Biochem Fu exam 1

DNA Polymerase

DNA Polymerase

enzyme- catalyzes DNA replication

RNA Polymerase

enzyme- Transcription

Hemoglobin

transport protein- transport O2 in blood

Myoglobin

transport protein- storage of oxygen

lactose permease

transport protein- transport lactose across cell membrane

collagen

structural - connective tissue

keratin

structural - hair, nails, horns ; contaminants of MS assay

myosin

protein for motion- muscle tissue ; thicker filament

actin

motion protein- muscle tissue, cell motility ; thinner filament

Luciferase

enzyme that works with cofactor Mg2+ to turn ATP (energy) and Luciferin to oxyluciferin and light. How fireflies light up.

General Structure of an amino acid:

carboxyl group, amino group, chiral center, r group. R group — (side chain) amino acids differ. Glycine is achiral, H for R group, smallest aa.

Exception to Amino acid Structure:

Proline- has a cyclic amino acid

Nonpolar Aliphatic R Groups: (7)

Glycine-G, Alanine-A Proline-P Valine-V Leucine-L, Isoleucine-I, Methionine-M

ph: 7

Aromatic R groups: (3)

Phenylalanine- F, Tyrosine-Y, Tryptophan-W

Absorb UV light at 270-280 nm

Non-polar hydrophobic amino acid
Hydrophobic: normally buried inside the protein core.

Y and W absorb UV light more than F

W=4Y

Polar Uncharged R groups (5)

Serine-S, Threonine-T, Cysteine-C, Glutamine-Q, Asparagine-N

Amino acids side chains can form hydrogen bonds.
soluble in water

Cysteine can form disulfide bonds

Positively Charged R Groups (3)

Lysine-K, Arginine-R, Histidine-H

Histidine has imidazole group

Ampholytes

substance with dual acid and base nature

phosphorylation

most common type of regulatory modification- involved in protein activity

Peptide Bonds

small condensation products of amino acids
small compared to proteins

• Mw < 10 kDa

Peptide ends (not same)

Amino-terminal end and carboxyl terminal end. Name/Number starting with amino terminus.

Average molecular weight of amino acids

138da

smaller amino acids predominant in proteins: 128da

Protein Extraction

grinding w/wo liquid nitrogen
grinding with sand
bead beater
sonication
french press
buffer w/wo mild detergent

protein precipitation

ammonium sulfate- add saturated solution to sample with buffer and protein will precipitate out

dialysis

selectively permeable membrane- large enzyme molecules cannot pass through pores in membrane

small molecules pass through and equilateral across membrane

SDS Page

SDS- sodium dodecyl phosphate-detergent- binds and unfolds all proteins, gives uniform negative charge no matter shape, rate of movement will depend on size, so smaller molecules will move faster.'

The bound SDS will contributes a negative charge, unmask the intrinsic charge of the protein. Therefore, all protein will have a similar charge to mass ratio. Migration purely based on the molecular weight.

Protein Purification- metal binding

Purification of his-tagged protein.

lyse bacteria cells to release
protein- incubate with nickel NTA agarose beads- wash with salt solution-elute using imidazole competitor- sds page

Fragmenting Polypeptide Chains: Trypsin

trypsin predominantly cleaves proteins at the carboxyl side (or "C-terminal side") of the amino acids lysine and arginine except when either is bound to a C-terminal proline, although large-scale mass spectrometry data suggest cleavage occurs even with proline.

Fragmenting Polypeptide Chains- Submaxillary protease

Cleaves Agr- c terminus

Chymotrypsin

Cleaves Phe,Trp,Tyr- Cterminus

globular protein

S aureus

cleaves Asp, Glu- c terminus

Asp-N-Protease

cleaves Asp, Glu- N terminus

Endoprotease lys C

cleaves lys- c terminus

Cyanogen bromide

cleaves methionine- c terminus

Levels of structure in proteins

Primary, secondary tertiary, quaternary

Primary structure

amino acid residues- sequence, peptide bond, and disulfide bond
polypeptide is made up of a series of linked planes at a carbons

secondary structure

local spatial arrangement of the peptide backbone
two common: a helix, b sheet

irregular arrangement is called random coil

a helix

stabilized by hydrogen bonds between nearby residues.

Helical backbone is held together by hydrogen bonds between the backbone amides of an n and n+4 amino acids

Right-handed helix with 3.6 residues (5.4 Å) per turn

Peptide bonds are aligned roughly parallel with the helical axis

Side chains point out and are roughly perpendicular with the helical axis

too small to fit anything "inside"
happens to fit well in the major groove of dsDNA

Affects on a helix stability

Not all polypeptide sequences adopt a-helical structures

Small hydrophobic residues such as Ala and Leu are strong helix formers

Pro acts as a helix breaker because the rotation around the N-Ca bond is impossible

Gly acts as a helix breaker because the tiny R-group supports other conformations

Attractive or repulsive interactions between side chains 3-4 amino acids apart will affect formation

b sheet

stabilized by hydrogen bonds between adjacent segments that may not be near by.

Parallel or antiparallel orientation of two chains within a sheet are possible
In parallel b sheets the H-bonded strands run in the same direction
Resulting in bent H-bonds (weaker)
In antiparallel b sheets the H-bonded strands run in opposite directions
Resulting in linear H-bonds (stronger)

silk fibroin

main protein in silk for moths and spiders

antiparallel B sheet structure

small side chains (Ala + Gly)= close packing of sheets

structure stabilized by
hydrogen bonding within sheets
and London dispersion interactions between sheets

spider silk is extremly strong, composite material-crystalline and rubber like parts

X-ray crystallography

Measure the locations and intensities of spots produced by a beam of X-ray

Steps needed
Purify the protein
Crystallize the protein
Collect diffraction data
Calculate electron density
Fit residues into density
Pros
No size limits
Well-established
Cons
Difficult for membrane proteins
Cannot see hydrogens

Biomolecular NMR

Nucleic magnetic resonance: manifestation of nuclear spin angular momentum.

Static magnetic field is applied: nuclear spin generates dipoles

Steps needed
Purify the protein
Dissolve the protein
Collect NMR data
Assign NMR signals
Calculate the structure
Pros
No need to crystallize the protein
Can see many hydrogens
Cons
Difficult for insoluble proteins
Works best with small proteins

denaturation

Loss of structural integrity with accompanying loss of activity

Proteins can be denatured by:
heat or cold
pH extremes
organic solvents
chaotropic agents: urea and guanidinium hydrochloride

Ribonuclease refolding experiment

Ribonuclease is a small protein that contains 8 cysteines linked via four disulfide bonds

Urea in the presence of 2-mercaptoethanol fully denatures ribonuclease

When urea and 2-mercaptoethanol are removed How, the protein spontaneously refolds, and the correct disulfide bonds are reformed

The sequence alone determines the native conformation

Quite "simple" experiment, but so important it earned Chris Anfinsen the 1972 Chemistry Nobel Prize

Chaperone Proteins prevent misfolding

1. prevent aggregation of unfolded peptides, Hsp70 bind to regions of unfolded peptide that are rich in hydrophobic residues to protect from denaturation from heat and

2. Prevent new peptide being synthesized

3. Block the folding of certain proteins until they are translocated across the membrane
4. Facilitate quaternary assembly

protein misfolding

basis of numerous human diseases

Amyloidoses: Alzheimer disease, Huntington disease, Parkinson diseases
Proteolytic cleavage of this larger protein leaves the relatively unstable amyloid-β peptide, which loses its α-helical structure. It can then assemble slowly into amyloid fibrils

Formation of disease-causing amyloid fibrils.

The aromatic side chains shown here play a significant role in stabilizing the amyloid structure.

Protein Binding

interaction strength can be expressed as Ka (units M^-1) or Kd (units M) Kd=1/Ka

Strong binding- Kd<10nM
weak binding - Kd> 10uM

Lock and key model

model by Emil fisher- assumes complementary surfaces are preformed

High specificity

proteins typically have it and only bind to certain ligands; explained by the complementary of the binding site and the ligand

Complementary in (high specificity)

size, shape, charge, or hydrophobic/hydrophilic character

Induced fit

Conformational changes may occur upon ligand binding

Induced fit allows for tighter binding of the ligand

Induced fit allows for high affinity for different ligands

Both the ligand and the protein can change their conformations

• myoglobin- Heme binding to protein. The bound O2 is hydrogen-bonded to the distal His, His E7 (His64), further facilitating the binding of O2.

Allosteric Protein

Binding of a ligand to one site affects the binding properties of a different site, on the same protein

Can be positive or negative

homotrophic

Normal ligand of the protein is the allosteric regulator

heterotrophic

Different ligand affects binding of the normal ligand

cooperativity

positive homotropic regulation

• hemoglobin- is a tetramer of two subunits (a2b2)

• Each subunit is similar to myoglobin

hemoglobin T state

T=tense state
more interactions, more stable
lower affinity for oxygen.

oxygen binding triggers a conformational change from T to R

hemoglobin R state

R=relaxed state
fewer interactions, more flexible
higher affinity for O2

T-R conformational change

O2 binding triggers conformational change

involves breaking ion pairs between the α1-b2 interface

pH effect on O2 Binding to hemoglobin(Hb)

Actively metabolizing tissues generate H+, lowering the pH of the blood near the tissues relative to the lungs

Hb Affinity for oxygen depends on the pH

H+ binds to Hb and stabilizes the T state

Protonates His146 which then forms a salt bridge with Asp94
Leads to the release of O2 (in the tissues)

Bohr Effect

The pH difference between lungs and metabolic tissues increases efficiency of the O2 transport

2,3-Bisphosphoglycerate regulates O2 binding

Negative heterotropic regulator of Hb function

Present at mM concentrations in erythrocytes

Produced from an intermediate in glycolysis

Small negatively charged molecule,
binds to the positively charged
central cavity of Hb
Stabilizes the T states

sickle cell anemia

due to a mutation in hemoglobin

Glu6 --Val in the B chain of Hb
The new Valine side chain can bind to a different Hb molecule to form a strand
This sickles the red blood cells
Untreated homozygous individuals generally die in childhood
Heterozygous individuals exhibit a resistance to malaria

Formation of Hb strands

deoxyhemoglobin S has a hydrophobic patch on its surface, which causes the molecules to aggregate into strands that align into insoluble fibers.

Anemia is a condition in which you don't have enough healthy red blood cells to carry adequate oxygen to your tissues.

enzymes

Enzymes are catalysts
Increase reaction rates without being used up

Most enzymes are globular proteins

However, some RNA (ribozymes and ribosomal RNA) also catalyze reactions

Study of enzymatic processes is the oldest field of biochemistry, dating back to late 1700s
Study of enzymes has dominated biochemistry in the past and continues to do so

Holoenzyme:

complete function including coenzyme and metal ions

Apoenzyme:

protein part of the enzyme

Enzyme Substrate complex

enzmyes act by binding substrates

michaelis complex

The noncovalent enzyme substrate complex

Enzymatic Catalysis

Enzymes do not affect equilibrium (ΔG)
Slow reactions face significant activation barriers (ΔG‡) that must be surmounted during the reaction
Enzymes increase reaction rates (k) by decreasing ΔG‡

oxidoreductases

transfer of electrons (hydride ions or H atoms)

transferases

group transfer reactions

hydrolases

hydrolysis reactions (transfer of functional groups to water)

lysases

cleavage of c-o,c,n bonds, or other bonds by elimination, leaving double bonds or rings, or addition of groups to double bonds

isomrases

transfer of groups within molecules to yeild isomeric forms

ligases

formation of c-c,o,n,s bonds via condensation reactions coupled to cleavage of ATP or similar cofactor

Nonlinear Michaelis-Menten plot

should be used to calculate parameters Km and Vmax

Linearized double-reciprocal plot

good for analysis of two-substrate data or inhibition

enzyme inhibitors

compounds that decrease enzyme’s activity

irreversible inhibitors (enzyme inhibition)

React with the enzyme

One inhibitor molecule can permanently shut off one enzyme molecule

They are often powerful toxins but also may be used as drugs

Reversible inhibitors (enzyme inhibition)

bind to and can dissociate from the enzyme

They are often structural analogs of substrates or products

They are often used as drugs to slow down a specific enzyme

Reversible inhibitor binding (enzyme inhibition)

to the free enzyme and prevent the binding of the substrate
to the enzyme-substrate complex and prevent the reaction

competitive inhibition

Competes with substrate for binding

Binds active site
Does not affect catalysis


No change in Vmax; apparent increase in KM
Lineweaver-Burk: lines intersect at the y-axis

uncompetitive inhibition

Uncompetitive inhibitors bind at separate site, but bind only to the ES complex

mixed inhibition

Binds enzyme with or without substrate

Binds to regulatory site
Inhibits both substrate binding and catalysis

Decrease in Vmax; apparent change in KM

Lineweaver-Burk: lines intersect left from the y-axis

Noncompetitive inhibitors are mixed inhibitors such that there is no change in KM

hexokinase

undergoes induced fit on substrate binding

u shape

Catalytic active form
The ends pinch towards each other after binding of D-glucose.

acid base catalysis

Charged intermediates are stabilized by transfer of protons to or from the substrate or intermediate to form a species that breaks down more readily to products.

• H3O+ (specific acid catalysis) or HA (general acid catalysis)

covalent catalysis

A transient covalent bond between the enzyme and the substrate
Changes the reaction Pathway


Requires a nucleophile on the enzyme

Can be a reactive serine, thiolate, amine, or carboxylate