proteins and enzymes 2

why do proteins have diverse structures and functions

different amino acid sequences with different side chains

common features of ⍺-amino acids

⍺-carbon (chiral centre) with attachments:

an amine group (-NH₂) which can be protonated

a carboxyl group (-COOH) which can be deprotonated

a hydrogen atom

a side chain of R-group

what determines amino acid properties

the R group (side chain)

why is the ⍺-carbon usually chiral

it is attached to four different groups

which amino acid is not chiral

Glycine

what is unusual about proline

its side chain forms a ring with the amine group

at low pH what form does the amino group take

protonated NH₃⁺ (and COOH)

at high pH what form does the carboxyl group take

deprotonated COO^- (and NH₂)

zwitterion

at neutral pH, amino acid: NH₃⁺ and COO^-

enantiomers

non-superimposable mirror images - stereoisomers

which stereoisomer is used in proteins

L amino acids

why are ⍺-helices usually right handed

proteins contain L amino acids

Fischer projection

drawn with carboxyl group on top and side chain on the bottom

how to identify L amino acids in Fischer projections

amine group on the left

where were D amino acids originally thought to only exist in

bacteria - but are now known to occur across plants, invertebrates and vertebrates including mammals

what important free D amino acid acts in mammalian brains

D-serine

D-serine

found in mammalian brains

important signalingi molecules - could serve as early biomarkers

what receptor does D-serine act on

NMDA receptor

how do D-AA-containing peptides (DAACPs) arise

spontaneous isomerisation - certain amino acids, especially aspartic acid, can convert non-enzymatically into heir D form in aging, this can disrupt protein structure and function, D-Asp has been detected in ⍺A crystallin from cataract patient’s eye lenses, D-Asp also occurs in β-amyloid in the brains of Alzheimer’s patients

enzyme-catalysed post-translational modifications (PTMs) - Dermorphin, an opioid peptide from frog skin, contains D-Ala even though its gene encodes L-Ala, peptide isomerase activity has been found in frog skin secretions

non-ribosomal peptide synthesis - bacteria incorporate D-Ala and D-Glu onto their cell walls using specific enzymes, many antibiotics with D-AAs, such as penicillin (contains D-Val) and gramicidin S (contains D-Phe) are made by step by step specialised non-ribosomal peptide synthesases

which bacterial cell wall amino acids are D forms

D-Ala and D-Glu

what disease-associated protein contains D-Asp

β-amyloid

number of common amino acids

20

essential amino acids

must be obtained from diet:

• Histidine

• Isoleucine

• Leucine

• Lysine

• Methionine

• Phenylalanine

• Threonine

• Tryptophan

• Valine

conditionally essential amino acids (essential under certain circumstances - illness, injury, stress)

• arginine

• asparagine

• glutamine

• glycine

• proline

• serine

• tyrosine

non-essential amino acids

this enrich the body is capable of synthesising

phenylketonuria

an inherited metabolic disorder where ether body cannot properly convert the amino acid phenylalanine into tyrosine due to a deficiency in the enzyme phenylalanine hydroxylase (so tyrosine is conditionally essential)

this leads to a deficiency in the enzyme phenylalanine and low levels of tyrosine

the main treatments is a lifetime diet with very limited intake of foods with phenylalanine

non-proteinogenic amino acids (npAAs)

amino acids not incorporated into proteins

what percentage of total amine acids known to exist in nature do the 20 in proteins (plus selenocysteine and pyrrolysine) make up

~2%

what is L-DOPA derived from

tyrosine (and then L-DOPA is converted to dopamine in neurones)

what neurotransmitter is produced from L-DOPA

dopamine

what disease is treated with L-DOPA

Parkinson’s disease

felinine

found in urine of cats - associated with the characteristic cat urinary odour

derived from 3-MBCG with the help of cauxin

amino acid properties categories

hydrophobic

polar

positively/negatively charged

aromatic

aliphatic

small

tiny

C-β branching

negatively charged amino acids

glutamic acid - Glu - E

aspartic acid - Asp - D

positively charged amino acids

Histidine - His - H

Lysine - Lys - K

Arginine - Arg - R

small amino acids

Aspartic acid - Asp - D

Proline - Pro - P

Asparagine - Asn - N

Threonine - Thr - T

Valine - Val - V

tiny amino acids

Serine - Ser - S

Alanine - Ala - A

Glycine - Gly - G

Cysteine - Cys - C

small and tiny amino acids

C-β branching

Threonine - Thr - T

Valine - Val - V

Isoleucine - Ile - I

aliphatic amino acids

Valine - Val - V

Isoleucine - Ile - I

Leucine - Leu - I

essential amino acids

Histidine - His - H

Isoleucine - Ile - I

Leucine - Leu - L

Lysine - Lys - K

Methionine - Met - M

Phenylalanine - Phe - F

Threonine - Thr - T

Tryptophan - Trp - W

Valine - Val - V

(Hot Wet MILF Takes Kielbasa)

hydrophobic amino acids

Methionine - Met - M

Isoleucine - Ile - I

Leucine - Leu - L

Phenylalanine - Phe - F

Valine - Val - V

Alanine - Ala - A

Glycine - Gly - G

Proline - Pro - P

Tryptophan - Trp - W

(MILF VAG Pisses Water)

E484K mutation

Glu → Lys subsitituoin at positon 484

which amino acids contain sulphur

M and C

amphipathic (have polar and non-polar character) amino acids

C

T

W

Y

K

H

(tiny wet cunts have yellow knots)

which amino cid is tiny and flexible and hwy does that make it structurally important

Glycine - fits into tight turns inaccessible to other residues

which amino acids are branched-chain amino acids (and also aliphatic)

V

L

I

(M not classified as aliphatic as it is very small)

which amino acids contain hydroxyl groups (polar, uncharged)

S

T

which amino acids contain amide groups (polar, uncharged)

N

Q

polar, uncharged amino acids

S (hydroxyl group)

T (hydroxyl group)

N (amide group)

Q (amide group)

acidic amino acids (negatively charged)

D

E

basic amino acids (positively charged)

K (second amino group)

R (guandino group)

H (imidazole group)

where are polar and charged amino acids found

usually on surface of proteins, when buried in the protein they are involved in salt bridges

why is histidine special

pK_a near physiological pH - this means it is important in enzymes as it can serve as a proton donor/acceptor

aromatic amino acids (relatively non-polar and participate in hydrophobic interactions)

F

Y

W

Y & W are significantly more polar than F because of the hydroxyl group in Y and the nitrogen of the W indole ring

Y & W and to a much lesser extent F, absorb ultraviolet lights - accounts for the characteristic strong absorbance o flight by most proteins at a wavelength of 280nm

wavelength of light all AAs absorb due to the peptide bond

190-220nm

proline structure

sidechain forms a cyclic ring with backbone nitrogen - a five-membered nitrogen-containing ring

also has a NH₂⁺ rather than an NH₃⁺ group in its isolated form at physiological pH

how many times is the proline side chain connected to the backbone

twice - this ring structure holds proline residues in a rigid conformation, reducing the structural flexibility of polypeptide regions contains proline

effect of proline on flexibility

reduces flexibility

why is proline often found in turns

rigid strutcure favours turns - meaning prolines are often found on the protein surface

which peptide bond configuration is more common generally

trans

which amino acid forms cis peptide bonds

proline - ~6% of the bonds are in cis configuration

what bond forms between two cysteines

disulphide (cysteine is readily oxidised to form covertly linked dimeric amino acid called cystine)

cystine

the oxidised cysteine dimer

role of disulphide bonds

covalent stabilisation of protein structure

PTMs

post translational modifications - chemical modifications after translation catalysed by specific enzymes which target specific residues

common PTMs

phosphorylation (on S, T, Y residues) —-

hydroxylation (P, K)

acetylation (S, T, K) —-

methylation (K, E)

carboxylation (E)

AMPylation (T, Y)

N- or O-glycosylation (S, T, N) —-

lipidation (C, S, K)

disulphide bond formation (C)

ubiquitylation (K) —-

SUMO-ylation (K)

proteolysis and deamination (N, Q) - irreversible PTMs —-

enzyme which adds phosphate groups (phosphorylation)

kinase

enzyme which removes phosphate groups (dephosphorylation)

phosphatase

effect of histone acetylation

opens chromatin

enzyme in histone acetylation

acetyl transferases transfer acetyl groups from acetyl-CoA to lysine residues on histones, neutralising their positive charge - this weakens the DNA, leading to a less condensed and more accessible chromatin structure

enzyme which removes acetyl groups from histones

histone acetyl detransferases - removes acetyl groups form lysine,, causing the chromatin to compact

erythropoietin glycosylation

the blood-stimulating hormone erythropoietin has N- and O- linked oligosaccharides contributing to its function and stability

ubiquitylation

involves the addition of a small protein called ubiquitin on to other proteins

involves a large family of proteins, the E2 and E3 ligases

deubiquitinating enzymes reverse this process

most associated with protein degradation

proteases

enzymatically hydrolyse peptide bonds in substrate proteins, resulting in a widespread, irreversible PTM if the protein’s structure and biological function

so the proteins re irreversibly cleaved

proteolysis of prothrombin

cleaved at two sites and converted to active thrombin

prothrombin is rich in 𝛾-carboxyglutamate which can bind to Ca²⁺ ions, crucial for the activation of prothrombin to thrombin

collagens

proteins that form fibrous structures and are abundant in the body - present in skin, blood vessel walls, bones, teeth and more

collagen molecule

three polypeptide chains with many repeating Gly-X-Y sequences where X is often proline and Y is often hydroxyproline

the three chains coil together in a tight triple helix

every third residue passes though the crowded centre of the hex, where only GLy can fit

the bulky, rigid proline and hydroxyproline residues give the entire structure its strength and stability

main repeating collagen sequence

Gly-X-Y

where X is often porcine and Y is often hydroxyproline

why si glycine essential in collagen

only residue small enough for triple helix centre

hydroxyproline generation

by PTM of proline by the enzyme proline hydroxylase which requires vitamin C (ascorbic acid) for activity

vitamin C deficiency causes

lower levels of hydroxyproline and therefore collagen fibres cannot form, leading to scurvy

scurvy

characterised by fragile blood vessels, skin lesions, poor wound healing and if untreated, death

vitamin C deficiency

21st and 22nd amino acid

Selenocysteine - Sec - U

Pyrrolysine - Pyl - O

stop codon which encodes selenocysteine

UGA

stop codon which encodes pyrrolysine

UAG

where is selenocysteine found

in active sites of a small number of selenoproteins in archea, bacteria and eukaryotes

where is pyrrolysine found

in a small number of row methanogenic archea and bacteria

Pyl synthesis and incorporation

from Lys by three enzymes

ligated directly to tRNA^Pyl and inserted into proteins in response to UAG codons

Sec synthesis and incorporation

synthesised from Set on its cognate tRNA (tRNA^Sec) by one enzyme in bacteria nd two in archaea and eukaryotes

during translation, Sec-tRNA^Sec is delivered to the ribosome by a specific elongation factor (via another protein in archaea and eukaryotes) that requires a characteristic stem-loop structure in the mRNA to actively recode an inframe UGA from stop codon to Sec sense codon

pK_a

pH where acid is 50% dissociated

relationship between pH and pK_a

pH = pK_a + log₁₀(A^-/HA)

the lower the pK_a, the … the acid

stronger - and the more easily it dissociates

if pH < pK_a, which form dominates

protonated form

if pH > pK_a, which form dominates

deprotonates form

zwitterion

molecule with both positive and negative charges but net charge 0

alanine net charge at low pH

+1 - acid dissociation constant at ~2 for the -COOH group so above 2 it becomes COO^-

alanine net charge at high pH

-1- acid dissociation constant at ~9.5 for the NH₃⁺ so above 9.5 it is NH₂

isoelectric point (pl)

pH where net charge = 0

pK_a of side chain of aspartic acid

~4