional groups- ex. Hexokinase- glycolysis.
Hydrolasecleave
bonds
with
watervhr
phosphatase.
Dephosphorylate erk kinases
Lyases- forms double bonds by elimination to generate double bonds.
Isomerase- phosphoglucose isomerase Ligases- ligates individual molecules together to form larger molecules.
Geometric specificity
Concerned with the shape and the aa functional groups lining the
interacting surfaces of both the enzyme and the substrate.
Stereospecificity- is prochiral- is chiral in one reaction. Will only bind in
a specific manner or orientation.
Chymotrypsin can break the bond in peptide and ester
Cofactors- two categories- metal ions and coenzymes
o Proteins without cofactor- apoprotein, apoenzyme
o With cofactor- holoprotein, holoenzyme
Metal ions
o Protein kinase phosphorylates substrate
Hexokinase.
Needs magnesium ion to help stabilize the binding of atp.
Is needed for both stabilizing and neutralizing charge
Many other ions like copper, mg, zn are common enzymes.
Most kinases if not all need mg cofactor.
Ions are strongly bound noncovalently- are an integral part
of the enzyme.
Without ions wont fold properly.
Coenzymes
o Can be cosubstrates or prosthetic groups.

Cosubstrates serve as a substrate- have a transient interaction

Step on the enzyme nd then step off.
Enzyme only needs them when it is conduction and reation
Prosthetic groups
Type of coenzyme
Are permanently attached and integral part of the enzyme
Amino group of a lysine with a carboxyl group.
Heme group is a prosthetic group on hemoglobin- is bound.
o Prosthetic group on one enzyme can be a cosubstrate for another
enzyme.
Cosubstrates
o NAD+
o Oxidation of the oh group to the aldehyde.
o When oxidize, get more e. will receive the electrons- will become
reduced and accept electrons.
o Cosubstrate- will accept electrons.
o Nad- nicotinamide, adenine, dinucleotide- is two nucleotides.
o NADP- add phosphate group.
Prosthetic groups
o Accepts electrons- SDH
o Becomes reduced and can be regenerated by coq.
o FMNo
o
o
o

2.5b

Reactions grow through an energy barrier

Can have a transition state.
Enzymes try to reduce the activation energy of the transition state
Will not change the overall thermodynamics.
Allows us to compare reaction kinetics.
Try to inhibit the transition state
Thermal energy the molecules need
Arrhenious equation lowering the delta g will make the reaction go
faster
K = equilibrium constant
Small k = rate of reaction
Lower the free energy- difference between is delta g1 minus delta g2.

2.5c Catalytic mechanisms

Enzyme accelerate rxns by bringing reacting groups together and
make them ready for the rxn and stabilizing transition state.
Active sites of enzymes have charged and polar residues.
Histidine
o Mediates acid base catalysis
o Pka value is close to neutral so can be an acid or a base

o Asp/glu acidic residues can donate a proton.

Ser/cys/tyr act in mediating nucleophilic catalysis.
Arg/lys- substrate stabilization.
Active sites can harbor charged and polar residues. These can be used
as nucleophiles.
8.5- 50% protonated, 50- negatively charged. Ph7- will predominantly
be in that conformation
many residues in enzymes have their pk values altered so they can
participate.

Acid base catalysis- acid catalysis

acid is a proton donor
proton is usually donated to the substrate. Makes a carbanion like
transition state by lowering its activation energy. Ts decays into the
enol form by releasing one of the methyl protons to the bulk water.
Acid is regenerated by taking up a proton from water.
Base catalysis
o Proton acceptor
o Proton is usually stripped from the substrate. Will generate a ts
by lowering its activation energy by acid catalysis. Ts decays into
the enol form by virtue of the ability of the carbonyl group to
take up a proton from the bulk water.
o Conjugate acid then donates a proton to water.
Rnase
o Two residues- are his residues.
o Has ph dependence of rnase a activity.
o His 12 acts as a general base- abstracts a proton from oh in rna.
Promotes, na on on adjacent
o His119- general acid will cleave phosphoester bond.
o Reverse roles- his 119 acts as a base, takes proton from waterpromote its na. his 12 is acid- hydrolysis of cyclic phosphodiester
bond.
VHR structure
o A phosphatase
o Will mediate the dephosphorylation of a kinase.
o Needs phosphorylation to be active.
o pERK = is a small peptide derived from the erk kinase.
Phosphotyrosine residue. Active site harbors 3 residues- d92,
c124, r130.
o Know the letters, side chains.
o Active site contains h123.
VHR catalysis
o Arginine is stabilizing the erk. Without it wont be stable.
o Erk can transiently bind to active site.

o Key role in arginine stabilizing substrate in active site.

o Cysteine acts as a nucleophile.
o Thiolate anion- serves as a nucleophile and attacks the
phosphate. Allows the erk to disappear.
VHR conundrum
o Histidine is a preceding residue
o

2.6 Enzyme kinetics

Y is n order of magnitude greater than xFirst order reaction
Rate of decay is da/dt = -ka
-da/dt = dp/dt = ka = k (ao p )
k is in s-1.
Ao = initial concentration
lnA = -kt + ln(Ao)
second order kinetics
a binds to a ligand, l = p
rate of decay of a is dependent on conc of l at time t
k is second order- depends on two.
Is in m-1s-1.
If conc a and l are equal= -ka2
K(initial conc a p formed)
1/a = kt + 1/ao straight line. Slope is K, y int is 1/a0
unit is M-1s-1

Pseudo first order

Ligand is greater than a.
L will remain more or less constant.
So a goes to p as L is a constant
K prime= kL
Units of k prime is s^-1 .
Exponential decay curve.
Lna = -kprimet +lna0
Michaelis menten
Enzyme binds to a substrate and makes an intermediate es, that then decays
into product p and enzyme.

Eo- initial enzyme conc

e- free enzyme, initially will be 0. Some will bind to substrate. Remaining
unbound ones are e.
s- substrate at t 0
if rate depends on conc of single species is first ordered
if depends on two species- is second order.
k-2 is negligible. After make a product, usually wont go back to enzyme and
substrate.
So known conc of substrate. Will drop and product formed
Enzyme- total enzyme conc will drop
Product form- increase.
E0 = e+ es. Enzyme is in two states- bound or unbound.
Rate of making es = subtract k-1 es bc is backward reaction. Less
formation of es. Same for k+2es, decr amount of es
Enzyme will saturate substrate- after some time, will release a steady staterate of formation equals the decay. Concentration of es doesnt change at all.
At steady state- change with respect to es and time is 0.
Km = (k-1 + k+2) / k+1 dont have to memorize.
Velocity depends on k+2 and ES.
V= dp/dt = k+2 (ES)
Michaelis menten eq= Vmax (S) / Km+(S)
If we assume rate is at half- Km = s, is the equilibrium value of the
substrate at half rate. Enzyme can reach its max rate at x amount of s, lower
the x the more efficient the substrate is to binding.
Flaw- dont really know where v max is, rather will linearize
Invert mm eq.
1/V = km +s / vmax s lineweaver burk plot.
Get a straight line- for 1/v versus 1/s
When y=0 get -1/km
MM kinetics- catalytic constant
Assess the catalytic efficiency- kcat
Maximum velocity reached/ eo
The catalytic constant is equal to k+2 = is the number of cycles per unit
time. Is the frequency or heart rate. Compare two enzymes- greater turnover
number is better. Doesnt tell us how good an enzyme is bc maybe cant
bind substrate efficiency.

Need to understand at how good the enzyme is to bind substrate.

Km- how good binding substrate.
During initial burst phaseAt the beginning s is very small, and amount of free enzyme is equal to the
amount we added, small amount is actually used up.
Combine the equations- velocity is proportional to the product substrate
enzyme to kcat.
Kcat is per second, km is per molar.
Kcat/km = indicates how efficiently an enzyme can convert a substrate into
product. Is second order rate constant units of kcat/km
Upper limit of kcat/km = 10^9. Diffusion of in and out of enzymes limit it.
2.6b Enzyme inhibition
know mm and lineweaver.
Competitive- incr km
Uncompetitive- decr both
Mixed- decr vm, km alters
1.

Competitive inhibition
Inhibitor resembles the substrate. Action by binding to the substrate.
Competes (i) with s to bind to e for active site
Cant be converted to p
Ki = reactants/ products.
Will reduce the conc of available enzyme for substrate binding= Km is
increased. By alpha
Increased because inhibitor is competing with the substrate (thinks
that there is less enzymes- need much more substrate in order to
compete with inhibitor)
Alpha is greater than or equal to one.
Increased so multiply the term.
Once reach vmax- need a lot of s.
Lineweaver
o For competitive inhibition- increase by alpha. Multiply by alpha.
o Intercept is a constant. Incr inhibitor conc- have lines at 1/vmax
cross there.
Uncompetitive
o Inhibitor doesnt compete with substrate
o Substrate is noncompetitive
o Only binds to the ES, not the free enzyme!

o Only binds to the ES

o Must bind to a site other than the active site. Will bind to
anywhere on the enzyme other than the active site- allosteric
site.
o Competitive binds to active site, uncompe- allosteric site.
o At half maximum- if add uncompetitive- will bind to enzyme
substrate. Will reduce both km and vmax.
o In competitive inhibitor- can recover enzyme by adding more
substrate
o Here we cannot reverse the inhibitor- cannot recover es.
o Divide equation by alpha in order to reduce it by alpha prime.
o Dont memorize eq.
o Alpha prime greater than or equal to 1.
o Lineweaver
Replace 1 with alpha prime
Slope is the same for all inhibitor concentrations. Since
doesnt affect the km/vmax.
Will never intersect either at the x and y axis.
MIXED
o Non competitive.
o Mixed inhibitor binds to both E and ES .
o Will bind es at a allosteric site other than the active site.
o Inhibitor also binds to a allosteric site other than a active site
when binding to E also bc I can only bind to one site.
o Km and vmax are modulated. Some enzyme is locked- cant
participate in reaction so vmax is reduced.
o Divide and multiply km by alpha.
o Alpha > ap km increase
o Can only intersect at some part.
Know mm
Lineweaver- know
Learn how to derive competitive, uncomp, mixed.